Targeting the PBX1–BCL2L1 Axis as a Therapeutic Strategy in Colorectal Cancer

Targeting the PBX1–BCL2L1 Axis as a Therapeutic Strategy in Colorectal Cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Targeting the PBX1–BCL2L1 Axis as a Therapeutic Strategy in Colorectal Cancer Lingzhu Xie, Hao Lin, Ting Su, Rulan Deng, Jie Li, Ying Liu, Xuanhao Lin, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7305360/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Pre-B-cell leukemia homeobox 1 ( PBX1 ) is a transcription factor involved in diverse cellular processes, but its role in colorectal cancer (CRC) remains incompletely understood. In this study, we show that PBX1 is downregulated in CRC tissues and cell lines. Functional experiments revealed that PBX1 overexpression inhibits proliferation, migration, and invasion, but paradoxically suppresses apoptosis, suggesting a dual regulatory role. Transcriptome and CUT&Tag profiling identified BCL2L1 as a direct transcriptional target of PBX1. PBX1 binds the BCL2L1 promoter and enhances Bcl-xL expression, contributing to apoptotic resistance. BCL2L1 knockdown reversed the anti-apoptotic effects of PBX1 and restored apoptosis levels. Upon 5-fluorouracil (5-FU) treatment, PBX1 overexpression reduced cell viability, while concurrent BCL2L1 knockdown significantly enhanced drug sensitivity. In vivo, xenograft experiments demonstrated that PBX1 overexpression suppressed tumor growth, which was further augmented by BCL2L1 knockdown. These results underscore the dual role of PBX1 in simultaneously inhibiting tumor growth while promoting cell survival through the BCL2L1 –Bcl-xL axis. Targeting this pathway could offer a novel therapeutic strategy for enhancing CRC treatment efficacy by simultaneously inhibiting proliferation and restoring apoptotic sensitivity. Biological sciences/Cell biology/Mechanisms of disease Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Colorectal cancer (CRC) is the third most common cancer worldwide, representing 10.0% of all cancer cases, and the second leading cause of cancer-related deaths, contributing to 9.4% of total cancer deaths in 2020 [ 1 ]. Despite advancements in surgical techniques, chemotherapy, and targeted therapies, the recurrence and mortality rates for CRC remain high [ 2 ]. Tumor recurrence, driven by the complex interplay of molecular pathways regulating cell proliferation, survival, and apoptosis, continues to challenge effective treatment. Understanding the key molecular regulators involved in these processes is crucial for developing more effective therapies and improving patient outcomes. Among the transcription factors implicated in cancer biology, the Pre-B-cell leukemia homeobox 1 ( PBX1 ) gene has garnered attention for its dual role in various cancers[ 3 – 6 ]. PBX1 , a member of the TALE (three amino acid loop extension) homeodomain family, is recognized for its role in developmental processes and gene expression regulation[ 7 – 9 ]. Depending on the cellular and molecular context, PBX1 can act as either a tumor suppressor or an oncogene in different types of cancer. For instance, in pre-B cell acute lymphoblastic leukemia, PBX1 was first identified as an oncogene through the t(1;19) chromosomal translocation, which produces the E2A–PBX1 fusion protein that drives leukemogenesis [ 10 ]. Similarly, PBX1 has been shown to promote tumor progression, chemoresistance, and stemness in breast, ovarian, and clear cell renal carcinomas [ 11 , 12 ]. However, in CRC, PBX1 appears to play a distinct role. While PBX1 expression is frequently downregulated in CRC tissues, forced overexpression of PBX1 has been shown to suppress tumor cell proliferation, migration, and invasion, suggesting a tumor-suppressive function [ 13 ]. However, the precise mechanisms by which PBX1 influences CRC cell fate remain poorly understood. In addition to its role in cell proliferation, PBX1 has been implicated in regulating apoptosis across various cancers. In lung cancer, PBX1 silencing induces apoptosis through ROS production and inhibition of the STAT3–Bcl-2 –Survivin pathway [ 14 ]. In breast cancer, PBX1 is negatively regulated by the lncRNA uc.38, which induces apoptosis by downregulating Bcl-2 family proteins [ 15 ]. Moreover, in prostate cancer, PBX1 's stability, regulated by the deubiquitinase USP9x, plays a critical role in resistance to apoptosis, positioning PBX1 as a potential therapeutic target to overcome chemoresistance [ 16 ]. These studies underscore PBX1 's critical role in the regulation of apoptosis and suggests that similar mechanisms may exist in other cancers. Apoptosis, a crucial mechanism for maintaining cellular homeostasis and preventing tumor growth, is often disrupted in cancer, leading to tumor recurrence and resistance to treatment. The Bcl-2 family of proteins, particularly BCL2L1, plays a pivotal role in regulating apoptosis. BCL2L1 encodes two isoforms: Bcl-xS, which promotes apoptosis, and Bcl-xL, which inhibits apoptosis and supports cell survival[ 17 – 19 ]. These isoforms are generated by alternative splicing, resulting in structural differences that influence their roles in apoptosis regulation [ 20 ]. Bcl-xL, with its intact BH1, BH2, and C-terminal hydrophobic domain, prevents apoptosis by binding to and inhibiting pro-apoptotic proteins such as Bax and Bak[ 21 ]. In contrast, Bcl-xS, lacking key anti-apoptotic domains, allows apoptotic pathways to proceed [ 22 ]. While several upstream signaling pathways and splice factors have been shown to modulate the balance between Bcl-xL and Bcl-xS [ 17 , 23 ], the transcriptional regulation of BCL2L1, particularly in CRC, remains poorly defined. This gap in understanding is crucial, as apoptosis and proliferation are closely linked in tumor progression. Our study investigates how PBX1 influences cell both cell proliferation and apoptosis, focusing on its regulation of BCL2L1 . By defining the PBX1 – BCL2L1 axis, this research may provide new therapeutic insights into strategies that simultaneously suppress tumor growth and enhance apoptosis sensitivity in CRC. 2. Result 2.1 Downregulation of PBX1 Expression in Colorectal Cancer Tissues and Cells Analysis of both TCGA and Oncomine databases revealed a significant reduction in PBX1 mRNA expression in CRC tissues compared to normal tissues, suggesting a potential tumor-suppressive role for PBX1 in CRC. Specifically, TCGA RNA-Seq data[ 24 ] showed that PBX1 expression was significantly lower in CRC tissues than in adjacent normal tissues ( P < 0.001) (Fig. 1 A). Consistent with this, analysis of the "Skrzypczak Colorectal" and "Skrzypczak Colorectal 2" datasets [ 25 ], from Oncomine further confirmed PBX1 downregulation in CRC tumor tissues, with fold changes of -2.036 and − 2.147, and P-values of 4.40 × 10 − 8 and 1.03 × 10 − 6 , respectively (Fig. 1 B, 1 C). To validate these database findings at the clinical level, we analyzed tumor and adjacent non-tumor tissues from 50 CRC patients. qPCR and Western blot analyses confirmed that PBX1 expression was significantly lower in tumor tissues compared to adjacent normal tissues (Fig. 1 D-F), reinforcing the hypothesis that PBX1 may act as a tumor suppressor in CRC. To further substantiate this observation at the cellular level, we examined PBX1 expression in multiple CRC cell lines (HCT116, HT-29, LoVo, RKO, SW480, SW620) and the normal colon cell line CCD-18Co. The results demonstrated that PBX1 expression was significantly downregulated at both the mRNA and protein levels in CRC cell lines (Fig. 1 G-I), providing additional support for the idea that PBX1 underexpression is a consistent feature of CRC, occurring across both patient tissues and in vitro cellular models. 2.2 PBX1 generally functions as a tumor suppressor in CRC Building on the observed downregulation of PBX1 in CRC tissues and cell lines, we further investigated its functional role in CRC progression by modulating its expression in vitro. Knockdown of PBX1 in HCT116 and RKO cells using shRNA significantly decreased PBX1 mRNA and protein levels (Fig. 2 A-F), which led to a a significant increase in cell proliferation, colony formation, migration and invasion (Fig. 2 G-R). Conversely, overexpression of PBX1 in HCT116 and RKO cells using a lentiviral vector significantly increased PBX1 mRNA and protein levels (Fig. 3 A-F), which led to a significant suppression in cell proliferation, migration, invasion, and colony formation (Fig. 3 G-R). These results suggest that PBX1 generally functions as a tumor suppressor in CRC. 2.3 BCL2L1 as a Potential Direct Target of PBX1 To investigate the downstream genes and pathways regulated by PBX1, we performed RNA sequencing (RNA-seq) analysis in HCT116 cells following PBX1 knockdown. The pathways significantly affected are summarized in Fig. 4 A, while representative differentially expressed genes are shown in Fig. 4 B. As Fig. 4 A shown, PBX1 knockdown in HCT116 cells led to significant enrichment of gene sets related to apoptosis, cell proliferation, and metastasis. The top-ranked pathway was the “Apoptosis-Related Network due to Altered Notch3” (NES = 2.10, FDR = 0.053), suggesting that reduced PBX1 expression may enhance pro-apoptotic signaling. Additional pathways enriched in the knockdown condition included “LDL Influence on CD14 and TLR4” and “Influence of Laminopathies on Wnt Signaling”, both associated with immune regulation and proliferative activity [ 26 ], as well as “TGF-β signaling in thyroid cells” and “Type 2 Papillary Renal Cell Carcinoma”, which are linked to epithelial-mesenchymal transition and metastatic potential [ 27 – 29 ]. Interestingly, while prior study has reported that low PBX1 expression is associated with enhanced CRC proliferation and invasion, and that PBX1 overexpression can suppress tumor growth [ 13 ], our data revealed a paradoxical enrichment of apoptosis-related signaling upon PBX1 knockdown. This suggests that although PBX1 overexpression may inhibit proliferative capacity in some contexts, it could simultaneously promote tumor cell apoptosis. Therefore, strategies aiming to suppress tumor growth via PBX1 overexpression may carry the unintended consequence of inhibiting tumor cell apoptosis, potentially leading to an accumulation of quiescent, less active, and drug-resistant tumor cells.These results underscore the need for further investigation into the relationship between PBX1 expression and apoptotic regulation in cancer. To further investigate the regulatory mechanisms underlying the transcriptional changes observed upon PBX1 knockdown, we performed a CUT&Tag assay in HCT116 cells using a PBX1-specific antibody to map genome-wide PBX1 binding sites. Given PBX1's established role as a transcription factor, we aimed to identify genes that are not only differentially expressed following PBX1 depletion but also directly bound by PBX1 on chromatin. Figure 4 C shows that PBX1 binding peaks are predominantly located in intergenic regions (40.29%) and intronic regions (39.94%), while approximately 14.49% of binding events occur near transcription start sites (TSS), suggesting PBX1 binds both distal and proximal regulatory elements. To determine the regulatory context of PBX1-bound regions, we analyzed histone modification signals at these loci, focusing on H3K4me3, H3K27ac, and H3K4me1—canonical markers for promoters and enhancers. As shown in the top of Fig. 4 D, the majority of PBX1 binding sites exhibited strong H3K4me3 and H3K27ac enrichment, along with relatively low H3K4me1 levels. This epigenetic signature is characteristic of active promoters, whereas we observed no clear evidence of enhancer-like regions defined by high H3K4me1 and H3K27ac but low H3K4me3. Based on this chromatin landscape, we annotated PBX1 binding peaks to nearby promoters and compiled a list of 701 genes with PBX1 occupancy at their promoter regions across three independent biological replicates (Fig. 4 E). To further narrow down functionally relevant targets, we intersected this list with the 89 differentially expressed genes identified from RNA-seq analysis. This integrative approach yielded four high-confidence candidate genes that are both transcriptionally regulated by PBX1 and directly bound at their promoters: DUSP5 , AP3S1 , PLK2 , and BCL2L1 (Fig. 4 F). Among the four candidate genes identified— DUSP5, AP3S1, PLK2 , and BCL2L1 , several have been previously linked to tumor-related processes. DUSP5 has been shown to regulate MAPK/ERK signaling and influence tumor cell proliferation and migration [ 30 , 31 ]. PLK2 is involved in cell cycle regulation [ 32 , 33 ], while AP3S1 remains less well characterized in cancer biology. BCL2L1 , which encodes the anti-apoptotic protein Bcl-xL, is frequently upregulated in tumors and associated with enhanced cell survival [ 34 – 36 ]. In addition, Fig. 4 G shows that BCL2L1 displayed the most significant change, with an adjusted p-value of 6.253 × 10 − 8 , indicating it is highly responsive to PBX1 depletion. In parallel, CUT&Tag profiling confirmed that PBX1 is strongly enriched at the promoter region of BCL2L1 (Fig. 4 H), supporting the likelihood that BCL2L1 is a direct transcriptional target of PBX1 in colorectal cancer cells. 2.4 PBX1-Driven Upregulation of BCL2L1 Promoter Activity in CRC Cells To elucidate the regulatory mechanism by which PBX1 modulates BCL2L1 expression, we performed ChIP-qPCR and dual-luciferase reporter assays. As illustrated in Fig. 5 A, we first cloned a ~ 1719 bp fragment of the BCL2L1 promoter region into the pGL3-Basic vector to construct a full-length promoter reporter plasmid. Based on the CUT&Tag data indicating PBX1 binding intensity, this promoter region was further subdivided into three overlapping fragments (Fragments 1, 2, and 3), which were individually cloned into the pGL3-Basic vector to assess PBX1 ’s effect on discrete regulatory elements. Luciferase reporter assays demonstrated that the full-length BCL2L1 promoter construct exhibited robust promoter activity in both HCT116 and RKO cells, with approximately 65-fold and 45-fold activation, respectively, compared to the empty vector control (Fig. 5 B). Knockdown of PBX1 via shRNA significantly reduced the promoter activity in both cell lines (Fig. 5 C), whereas stable overexpression of PBX1 led to a marked increase in promoter activity (Fig. 5 D). To further localize the critical PBX1 -responsive region within the promoter, we assessed the activity of the three truncated promoter fragments. In HCT116 cells, knockdown of PBX1 resulted in a significant decrease in luciferase activity driven by Fragment 3, while Fragments 1 and 2 showed minimal changes (Fig. 5 E). Conversely, PBX1 overexpression significantly enhanced the activity of Fragment 3 (Fig. 5 F). Similar results were observed in RKO cells, where PBX1 knockdown suppressed (Fig. 5 G) and PBX1 overexpression enhanced (Fig. 5 H) the activity of Fragment 3, indicating that this region contains a critical PBX1-responsive element. To verify the binding of PBX1 to the BCL2L1 promoter in vivo and assess its impact on chromatin activation, we performed ChIP-qPCR targeting PBX1 and H3K27ac. In the ChIP-qPCR assay, we assessed the enrichment of PBX1 at this region, as well as its impact on chromatin activation status, marked by the histone modification H3K27ac [ 37 ]. Based on previous studies showing that transcription factor binding sites and histone modifications such as H3K27ac often occur in adjacent but non-overlapping regions within regulatory domains [ 38 , 39 ], we designed ChIP-qPCR primers to independently capture the peak enrichment sites for PBX1 and H3K27ac, respectively, according to our CUT&Tag and luciferase reporter assays results. As shown in Figs. 5 I–L, both PBX1 and H3K27ac were significantly enriched at the BCL2L1 promoter in HCT116 and RKO cells, with enrichment levels being notably higher in HCT116 cells. PBX1 knockdown led to a substantial reduction in PBX1 and H3K27ac occupancy at the promoter region (Figs. 5 M and 5 O), whereas PBX1 overexpression increased their enrichment (Figs. 5 N and 5 P), suggesting that PBX1 binding facilitates chromatin activation of the BCL2L1 promoter through H3K27 acetylation. Collectively, these results demonstrate that PBX1 directly binds to a critical enhancer region within the BCL2L1 promoter, enhancing its transcriptional activity by modulating local chromatin accessibility. 2.5 PBX1-Mediated Upregulation of Bcl-xL Suppresses Apoptosis To further clarify the regulatory effect of PBX1 on BCL2L1 expression, we conducted both knockdown and overexpression experiments in HCT116 and RKO colorectal cancer cells. Specifically, we assessed how changes in PBX1 expression influence the two major BCL2L1 transcript variants, which encode the protein isoforms Bcl-xL and Bcl-xS. As described in the Introduction, Bcl-xL functions as an anti-apoptotic protein, whereas Bcl-xS promotes apoptosis. We therefore measured the mRNA levels of Bcl-xL and Bcl-xS separately. As shown in Fig. 6 A and 6 C, PBX1 knockdown significantly reduced the expression of Bcl-xL, while Bcl-xS levels remained unchanged. Conversely, overexpression of PBX1 led to a marked increase in Bcl-xL mRNA, with only a modest upregulation of Bcl-xS observed in RKO cells (Fig. 6 B and 6 D). These results suggest that PBX1 predominantly regulates the expression of the anti-apoptotic isoform Bcl-xL at the mRNA level in colorectal cancer cells. Consistent with the mRNA-level findings, our long-term observations from repeated WB experiments revealed that CRC cells predominantly express the Bcl-xL protein isoform (~ 30 kDa). Even under overexposed blot conditions, the Bcl-xS isoform (~ 18 kDa) was undetectable in endogenous settings (Fig. 6 E). Only upon exogenous overexpression of Bcl-xS from a plasmid could we detect the corresponding protein band in HCT116 cells (Fig. 6 F). Taken together, these results suggest that in CRC cells, PBX1 primarily regulates the Bcl-xL isoform of BCL2L1 . Given the predominance of the Bcl-xL isoform in CRC cells, subsequent protein-level analyses focused specifically on Bcl-xL. As shown in Fig. 6 G and 6 H, PBX1 knockdown in HCT116 cells led to a reduction in Bcl-xL protein levels, accompanied by an increase in apoptotic cells. Conversely, PBX1 overexpression resulted in elevated Bcl-xL protein expression and a decrease in apoptosis in HCT116 (Fig. 6 I and 6 J). Similar results were observed in RKO cells, where PBX1 overexpression upregulated Bcl-xL protein and reduced apoptosis, while PBX1 knockdown produced the opposite effect (Fig. 6 K–N). Together, these findings indicate that PBX1 modulates apoptosis in colorectal cancer cells primarily through regulation of the anti-apoptotic isoform Bcl-xL. 2.6 Functional Interaction Between PBX1 and BCL2L1 in Regulating Apoptosis and Tumor Growth To further explore the functional relationship between PBX1 and BCL2L1 in regulating tumor cell fate, we examined the effects of BCL2L1 knockdown in the context of PBX1 overexpression. As shown in Figs. 7 A and 7 B, overexpression of PBX1 in HCT116 cells led to a marked increase in Bcl-xL protein levels, while silencing BCL2L1 (shBCL2L1) effectively diminished Bcl-xL expression, confirming that PBX1 enhances Bcl-xL expression through transcriptional upregulation. Similar results were observed in RKO cells (Figs. 7 C and 7 D), indicating that this regulatory mechanism is consistent across CRC models. Apoptosis assays (Figs. 7 E-H) demonstrated that PBX1 overexpression alone significantly suppressed apoptosis in HCT116 and RKO cells. However, this anti-apoptotic effect was reversed upon BCL2L1 knockdown, with apoptosis levels restored to baseline, indicating that PBX1 modulates apoptosis predominantly through BCL2L1 -dependent Bcl-xL upregulation. To evaluate the impact of this regulatory axis on chemotherapy sensitivity, we treated HCT116 and RKO cells with increasing doses of 5-fluorouracil (5-FU) and assessed cell viability. As shown in Fig. 7 I, dose-response curves revealed IC50 values of 37.84 µM for HCT116 cells and 35.87 µM for RKO cells after 24-hour exposure. Based on these results, we selected a treatment concentration of 10 µM—approximately one-quarter to one-half of the IC50—to induce measurable drug effects without excessive cytotoxicity[ 40 ]. At this concentration, PBX1 overexpression alone significantly reduced cell viability in HCT116 cells (Fig. 7 J), suggesting enhanced drug sensitivity. Importantly, combined PBX1 overexpression and BCL2L1 knockdown led to an even more pronounced reduction in cell viability, highlighting a synergistic inhibitory effect and reinforcing the functional relevance of the PBX1 – BCL2L1 axis under chemotherapeutic stress. A similar trend was observed in RKO cells (Fig. 7 K), reinforcing the functional relevance and generalizability of the PBX1–BCL2L1 regulatory axis in modulating chemoresistance across CRC models. To validate these findings in vivo, we established xenograft models in nude mice with HCT116 cells and divided them into three groups: control (empty-pCDH + shNC), PBX1 overexpression (PBX1-pCDH + shNC), and PBX1 overexpression combined with BCL2L1 knockdown (PBX1-pCDH + shBCL2L1). As shown in Fig. 7 L, the control group developed the largest tumors, the PBX1 overexpression group showed moderate suppression, and the combination group exhibited the smallest tumor size, indicating enhanced tumor suppression when both interventions were applied. Figure 7 M shows that there were no significant differences in body weight across groups during the experiment, suggesting no overt toxicity from the treatments. Tumor volume monitoring over time (Fig. 7 N) confirmed that tumors in the PBX1 overexpression combined with BCL2L1 knockdown group grew significantly slower than those in the other groups, while no significant difference was observed between the control and PBX1 -overexpressing groups alone. Final tumor images (Fig. 7 O) and corresponding tumor weights (Fig. 7 P) further supported this trend: the control group had the heaviest tumors, followed by the PBX1 overexpression group, with the PBX1 overexpression combined with BCL2L1 knockdown group displaying the lowest tumor burden. These results demonstrate that while PBX1 overexpression alone moderately suppresses tumor growth, the concurrent silencing of BCL2L1 substantially enhances this effect. This combinatorial approach underscores the therapeutic potential of targeting the PBX1 – BCL2L1 axis to simultaneously reduce tumor proliferation and overcome apoptotic resistance in colorectal cancer. 3. Discussion Given the central role of apoptosis evasion in cancer progression and therapeutic resistance, our study elucidates how the PBX1 – BCL2L1 axis contributes to apoptosis suppression and modulates tumor cell fate in CRC. We demonstrate that PBX1 overexpression inhibits CRC cell proliferation, migration, and invasion, indicating a tumor-suppressive role. However, PBX1 overexpression concurrently reduces apoptosis, potentially allowing cells to enter a quiescent, drug-resistant state. This dual effect complicates the therapeutic use of PBX1 overexpression alone, as it may restrict the efficacy of pro-apoptotic treatments. Our analysis of public datasets (TCGA and Oncomine) and patient samples confirmed that PBX1 is consistently downregulated in CRC tissues and cell lines, supporting previous findings that PBX1 often plays a tumor-suppressive role in solid tumors[ 13 ]. Functional assays revealed that PBX1 knockdown promotes proliferation, colony formation, and invasion, while PBX1 overexpression suppresses these oncogenic behaviors, reinforcing its role as a tumor suppressor in CRC. Mechanistically, transcriptomic and epigenomic profiling revealed BCL2L1 as a direct transcriptional target of PBX1. PBX1 binds the BCL2L1 promoter and enhances its transcription, supported by increased H3K27ac enrichment and and luciferase reporter activation. These results align with previous studies that demonstrate PBX1’s ability to regulate chromatin accessibility and gene expression at key loci [ 41 ]. Further isoform-specific analysis showed that PBX1 primarily upregulates Bcl-xL, the anti-apoptotic isoform, in a splicing environment biased toward Bcl-xL. This was validated by qPCR and western blotting, which detected endogenous Bcl-xL but not Bcl-xS in CRC cells. These findings suggest that PBX1 increases the overall transcription of BCL2L1 , and in the presence of a cell-intrinsic splicing bias, promotes Bcl-xL expression, contributing to apoptosis resistance. Functional assays confirmed that PBX1 knockdown decreased Bcl-xL levels and increased apoptosis, while overexpression of PBX1 elevated Bcl-xL and reduced apoptotic cell death. These findings align with prior studies highlighting the oncogenic role of Bcl-xL in tumor survival and drug resistance [ 42 ]. Importantly, knocking down BCL2L1 in PBX1 -overexpressing cells reversed PBX1 ’s anti-apoptotic effect, highlighting Bcl-xL as a key downstream effector of PBX1 . While PBX1 overexpression modestly reduced proliferation, it enhanced cell survival—a phenotype that was abolished when BCL2L1 was knocked down. This finding suggests that PBX1 maintains a pool of non-proliferative, apoptosis-resistant tumor cells that may evade therapy and contribute to relapse. Functional assays further supported this regulatory preference: PBX1 knockdown decreased Bcl-xL and significantly increased apoptosis, while PBX1 overexpression elevated Bcl-xL expression and suppressed apoptotic activity. Importantly, functional interaction experiments in PBX1 -overexpressing cells demonstrated that BCL2L1 knockdown reversed the anti-apoptotic effect of PBX1 , confirming that Bcl-xL is a key functional effector downstream of PBX1 . In addition, while PBX1 overexpression modestly suppressed proliferation, it simultaneously enhanced cell survival—an effect that was abrogated by BCL2L1 knockdown. This dual effect suggests that PBX1 may promote the persistence of non-proliferative, apoptosis-resistant tumor cells, which could contribute to treatment resistance or relapse if left unchecked. To assess the clinical relevance of this axis, we evaluated how PBX1 and BCL2L1 influence 5-FU chemotherapy response. PBX1 overexpression reduced cell viability under 5-FU exposure, and the combination of PBX1 overexpression with BCL2L1 knockdown further enhanced 5-FU cytotoxicity. These results suggest that targeting BCL2L1 can improve the therapeutic efficacy of PBX1 -based modulation. Similar patterns were observed in RKO cells, reinforcing the generalizability of the PBX1–BCL2L1 axis in CRC. In vivo, xenograft experiments confirmed that PBX1 overexpression suppresses tumor growth, and this effect was significantly augmented by BCL2L1 knockdown. Tumor volume and weight were lowest in the PBX1 overexpression combined with BCL2L1 knockdown group, supporting a synergistic effect in vivo. These findings underscore the cooperative role of PBX1 and BCL2L1 in modulating tumorigenesis and therapeutic sensitivity. Beyond BCL2L1 , we identified additional PBX1 -regulated targets such as DUSP5 and PLK2 , which are known to inhibit MAPK signaling and cell cycle progression, respectively [ 31 , 32 ]. These targets likely contribute to the anti-proliferative function of PBX1 and further establish its multifaceted role in CRC suppression. In summary, we propose a working model in which PBX1 simultaneously promotes anti-proliferative and anti-apoptotic pathways in CRC (Fig. 8 ). PBX1 directly enhances BCL2L1 transcription, leading to Bcl-xL–mediated apoptotic resistance. Concurrently, PBX1 activates tumor-suppressive genes like DUSP5 and PLK2 , inhibiting proliferation. This dual regulation creates a state in which tumor cells are growth-arrested yet resistant to apoptosis, potentially contributing to therapy resistance. Disrupting this balance by combining PBX1 overexpression with BCL2L1 knockdown effectively inhibits both proliferation and survival, thereby improving treatment efficacy. The PBX1 – BCL2L1 axis thus emerges as a promising dual-action therapeutic target in colorectal cancer. 4. Materials and Methods 4.1 Patients and tissues This study examined 50 cases of colonic adenocarcinoma and corresponding adjacent normal colonic mucosa, collected from patients who underwent surgical treatment at Shantou Central Hospital (Guangdong, China) between 2020 and 2022. All tissues were paraffin-embedded and diagnosedindependently by two pathologists. Patients who had received radiotherapy or chemotherapy prior to surgery were excluded. Ethical approval was obtained from the local ethics committee (Shantou University Medical College, SUMC-2020-14), and informed consent was acquired. 4.2 Cell Lines and Culture Conditions Human colorectal cancer cell lines, including HCT116, HT-29, LoVo, RKO, SW480, and SW620, and the normal colon cell line CCD-18Co, were obtained from ATCC (Manassas, VA, USA) or the Shanghai Cell Bank, Chinese Academy of Sciences (LoVo). Cells were maintained in cell line-specific media as follows: McCoy’s 5A Medium (for HCT116 and HT-29; Gibco, Cat. No. 16600082); F-12K Nutrient Mixture (for LoVo; Gibco, Cat. No. 21127022); Minimum Essential Medium (MEM) supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate (for RKO; Gibco, Cat. No. 11380037); Leibovitz’s L-15 Medium (for SW480 and SW620; Gibco, Cat. No. 11415064); MEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 1× non-essential amino acids (NEAA) (for CCD-18Co; Gibco, Cat. No. 11140050). All media were supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat. No. 10099141), and cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ (except for L-15, which was cultured in atmospheric air). 4.3 RNA Extraction, cDNA Synthesis, and qPCR Total RNA was extracted using RNAiso Plus (Takara Bio Inc., Shiga, Japan; Cat. No. 9109). First-strand cDNA synthesis was performed using the HiScript II Q RT SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China; Cat. No. R223-01). Quantitative PCR was conducted with the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China; Cat. No. Q331) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). β-actin was used as an internal control. Primer sequences are listed in Supplementary Table S1 . 4.4 Western Blot Analysis Proteins were resolved on 10% SDS-PAGE gels and transferred onto PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. ISEQ00010). Membranes were incubated with primary antibodies followed by HRP-conjugated secondary antibodies. Signals were visualized using enhanced chemiluminescent detection reagents (Yeasen Biotechnology, Shanghai, China; Cat. No. 36208ES76). The primary antibodies used included: Rabbit anti-PBX1 (Proteintech Group, Wuhan, China; Cat. No. 18204-1-AP; dilution 1:500), Mouse anti-Bcl-xS/L (Santa Cruz Biotechnology, Dallas, TX, USA; Cat. No. sc-271121; dilution 1:200), Mouse anti-β-actin (Proteintech Group, Wuhan, China; Cat. No. 66009-1-Ig; dilution 1:10000). The secondary antibodies used were: HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 31430; dilution 1:5000), HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 31460; dilution 1:10000). 4.5 Database Analysis PBX1 expression in CRC was assessed using TCGA and Oncomine databases. TCGA RNA-seq data were analyzed using DESeq2 (v1.46.0), identifying significantly differentially expressed genes (adjusted p < 0.05, |fold change| ≥ 2). Oncomine Database Analysis: We queried the database to analyze PBX1 mRNA expression in CRC patients. By comparing multiple datasets provided by Oncomine ( www.oncomine.org , accessed on 1 March 2021), downloaded in March 2021, and data were analyzed with thresholds of p 2. 4.6 Cell Transfections For stable gene modulation, lentiviral transduction was employed to achieve PBX1 overexpression and shRNA-mediated knockdown. Lentiviral particles were generated by co-transfecting packaging plasmids and expression vectors into HEK293T cells using standard calcium phosphate or lipid-based methods. Target CRC cells were infected with viral supernatants in the presence of 8 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. H9268) for 24 hours. Infected cells were selected using puromycin (1 µg/mL; Sangon Biotech, Shanghai, China; Cat. No. A610593) and/or blasticidin (8 µg/mL; Beyotime, Shanghai, China; Cat. No. ST018), or sorted by flow cytometry when applicable. The PBX1 overexpression plasmid was constructed by inserting the full-length coding sequence of PBX1 (NM_001204961.2) into the pCDH-CMV-EF1-copGFP lentiviral backbone (System Biosciences, Palo Alto, CA, USA). shRNA plasmids were obtained from GenePharma (Suzhou, China): shPBX1 was cloned into the LV3 (H1/GFP&Puro) backbone, targeting sequence: 5'-GTGGAGCATTCAGATTACA-3', and shBCL2L1 was cloned into the pLKO.1-blast backbone, targeting sequence: 5'-CCCTACCTGATTGGTGCAA-3'. 4.7 RNA Sequencing and Data Processing Total RNA from PBX1 -knockdown and control HCT116 cells was sequenced by BGI Genomics (Shenzhen, China) using the BGISEQ platform. Reads were mapped to GRCh38 using RSEM, and differential expression analysis was conducted using DESeq2 (v1.46.0). Gene set enrichment analysis (GSEA, v4.3.3) was performed with the MSigDB collection c2.cp.wikipathways.v2024.1. NES and FDR < 0.25 were considered significant. 4.8 CUT&Tag Assay and Data Processing CUT&Tag assays were conducted using the Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904; Vazyme Biotech, Nanjing, China), with a mild formaldehyde crosslinking step added prior to cell permeabilization. Library construction was performed via Tn5 tagmentation followed by PCR amplification using the reagents provided in the kit. Final libraries were sequenced on the BGISEQ platform (BGI Genomics, Shenzhen, China). Raw reads were aligned to the human genome using Bowtie2 (v2.3.5.1), and peak calling was performed with SEACR (Sparse Enrichment Analysis for CUT&RUN). Peak annotation and motif enrichment analyses were carried out using HOMER (v4.11). CUT&Tag experiments targeting PBX1 and H3K27ac were performed in-house. The following primary antibodies were used: anti-PBX1 (Abnova, H00005087-M01, mouse monoclonal, 1:50; Taipei, Taiwan), anti-H3K27ac (Invitrogen, 720096, rabbit polyclonal, 1:50; Carlsbad, CA, USA). Public ChIP-seq datasets for H3K4me3 (GEO: GSM1224663) and H3K4me1 (GEO: GSM1866700) were downloaded from the GEO database for comparative chromatin landscape profiling. 4.9 Cell Proliferation Assay Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; MedChemExpress, HY-K0301; Monmouth Junction, NJ, USA) following the manufacturer’s instructions. Cells were seeded in 96-well plates at appropriate densities according to cell line characteristics. Absorbance at 450 nm was measured daily using a microplate reader to generate cell growth curves. 4.10 Colony Formation Assay For colony formation assays, cells were seeded in 6-well plates at a density of 300 cells per well and cultured for 2 weeks. Colonies were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, C0121; Shanghai, China) for 30 minutes at room temperature. The colony-covered area was quantified using ImageJ software (version 1.46r; NIH, Bethesda, MD, USA) and compared to the control group. 4.11 Transwell Migration and Invasion Assay For cell migration and invasion assays, Transwell chambers (8-µm pore size; BD Falcon, 35309724; Franklin Lakes, NJ, USA) were used. For invasion assays, the upper chambers were pre-coated with Matrigel (BD Biosciences, 356234; San Jose, CA, USA). Cells were serum-starved for 12 hours, seeded into the upper chambers in serum-free medium, and allowed to migrate toward medium containing 20% fetal bovine serum in the lower chambers. After 24 hours of incubation, cells on the lower surface were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, C0121; Shanghai, China) for 30 minutes at room temperature. 4.12 Cell Apoptosis Assay Apoptosis was evaluated using the APC Annexin V Apoptosis Detection Kit (Tonbo Biosciences, 20-6410-KIT; San Diego, CA, USA) according to the manufacturer’s instructions. Cells were stained with APC Annexin V and 7-AAD, and analyzed by flow cytometry using a Celula Sparrow2040 flow cytometer (Celula Inc., Shanghai, China). The percentages of early and late apoptotic cells were quantified. 4.13 Chromatin immunoprecipitation (ChIP) Chromatin immunoprecipitation (ChIP) was performed using the EZ-ChIP™ Chromatin Immunoprecipitation Kit (Sigma-Aldrich, 17–611; St. Louis, MO, USA) according to the manufacturer’s instructions, as previously described [ 43 ]. Immunoprecipitated DNA was analyzed by quantitative PCR, and results were normalized to input DNA. The following primary antibodies were used: anti-PBX1 (Abnova, H00005087-M01, mouse monoclonal, 1:50; Taipei, Taiwan), anti-H3K27ac (Invitrogen, 720096, rabbit polyclonal, 1:50; Carlsbad, CA, USA), mouse IgG control (Millipore, 12-371B, 1:50; Burlington, MA, USA), rabbit IgG control (Millipore, CS200581, 1:50; Burlington, MA, USA). Primer sequences used for qPCR analysis are listed in Supplementary Table S2 . 4.14 Dual-luciferase reporter assay The BCL2L1 promoter (~ 1719 bp) and its truncated fragments (Fragment 1: 695 bp; Fragment 2: 638 bp; Fragment 3: 503 bp) were amplified using specific primers (sequences listed in Supplementary Table S3) and cloned into the pGL3-Basic vector (Promega, Madison, WI, USA) at the HindIII restriction site using the ClonExpress II One Step Cloning Kit (Vazyme, C116; Nanjing, China). For luciferase activity measurements, HCT116 and RKO cells were co-transfected with the constructed firefly luciferase plasmids and Renilla luciferase internal control plasmid (pRL-SV40, Promega, Madison, WI, USA) at a molar ratio of 100:1, using FuGENE® HD Transfection Reagent (Promega, E2311; Madison, WI, USA). After 48 hours, dual-luciferase activity was quantified using the Dual-Luciferase® Reporter Assay System (Vazyme, DD1205; Nanjing, China) according to the manufacturer’s protocol. Firefly luciferase activity was normalized to Renilla luciferase to control for transfection efficiency. 4.15 5-FU Treatment and Chemotherapy Sensitivity Assay To evaluate chemotherapy response, HCT116 and RKO cells were treated with 0–160 µM 5-fluorouracil (5-FU; MedChemExpress, HY-90006, Monmouth Junction, NJ, USA) for 24 hours. IC50 values were calculated using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Based on these values, a 10 µM concentration (approximately 1/4–1/2 of the IC50) was selected for subsequent cell viability assays. 4.16 Xenograft Tumorigenesis in Nude Mice HCT116 cells (1×10 7 ) stably expressing Empty-pCDH + shNC, PBX1-pCDH + shNC, PBX1-pCDH + shBCL2L1 were injected subcutaneously into 5-week-old male BALB/c nude mice (n = 5 per group; mice were randomly assigned to experimental groups using a random number table method; purchased from Guangdong Yaokang Biological Technology Co., Ltd., Guangdong, China). Tumor size and mouse weight were measured every 2–3 days. Tumor volume was calculated as (length × width 2 )/2. Mice were sacrificed at day 15, and tumors were photographed and weighed. Tumor measurements and outcome assessments were performed by investigators blinded to group allocation. All animal procedures were approved by the Institutional Animal Care and Use Committee of Shantou University Medical College ( SUMC-2022-289). 4.17 Statistical Analysis All statistical analyses were performed using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Each experiment with statistical comparison was conducted with at least three independent biological replicates. Data are expressed as mean ± standard deviation (SD). Homogeneity of variance was tested using Levene's test prior to t-test analysis. For comparisons with equal variance, standard Student’s t-test was applied; otherwise, Welch’s correction was used. One-way ANOVA followed by LSD post hoc test was used for multiple group comparisons. A P-value less than 0.05 was considered statistically significant. Detailed statistical methods and replicate numbers are provided in the corresponding figure legends. 4.18 Code availability No custom code was used in this study. All analyses were performed using standard bioinformatics packages and commercially available software. Abbreviations PBX1 Pre-B-cell leukemia homeobox 1 CRC colorectal cancer BCL2L1 BCL2 Like 1 H3K27ac Histone H3 lysine 27 acetylation H3K4me1 Histone H3 lysine 4 monomethylation H3K4me3 Histone H3 lysine 4 trimethylation Bcl-xL B-cell lymphoma-extra large TALE Three amino acid loop extension Bcl-xS B-cell lymphoma-extra small ATCC American Type Culture Collection TCGA The Cancer Genome Atlas BGI Beijing Genomics Institute RSEM RNA-Seq by Expectation Maximization CUT&RUN Cleavage Under Targets and Release Using Nuclease SEACR Sparse Enrichment Analysis for CUT&RUN HOMER Hypergeometric Optimization of Motif EnRichment ChIP Chromatin immunoprecipitation. Declarations Competing Interests Statement: The authors declare no competing interests. This research was supported by the Natural Science Foundation of Guangdong Province (Grant No. 2023A1515010434) and the National Natural Science Foundation of China (NSFC, Grant No. 32000456). Acknowledgments The authors have no specific acknowledgments for this study. Competing interests The authors declare that they have no competing interests. Authors’ contributions Hao Lin and Ting Su contributed equally to this work and share first authorship. Hao Lin and Lingzhu Xie designed the study and supervised the project. Hao Lin and Xuanhao Lin collected and analyzed patient samples and clinical data. Ting Su, Rulan Deng, Jie Li, Ying Liu, Qiaoling Ke, Lele Meng, and Yijing Luo performed the experiments and collected data. Xuhong Song and Bin Liang conducted statistical analyses. Hao Lin, and Lingzhu Xie wrote the manuscript. Dongyang Huang and Lingzhu Xie provided overall supervision and revised the manuscript. All authors read and approved the final manuscript. Ethics approval and consent to participate This study was approved by the Ethics Committee of Shantou University Medical College, China (Approval No. SUMC-2020-14). Written informed consent was obtained from all patients for tissue collection and analysis. All animal procedures were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Shantou University Medical College (Approval No. SUMC-2022-289). Funding This research was supported by the Natural Science Foundation of Guangdong Province (Grant No. 2023A1515010434) and the National Natural Science Foundation of China (NSFC, Grant No. 32000456). Availability of data The datasets used and analysed are available from the corresponding authors upon reasonable request. References Sung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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J Exp Clin Cancer Res 38, 213–28 (2019). https://doi.org/10.1186/s13046-019-1217-9 Additional Declarations (Not answered) Supplementary Files 20250513Supplementaryfiguresandtables.doc Supplementary figures and tables Originalfulllengthwesternblots.pdf Original western blots Cite Share Download PDF Status: Under Review Version 1 posted Unknown event 21 Jan, 2026 Editorial decision: revise 11 Sep, 2025 Review # 2 received at journal 03 Sep, 2025 Review # 1 received at journal 29 Aug, 2025 Reviewer # 2 agreed at journal 21 Aug, 2025 Reviewer # 1 agreed at journal 21 Aug, 2025 Reviewers invited by journal 14 Aug, 2025 Submission checks completed at journal 06 Aug, 2025 First submitted to journal 05 Aug, 2025 Editor assigned by journal 05 Aug, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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College","correspondingAuthor":false,"prefix":"","firstName":"Yijing","middleName":"","lastName":"Luo","suffix":""},{"id":500783870,"identity":"17b8af10-8e2a-49aa-b612-f476cbe3d937","order_by":9,"name":"Lele Meng","email":"","orcid":"","institution":"Shantou University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Lele","middleName":"","lastName":"Meng","suffix":""},{"id":500783871,"identity":"61ca32c2-688e-4a30-bfb4-281074a26dc4","order_by":10,"name":"Bin Liang","email":"","orcid":"","institution":"Shantou University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Bin","middleName":"","lastName":"Liang","suffix":""},{"id":500783872,"identity":"5bf1ccf3-9d65-4257-aff7-ab24a209b231","order_by":11,"name":"Xuhong Song","email":"","orcid":"","institution":"Shantou University Medical College","correspondingAuthor":false,"prefix":"","firstName":"Xuhong","middleName":"","lastName":"Song","suffix":""},{"id":500783873,"identity":"2c10ca72-cc7a-4d0f-9952-2add8615a9d6","order_by":12,"name":"Dongyang Huang","email":"","orcid":"","institution":"Shantou Medical College","correspondingAuthor":false,"prefix":"","firstName":"Dongyang","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2025-08-06 03:55:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7305360/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7305360/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":89675171,"identity":"9e5df9e9-9570-47be-bcd4-94e9ad22ed55","added_by":"auto","created_at":"2025-08-22 13:31:30","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2407641,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e is downregulated in CRC tissues and cell lines.\u003c/strong\u003e (A) \u003cem\u003ePBX1\u003c/em\u003e mRNA expression levels in CRC vs. normal tissues analyzed from TCGA RNA-seq data. (B-C) \u003cem\u003ePBX1\u003c/em\u003e expression in Oncomine datasets 'Skrzypczak Colorectal' and 'Skrzypczak Colorectal 2'. (D-F) qPCR and western blot showing lower \u003cem\u003ePBX1\u003c/em\u003e expression in CRC tumor tissues vs. adjacent normal tissues from 50 patients. (G-I) \u003cem\u003ePBX1\u003c/em\u003e expression in normal colon cells and CRC cell lines. Significance for data D, F, G and I were determined by the independent samples t-test. Data are shown as mean ± S.D., n \u0026gt;= 3. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure101.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/8609fb16be1b244e2f617b24.png"},{"id":89675174,"identity":"41397bea-ca48-4986-94b6-efd6656fb98c","added_by":"auto","created_at":"2025-08-22 13:31:30","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":6442926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e knockdown promotes malignant phenotypes in CRC cells.\u003c/strong\u003e (A-F) Confirmation of \u003cem\u003ePBX1\u003c/em\u003e knockdown in HCT116 and RKO cells by qPCR and western blot. Functional assays show that \u003cem\u003ePBX1\u003c/em\u003eknockdown significantly enhances cell proliferation (G-H), colony formation (I-L), migration and invasion (M-R) in HCT116 and RKO cells. Significance for all data was determined by the independent samples t-test. Data are shown as mean ± S.D., n \u0026gt;= 3. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure201.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/926bdd72067196a38e5e6063.png"},{"id":89675974,"identity":"3cda7ef4-0490-4123-9a31-404d4ec038fe","added_by":"auto","created_at":"2025-08-22 13:39:30","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":6528383,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e overexpression suppresses CRC progression.\u003c/strong\u003e (A-F) Confirmation of \u003cem\u003ePBX1\u003c/em\u003e overexpression by qPCR and western blot in HCT116 and RKO cells. Functional assays indicate reduced proliferation (G-H) , colony formation (I-L), migration and invasion (M-R) upon \u003cem\u003ePBX1\u003c/em\u003e overexpression in HCT116 and RKO cells. Significance for all data was determined by the independent samples t-test. Data are shown as mean ± S.D., n \u0026gt;= 3. *\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure301.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/96cff831061e49aae6391e59.png"},{"id":89675178,"identity":"51e313b9-43dc-403b-9a5e-c8136c9e1b49","added_by":"auto","created_at":"2025-08-22 13:31:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":4026228,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIntegrated transcriptomic and epigenomic profiling identifies \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBCL2L1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e as a potential \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e target.\u003c/strong\u003e (A) GSEA shows enrichment of apoptosis- and proliferation-related pathways after \u003cem\u003ePBX1\u003c/em\u003eknockdown. (B) Volcano plot of differentially expressed genes following \u003cem\u003ePBX1\u003c/em\u003eknockdown. (C) Genomic distribution of \u003cem\u003ePBX1\u003c/em\u003e binding sites identified by CUT\u0026amp;Tag. (D) Histone modification profiles at \u003cem\u003ePBX1\u003c/em\u003e-bound regions show promoter-associated chromatin marks. (E) Venn diagram indicating 701 genes with PBX1 promoter binding. (F) Overlap between CUT\u0026amp;Tag and RNA-seq reveals four direct PBX1 targets. (G) Heatmap showing the expression levels of four direct PBX1 target genes; \u003cem\u003eBCL2L1\u003c/em\u003e shows the lowest adjusted p-value. (H) CUT\u0026amp;Tag profile shows PBX1 binding at \u003cem\u003eBCL2L1\u003c/em\u003e promoter.\u003c/p\u003e","description":"","filename":"figure401.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/9b941cd047390b2517909339.png"},{"id":89675183,"identity":"cc9a3487-2869-4b4c-8feb-192687059a59","added_by":"auto","created_at":"2025-08-22 13:31:30","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":2202712,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e activates \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBCL2L1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e promoter activity by direct binding and chromatin activation.\u003c/strong\u003e(A) Diagram of the \u003cem\u003eBCL2L1\u003c/em\u003e promoter region used for luciferase reporter assay and ChIP-qPCR. (B) Luciferase reporter activity of the full-length \u003cem\u003eBCL2L1\u003c/em\u003epromoter in HCT116 and RKO cells. (C) Luciferase activity of the \u003cem\u003eBCL2L1\u003c/em\u003epromoter after PBX1 knockdown in HCT116 and RKO cells. (D) Luciferase activity of the \u003cem\u003eBCL2L1\u003c/em\u003e promoter after PBX1 overexpression in HCT116 and RKO cells. (E–H) Fragment-specific luciferase assays in HCT116 and RKO cells. PBX1 knockdown (E, G) markedly reduced the luciferase activity driven by Fragment 3, while PBX1 overexpression (F, H) significantly enhanced the activity of Fragment 3. (I–L) ChIP-qPCR analysis of PBX1 and H3K27ac enrichment at the BCL2L1 promoter region in HCT116 and RKO cells. (M, O) PBX1 knockdown reduced the enrichment of PBX1 and H3K27ac at the \u003cem\u003eBCL2L1 \u003c/em\u003epromoter in HCT116 and RKO cells. (N, P) PBX1 overexpression increased PBX1 and H3K27ac enrichment at the \u003cem\u003eBCL2L1\u003c/em\u003e promoter. Significance for all data was determined by the independent samples t-test. Data are shown as mean ± S.D., n = 3. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"figure501.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/3a9ea19996150f63bdb79931.png"},{"id":89675181,"identity":"9fc46dc3-d253-4489-b635-0652792a6db0","added_by":"auto","created_at":"2025-08-22 13:31:30","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4454436,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e regulates isoform-specific expression of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eBCL2L1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eand modulates apoptosis.\u003c/strong\u003e (A, C) \u003cem\u003ePBX1\u003c/em\u003eknockdown decreases Bcl-xL mRNA in HCT116 (A) and RKO (C) cells; (B, D) overexpression increases Bcl-xL mRNA in HCT116 (B) and RKO (D) cells, with little change in Bcl-xS. (E) Western blot confirms predominant expression of Bcl-xL in CRC cells; (F) Bcl-xS detectable only upon exogenous overexpression. (G-H) \u003cem\u003ePBX1\u003c/em\u003eknockdown reduces Bcl-xL protein and increases apoptosis in HCT116 cells; (I-J) Overexpression of \u003cem\u003ePBX1\u003c/em\u003e increases Bcl-xL and decreases apoptosis in HCT116 cells. (K-L) \u003cem\u003ePBX1\u003c/em\u003e knockdown reduces Bcl-xL protein and increases apoptosis in RKO cells; (M-N) Overexpression of \u003cem\u003ePBX1\u003c/em\u003e increases Bcl-xL and decreases apoptosis in RKO cells. Significance for all data was determined by the independent samples t-test. Data are shown as mean ± S.D., n \u0026gt;= 3. *\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"figure601.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/9e305ab30913ccb264e22ab5.png"},{"id":89675204,"identity":"aaeec645-3565-4338-8c08-edc398c8cdea","added_by":"auto","created_at":"2025-08-22 13:31:31","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":6030776,"visible":true,"origin":"","legend":"","description":"","filename":"figure701.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/ed022e0ba9da3eef457291d9.png"},{"id":89675987,"identity":"28ee7ed3-c7e2-46a3-a06e-3bd250d573dd","added_by":"auto","created_at":"2025-08-22 13:39:31","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2113968,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWorking model of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ePBX1\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e dual regulatory roles in colorectal cancer.\u003c/strong\u003e \u003cem\u003ePBX1\u003c/em\u003e activates \u003cem\u003eBCL2L1\u003c/em\u003e transcription to promote expression of anti-apoptotic Bcl-xL, suppressing apoptosis and promoting the survival of quiescent or drug-resistant cells. Simultaneously, \u003cem\u003ePBX1\u003c/em\u003einduces tumor suppressor genes (\u003cem\u003eDUSP5\u003c/em\u003e and \u003cem\u003ePLK2\u003c/em\u003e), which inhibit proliferation. Therapeutically, co-targeting \u003cem\u003ePBX1\u003c/em\u003e and \u003cem\u003eBCL2L1\u003c/em\u003e may overcome this balance and enhance treatment efficacy.\u003c/p\u003e","description":"","filename":"figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/9d312da318039125b9f57d10.png"},{"id":100788732,"identity":"3b978ba2-e90b-4591-90c2-a9e330578787","added_by":"auto","created_at":"2026-01-21 12:06:48","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":33175651,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/8256f28c-573f-4f22-9151-7c6e37e5023a.pdf"},{"id":89675172,"identity":"c4a76f32-953a-48ba-bf67-876cf47233a8","added_by":"auto","created_at":"2025-08-22 13:31:30","extension":"doc","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1067190,"visible":true,"origin":"","legend":"Supplementary figures and tables","description":"","filename":"20250513Supplementaryfiguresandtables.doc","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/4dc1308f82b1309f8da0547a.doc"},{"id":89675976,"identity":"e048194a-34cb-47dd-95d7-76cbb64af236","added_by":"auto","created_at":"2025-08-22 13:39:30","extension":"pdf","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":8946369,"visible":true,"origin":"","legend":"Original western blots","description":"","filename":"Originalfulllengthwesternblots.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7305360/v1/a39d09f1b5c00de32f5d6015.pdf"}],"financialInterests":"(Not answered)","formattedTitle":"\u003cp\u003eTargeting the \u003cem\u003ePBX1–BCL2L1\u003c/em\u003e Axis as a Therapeutic Strategy in Colorectal Cancer\u003c/p\u003e","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eColorectal cancer (CRC) is the third most common cancer worldwide, representing 10.0% of all cancer cases, and the second leading cause of cancer-related deaths, contributing to 9.4% of total cancer deaths in 2020 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Despite advancements in surgical techniques, chemotherapy, and targeted therapies, the recurrence and mortality rates for CRC remain high [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Tumor recurrence, driven by the complex interplay of molecular pathways regulating cell proliferation, survival, and apoptosis, continues to challenge effective treatment. Understanding the key molecular regulators involved in these processes is crucial for developing more effective therapies and improving patient outcomes.\u003c/p\u003e\u003cp\u003eAmong the transcription factors implicated in cancer biology, the \u003cem\u003ePre-B-cell leukemia homeobox 1\u003c/em\u003e (\u003cem\u003ePBX1\u003c/em\u003e) gene has garnered attention for its dual role in various cancers[\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003ePBX1\u003c/em\u003e, a member of the TALE (three amino acid loop extension) homeodomain family, is recognized for its role in developmental processes and gene expression regulation[\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Depending on the cellular and molecular context, \u003cem\u003ePBX1\u003c/em\u003e can act as either a tumor suppressor or an oncogene in different types of cancer. For instance, in pre-B cell acute lymphoblastic leukemia, \u003cem\u003ePBX1\u003c/em\u003e was first identified as an oncogene through the t(1;19) chromosomal translocation, which produces the E2A\u0026ndash;PBX1 fusion protein that drives leukemogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Similarly, \u003cem\u003ePBX1\u003c/em\u003e has been shown to promote tumor progression, chemoresistance, and stemness in breast, ovarian, and clear cell renal carcinomas [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. However, in CRC, \u003cem\u003ePBX1\u003c/em\u003e appears to play a distinct role. While \u003cem\u003ePBX1\u003c/em\u003e expression is frequently downregulated in CRC tissues, forced overexpression of \u003cem\u003ePBX1\u003c/em\u003e has been shown to suppress tumor cell proliferation, migration, and invasion, suggesting a tumor-suppressive function [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the precise mechanisms by which \u003cem\u003ePBX1\u003c/em\u003e influences CRC cell fate remain poorly understood.\u003c/p\u003e\u003cp\u003eIn addition to its role in cell proliferation, \u003cem\u003ePBX1\u003c/em\u003e has been implicated in regulating apoptosis across various cancers. In lung cancer, \u003cem\u003ePBX1\u003c/em\u003e silencing induces apoptosis through ROS production and inhibition of the \u003cem\u003eSTAT3\u0026ndash;Bcl-2\u003c/em\u003e\u0026ndash;Survivin pathway [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. In breast cancer, \u003cem\u003ePBX1\u003c/em\u003e is negatively regulated by the lncRNA uc.38, which induces apoptosis by downregulating Bcl-2 family proteins [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Moreover, in prostate cancer, \u003cem\u003ePBX1\u003c/em\u003e's stability, regulated by the deubiquitinase USP9x, plays a critical role in resistance to apoptosis, positioning \u003cem\u003ePBX1\u003c/em\u003e as a potential therapeutic target to overcome chemoresistance [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. These studies underscore \u003cem\u003ePBX1\u003c/em\u003e's critical role in the regulation of apoptosis and suggests that similar mechanisms may exist in other cancers.\u003c/p\u003e\u003cp\u003eApoptosis, a crucial mechanism for maintaining cellular homeostasis and preventing tumor growth, is often disrupted in cancer, leading to tumor recurrence and resistance to treatment. The Bcl-2 family of proteins, particularly BCL2L1, plays a pivotal role in regulating apoptosis. BCL2L1 encodes two isoforms: Bcl-xS, which promotes apoptosis, and Bcl-xL, which inhibits apoptosis and supports cell survival[\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. These isoforms are generated by alternative splicing, resulting in structural differences that influence their roles in apoptosis regulation [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Bcl-xL, with its intact BH1, BH2, and C-terminal hydrophobic domain, prevents apoptosis by binding to and inhibiting pro-apoptotic proteins such as Bax and Bak[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast, Bcl-xS, lacking key anti-apoptotic domains, allows apoptotic pathways to proceed [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While several upstream signaling pathways and splice factors have been shown to modulate the balance between Bcl-xL and Bcl-xS [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], the transcriptional regulation of BCL2L1, particularly in CRC, remains poorly defined.\u003c/p\u003e\u003cp\u003e\u003cul\u003e\u003cli\u003e\u003cp\u003eThis gap in understanding is crucial, as apoptosis and proliferation are closely linked in tumor progression. Our study investigates how \u003cem\u003ePBX1\u003c/em\u003e influences cell both cell proliferation and apoptosis, focusing on its regulation of \u003cem\u003eBCL2L1\u003c/em\u003e. By defining the \u003cem\u003ePBX1\u003c/em\u003e\u0026ndash;\u003cem\u003eBCL2L1\u003c/em\u003e axis, this research may provide new therapeutic insights into strategies that simultaneously suppress tumor growth and enhance apoptosis sensitivity in CRC.\u003c/p\u003e\u003c/li\u003e\u003c/ul\u003e\u003c/p\u003e"},{"header":"2. Result","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Downregulation of PBX1 Expression in Colorectal Cancer Tissues and Cells\u003c/h2\u003e\u003cp\u003eAnalysis of both TCGA and Oncomine databases revealed a significant reduction in \u003cem\u003ePBX1\u003c/em\u003e mRNA expression in CRC tissues compared to normal tissues, suggesting a potential tumor-suppressive role for \u003cem\u003ePBX1\u003c/em\u003e in CRC. Specifically, TCGA RNA-Seq data[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] showed that \u003cem\u003ePBX1\u003c/em\u003e expression was significantly lower in CRC tissues than in adjacent normal tissues (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Consistent with this, analysis of the \"Skrzypczak Colorectal\" and \"Skrzypczak Colorectal 2\" datasets [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], from Oncomine further confirmed \u003cem\u003ePBX1\u003c/em\u003e downregulation in CRC tumor tissues, with fold changes of -2.036 and \u0026minus;\u0026thinsp;2.147, and P-values of 4.40 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e and 1.03 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;6\u003c/sup\u003e, respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB, \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). To validate these database findings at the clinical level, we analyzed tumor and adjacent non-tumor tissues from 50 CRC patients. qPCR and Western blot analyses confirmed that \u003cem\u003ePBX1\u003c/em\u003e expression was significantly lower in tumor tissues compared to adjacent normal tissues (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F), reinforcing the hypothesis that \u003cem\u003ePBX1\u003c/em\u003e may act as a tumor suppressor in CRC. To further substantiate this observation at the cellular level, we examined \u003cem\u003ePBX1\u003c/em\u003e expression in multiple CRC cell lines (HCT116, HT-29, LoVo, RKO, SW480, SW620) and the normal colon cell line CCD-18Co. The results demonstrated that \u003cem\u003ePBX1\u003c/em\u003e expression was significantly downregulated at both the mRNA and protein levels in CRC cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-I), providing additional support for the idea that \u003cem\u003ePBX1\u003c/em\u003e underexpression is a consistent feature of CRC, occurring across both patient tissues and in vitro cellular models.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 \u003cem\u003ePBX1\u003c/em\u003e generally functions as a tumor suppressor in CRC\u003c/h2\u003e\u003cp\u003eBuilding on the observed downregulation of \u003cem\u003ePBX1\u003c/em\u003e in CRC tissues and cell lines, we further investigated its functional role in CRC progression by modulating its expression in vitro. Knockdown of \u003cem\u003ePBX1\u003c/em\u003e in HCT116 and RKO cells using shRNA significantly decreased \u003cem\u003ePBX1\u003c/em\u003e mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-F), which led to a a significant increase in cell proliferation, colony formation, migration and invasion (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-R). Conversely, overexpression of \u003cem\u003ePBX1\u003c/em\u003e in HCT116 and RKO cells using a lentiviral vector significantly increased \u003cem\u003ePBX1\u003c/em\u003e mRNA and protein levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F), which led to a significant suppression in cell proliferation, migration, invasion, and colony formation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-R). These results suggest that \u003cem\u003ePBX1\u003c/em\u003e generally functions as a tumor suppressor in CRC.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 \u003cem\u003eBCL2L1\u003c/em\u003e as a Potential Direct Target of PBX1\u003c/h2\u003e\u003cp\u003eTo investigate the downstream genes and pathways regulated by PBX1, we performed RNA sequencing (RNA-seq) analysis in HCT116 cells following \u003cem\u003ePBX1\u003c/em\u003e knockdown. The pathways significantly affected are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, while representative differentially expressed genes are shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB. As Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA shown, \u003cem\u003ePBX1\u003c/em\u003e knockdown in HCT116 cells led to significant enrichment of gene sets related to apoptosis, cell proliferation, and metastasis. The top-ranked pathway was the \u0026ldquo;Apoptosis-Related Network due to Altered Notch3\u0026rdquo; (NES\u0026thinsp;=\u0026thinsp;2.10, FDR\u0026thinsp;=\u0026thinsp;0.053), suggesting that reduced \u003cem\u003ePBX1\u003c/em\u003e expression may enhance pro-apoptotic signaling. Additional pathways enriched in the knockdown condition included \u0026ldquo;LDL Influence on CD14 and TLR4\u0026rdquo; and \u0026ldquo;Influence of Laminopathies on Wnt Signaling\u0026rdquo;, both associated with immune regulation and proliferative activity [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], as well as \u0026ldquo;TGF-β signaling in thyroid cells\u0026rdquo; and \u0026ldquo;Type 2 Papillary Renal Cell Carcinoma\u0026rdquo;, which are linked to epithelial-mesenchymal transition and metastatic potential [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Interestingly, while prior study has reported that low \u003cem\u003ePBX1\u003c/em\u003e expression is associated with enhanced CRC proliferation and invasion, and that \u003cem\u003ePBX1\u003c/em\u003e overexpression can suppress tumor growth [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], our data revealed a paradoxical enrichment of apoptosis-related signaling upon \u003cem\u003ePBX1\u003c/em\u003e knockdown. This suggests that although \u003cem\u003ePBX1\u003c/em\u003e overexpression may inhibit proliferative capacity in some contexts, it could simultaneously promote tumor cell apoptosis. Therefore, strategies aiming to suppress tumor growth via \u003cem\u003ePBX1\u003c/em\u003e overexpression may carry the unintended consequence of inhibiting tumor cell apoptosis, potentially leading to an accumulation of quiescent, less active, and drug-resistant tumor cells.These results underscore the need for further investigation into the relationship between \u003cem\u003ePBX1\u003c/em\u003e expression and apoptotic regulation in cancer.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo further investigate the regulatory mechanisms underlying the transcriptional changes observed upon \u003cem\u003ePBX1\u003c/em\u003e knockdown, we performed a CUT\u0026amp;Tag assay in HCT116 cells using a PBX1-specific antibody to map genome-wide PBX1 binding sites. Given PBX1's established role as a transcription factor, we aimed to identify genes that are not only differentially expressed following \u003cem\u003ePBX1\u003c/em\u003e depletion but also directly bound by PBX1 on chromatin.\u003c/p\u003e\u003cp\u003eFigure \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC shows that PBX1 binding peaks are predominantly located in intergenic regions (40.29%) and intronic regions (39.94%), while approximately 14.49% of binding events occur near transcription start sites (TSS), suggesting PBX1 binds both distal and proximal regulatory elements.\u003c/p\u003e\u003cp\u003eTo determine the regulatory context of PBX1-bound regions, we analyzed histone modification signals at these loci, focusing on H3K4me3, H3K27ac, and H3K4me1\u0026mdash;canonical markers for promoters and enhancers. As shown in the top of Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD, the majority of PBX1 binding sites exhibited strong H3K4me3 and H3K27ac enrichment, along with relatively low H3K4me1 levels. This epigenetic signature is characteristic of active promoters, whereas we observed no clear evidence of enhancer-like regions defined by high H3K4me1 and H3K27ac but low H3K4me3.\u003c/p\u003e\u003cp\u003eBased on this chromatin landscape, we annotated PBX1 binding peaks to nearby promoters and compiled a list of 701 genes with PBX1 occupancy at their promoter regions across three independent biological replicates (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). To further narrow down functionally relevant targets, we intersected this list with the 89 differentially expressed genes identified from RNA-seq analysis. This integrative approach yielded four high-confidence candidate genes that are both transcriptionally regulated by PBX1 and directly bound at their promoters: \u003cem\u003eDUSP5\u003c/em\u003e, \u003cem\u003eAP3S1\u003c/em\u003e, \u003cem\u003ePLK2\u003c/em\u003e, and \u003cem\u003eBCL2L1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF).\u003c/p\u003e\u003cp\u003eAmong the four candidate genes identified\u0026mdash;\u003cem\u003eDUSP5, AP3S1, PLK2\u003c/em\u003e, and \u003cem\u003eBCL2L1\u003c/em\u003e, several have been previously linked to tumor-related processes. \u003cem\u003eDUSP5\u003c/em\u003e has been shown to regulate MAPK/ERK signaling and influence tumor cell proliferation and migration [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. \u003cem\u003ePLK2\u003c/em\u003e is involved in cell cycle regulation [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], while \u003cem\u003eAP3S1\u003c/em\u003e remains less well characterized in cancer biology. \u003cem\u003eBCL2L1\u003c/em\u003e, which encodes the anti-apoptotic protein Bcl-xL, is frequently upregulated in tumors and associated with enhanced cell survival [\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In addition, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG shows that \u003cem\u003eBCL2L1\u003c/em\u003e displayed the most significant change, with an adjusted p-value of 6.253 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e, indicating it is highly responsive to \u003cem\u003ePBX1\u003c/em\u003e depletion. In parallel, CUT\u0026amp;Tag profiling confirmed that PBX1 is strongly enriched at the promoter region of \u003cem\u003eBCL2L1\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH), supporting the likelihood that \u003cem\u003eBCL2L1\u003c/em\u003e is a direct transcriptional target of PBX1 in colorectal cancer cells.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 PBX1-Driven Upregulation of \u003cem\u003eBCL2L1\u003c/em\u003e Promoter Activity in CRC Cells\u003c/h2\u003e\u003cp\u003eTo elucidate the regulatory mechanism by which \u003cem\u003ePBX1\u003c/em\u003e modulates \u003cem\u003eBCL2L1\u003c/em\u003e expression, we performed ChIP-qPCR and dual-luciferase reporter assays. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA, we first cloned a\u0026thinsp;~\u0026thinsp;1719 bp fragment of the \u003cem\u003eBCL2L1\u003c/em\u003e promoter region into the pGL3-Basic vector to construct a full-length promoter reporter plasmid. Based on the CUT\u0026amp;Tag data indicating \u003cem\u003ePBX1\u003c/em\u003e binding intensity, this promoter region was further subdivided into three overlapping fragments (Fragments 1, 2, and 3), which were individually cloned into the pGL3-Basic vector to assess \u003cem\u003ePBX1\u003c/em\u003e\u0026rsquo;s effect on discrete regulatory elements.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eLuciferase reporter assays demonstrated that the full-length \u003cem\u003eBCL2L1\u003c/em\u003e promoter construct exhibited robust promoter activity in both HCT116 and RKO cells, with approximately 65-fold and 45-fold activation, respectively, compared to the empty vector control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Knockdown of \u003cem\u003ePBX1\u003c/em\u003e via shRNA significantly reduced the promoter activity in both cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC), whereas stable overexpression of \u003cem\u003ePBX1\u003c/em\u003e led to a marked increase in promoter activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003eTo further localize the critical \u003cem\u003ePBX1\u003c/em\u003e-responsive region within the promoter, we assessed the activity of the three truncated promoter fragments. In HCT116 cells, knockdown of \u003cem\u003ePBX1\u003c/em\u003e resulted in a significant decrease in luciferase activity driven by Fragment 3, while Fragments 1 and 2 showed minimal changes (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Conversely, PBX1 overexpression significantly enhanced the activity of Fragment 3 (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). Similar results were observed in RKO cells, where \u003cem\u003ePBX1\u003c/em\u003e knockdown suppressed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG) and \u003cem\u003ePBX1\u003c/em\u003e overexpression enhanced (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eH) the activity of Fragment 3, indicating that this region contains a critical PBX1-responsive element.\u003c/p\u003e\u003cp\u003eTo verify the binding of PBX1 to the BCL2L1 promoter in vivo and assess its impact on chromatin activation, we performed ChIP-qPCR targeting PBX1 and H3K27ac. In the ChIP-qPCR assay, we assessed the enrichment of PBX1 at this region, as well as its impact on chromatin activation status, marked by the histone modification H3K27ac [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Based on previous studies showing that transcription factor binding sites and histone modifications such as H3K27ac often occur in adjacent but non-overlapping regions within regulatory domains [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], we designed ChIP-qPCR primers to independently capture the peak enrichment sites for PBX1 and H3K27ac, respectively, according to our CUT\u0026amp;Tag and luciferase reporter assays results.\u003c/p\u003e\u003cp\u003eAs shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eI\u0026ndash;L, both PBX1 and H3K27ac were significantly enriched at the BCL2L1 promoter in HCT116 and RKO cells, with enrichment levels being notably higher in HCT116 cells. PBX1 knockdown led to a substantial reduction in PBX1 and H3K27ac occupancy at the promoter region (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eM and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eO), whereas PBX1 overexpression increased their enrichment (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eN and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eP), suggesting that PBX1 binding facilitates chromatin activation of the BCL2L1 promoter through H3K27 acetylation.\u003c/p\u003e\u003cp\u003eCollectively, these results demonstrate that PBX1 directly binds to a critical enhancer region within the BCL2L1 promoter, enhancing its transcriptional activity by modulating local chromatin accessibility.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 PBX1-Mediated Upregulation of Bcl-xL Suppresses Apoptosis\u003c/h2\u003e\u003cp\u003eTo further clarify the regulatory effect of \u003cem\u003ePBX1\u003c/em\u003e on \u003cem\u003eBCL2L1\u003c/em\u003e expression, we conducted both knockdown and overexpression experiments in HCT116 and RKO colorectal cancer cells. Specifically, we assessed how changes in \u003cem\u003ePBX1\u003c/em\u003e expression influence the two major \u003cem\u003eBCL2L1\u003c/em\u003e transcript variants, which encode the protein isoforms Bcl-xL and Bcl-xS. As described in the Introduction, Bcl-xL functions as an anti-apoptotic protein, whereas Bcl-xS promotes apoptosis.\u003c/p\u003e\u003cp\u003eWe therefore measured the mRNA levels of Bcl-xL and Bcl-xS separately. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC, \u003cem\u003ePBX1\u003c/em\u003e knockdown significantly reduced the expression of Bcl-xL, while Bcl-xS levels remained unchanged. Conversely, overexpression of \u003cem\u003ePBX1\u003c/em\u003e led to a marked increase in Bcl-xL mRNA, with only a modest upregulation of Bcl-xS observed in RKO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results suggest that PBX1 predominantly regulates the expression of the anti-apoptotic isoform Bcl-xL at the mRNA level in colorectal cancer cells. Consistent with the mRNA-level findings, our long-term observations from repeated WB experiments revealed that CRC cells predominantly express the Bcl-xL protein isoform (~\u0026thinsp;30 kDa). Even under overexposed blot conditions, the Bcl-xS isoform (~\u0026thinsp;18 kDa) was undetectable in endogenous settings (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE). Only upon exogenous overexpression of Bcl-xS from a plasmid could we detect the corresponding protein band in HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF). Taken together, these results suggest that in CRC cells, \u003cem\u003ePBX1\u003c/em\u003e primarily regulates the Bcl-xL isoform of \u003cem\u003eBCL2L1\u003c/em\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eGiven the predominance of the Bcl-xL isoform in CRC cells, subsequent protein-level analyses focused specifically on Bcl-xL. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eH, \u003cem\u003ePBX1\u003c/em\u003e knockdown in HCT116 cells led to a reduction in Bcl-xL protein levels, accompanied by an increase in apoptotic cells. Conversely, \u003cem\u003ePBX1\u003c/em\u003e overexpression resulted in elevated Bcl-xL protein expression and a decrease in apoptosis in HCT116 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eI and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eJ). Similar results were observed in RKO cells, where \u003cem\u003ePBX1\u003c/em\u003e overexpression upregulated Bcl-xL protein and reduced apoptosis, while \u003cem\u003ePBX1\u003c/em\u003e knockdown produced the opposite effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eK\u0026ndash;N). Together, these findings indicate that \u003cem\u003ePBX1\u003c/em\u003e modulates apoptosis in colorectal cancer cells primarily through regulation of the anti-apoptotic isoform Bcl-xL.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Functional Interaction Between PBX1 and \u003cem\u003eBCL2L1\u003c/em\u003e in Regulating Apoptosis and Tumor Growth\u003c/h2\u003e\u003cp\u003eTo further explore the functional relationship between PBX1 and \u003cem\u003eBCL2L1\u003c/em\u003e in regulating tumor cell fate, we examined the effects of \u003cem\u003eBCL2L1\u003c/em\u003e knockdown in the context of \u003cem\u003ePBX1\u003c/em\u003e overexpression. As shown in Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, overexpression of \u003cem\u003ePBX1\u003c/em\u003e in HCT116 cells led to a marked increase in Bcl-xL protein levels, while silencing \u003cem\u003eBCL2L1\u003c/em\u003e (shBCL2L1) effectively diminished Bcl-xL expression, confirming that \u003cem\u003ePBX1\u003c/em\u003e enhances Bcl-xL expression through transcriptional upregulation. Similar results were observed in RKO cells (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC and \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD), indicating that this regulatory mechanism is consistent across CRC models.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eApoptosis assays (Figs.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE-H) demonstrated that \u003cem\u003ePBX1\u003c/em\u003e overexpression alone significantly suppressed apoptosis in HCT116 and RKO cells. However, this anti-apoptotic effect was reversed upon \u003cem\u003eBCL2L1\u003c/em\u003e knockdown, with apoptosis levels restored to baseline, indicating that \u003cem\u003ePBX1\u003c/em\u003e modulates apoptosis predominantly through \u003cem\u003eBCL2L1\u003c/em\u003e-dependent Bcl-xL upregulation.\u003c/p\u003e\u003cp\u003eTo evaluate the impact of this regulatory axis on chemotherapy sensitivity, we treated HCT116 and RKO cells with increasing doses of 5-fluorouracil (5-FU) and assessed cell viability. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI, dose-response curves revealed IC50 values of 37.84 \u0026micro;M for HCT116 cells and 35.87 \u0026micro;M for RKO cells after 24-hour exposure. Based on these results, we selected a treatment concentration of 10 \u0026micro;M\u0026mdash;approximately one-quarter to one-half of the IC50\u0026mdash;to induce measurable drug effects without excessive cytotoxicity[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. At this concentration, \u003cem\u003ePBX1\u003c/em\u003e overexpression alone significantly reduced cell viability in HCT116 cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ), suggesting enhanced drug sensitivity. Importantly, combined \u003cem\u003ePBX1\u003c/em\u003e overexpression and \u003cem\u003eBCL2L1\u003c/em\u003e knockdown led to an even more pronounced reduction in cell viability, highlighting a synergistic inhibitory effect and reinforcing the functional relevance of the \u003cem\u003ePBX1\u003c/em\u003e\u0026ndash;\u003cem\u003eBCL2L1\u003c/em\u003e axis under chemotherapeutic stress. A similar trend was observed in RKO cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eK), reinforcing the functional relevance and generalizability of the PBX1\u0026ndash;BCL2L1 regulatory axis in modulating chemoresistance across CRC models.\u003c/p\u003e\u003cp\u003eTo validate these findings in vivo, we established xenograft models in nude mice with HCT116 cells and divided them into three groups: control (empty-pCDH\u0026thinsp;+\u0026thinsp;shNC), \u003cem\u003ePBX1\u003c/em\u003e overexpression (PBX1-pCDH\u0026thinsp;+\u0026thinsp;shNC), and \u003cem\u003ePBX1\u003c/em\u003e overexpression combined with \u003cem\u003eBCL2L1\u003c/em\u003e knockdown (PBX1-pCDH\u0026thinsp;+\u0026thinsp;shBCL2L1). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eL, the control group developed the largest tumors, the \u003cem\u003ePBX1\u003c/em\u003e overexpression group showed moderate suppression, and the combination group exhibited the smallest tumor size, indicating enhanced tumor suppression when both interventions were applied. Figure\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eM shows that there were no significant differences in body weight across groups during the experiment, suggesting no overt toxicity from the treatments.\u003c/p\u003e\u003cp\u003eTumor volume monitoring over time (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eN) confirmed that tumors in the \u003cem\u003ePBX1\u003c/em\u003e overexpression combined with \u003cem\u003eBCL2L1\u003c/em\u003e knockdown group grew significantly slower than those in the other groups, while no significant difference was observed between the control and \u003cem\u003ePBX1\u003c/em\u003e-overexpressing groups alone. Final tumor images (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eO) and corresponding tumor weights (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eP) further supported this trend: the control group had the heaviest tumors, followed by the \u003cem\u003ePBX1\u003c/em\u003e overexpression group, with the \u003cem\u003ePBX1\u003c/em\u003e overexpression combined with \u003cem\u003eBCL2L1\u003c/em\u003e knockdown group displaying the lowest tumor burden.\u003c/p\u003e\u003cp\u003eThese results demonstrate that while \u003cem\u003ePBX1\u003c/em\u003e overexpression alone moderately suppresses tumor growth, the concurrent silencing of \u003cem\u003eBCL2L1\u003c/em\u003e substantially enhances this effect. This combinatorial approach underscores the therapeutic potential of targeting the \u003cem\u003ePBX1\u003c/em\u003e\u0026ndash;\u003cem\u003eBCL2L1\u003c/em\u003e axis to simultaneously reduce tumor proliferation and overcome apoptotic resistance in colorectal cancer.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eGiven the central role of apoptosis evasion in cancer progression and therapeutic resistance, our study elucidates how the \u003cem\u003ePBX1\u003c/em\u003e\u0026ndash;\u003cem\u003eBCL2L1\u003c/em\u003e axis contributes to apoptosis suppression and modulates tumor cell fate in CRC. We demonstrate that \u003cem\u003ePBX1\u003c/em\u003e overexpression inhibits CRC cell proliferation, migration, and invasion, indicating a tumor-suppressive role. However, \u003cem\u003ePBX1\u003c/em\u003e overexpression concurrently reduces apoptosis, potentially allowing cells to enter a quiescent, drug-resistant state. This dual effect complicates the therapeutic use of \u003cem\u003ePBX1\u003c/em\u003e overexpression alone, as it may restrict the efficacy of pro-apoptotic treatments.\u003c/p\u003e\n\u003cp\u003eOur analysis of public datasets (TCGA and Oncomine) and patient samples confirmed that \u003cem\u003ePBX1\u003c/em\u003e is consistently downregulated in CRC tissues and cell lines, supporting previous findings that \u003cem\u003ePBX1\u003c/em\u003e often plays a tumor-suppressive role in solid tumors[\u003cspan class=\"CitationRef\"\u003e13\u003c/span\u003e]. Functional assays revealed that \u003cem\u003ePBX1\u003c/em\u003e knockdown promotes proliferation, colony formation, and invasion, while \u003cem\u003ePBX1\u003c/em\u003e overexpression suppresses these oncogenic behaviors, reinforcing its role as a tumor suppressor in CRC.\u003c/p\u003e\n\u003cp\u003eMechanistically, transcriptomic and epigenomic profiling revealed \u003cem\u003eBCL2L1\u003c/em\u003e as a direct transcriptional target of PBX1. PBX1 binds the \u003cem\u003eBCL2L1\u003c/em\u003e promoter and enhances its transcription, supported by increased H3K27ac enrichment and and luciferase reporter activation. These results align with previous studies that demonstrate PBX1\u0026rsquo;s ability to regulate chromatin accessibility and gene expression at key loci [\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e]. Further isoform-specific analysis showed that PBX1 primarily upregulates Bcl-xL, the anti-apoptotic isoform, in a splicing environment biased toward Bcl-xL. This was validated by qPCR and western blotting, which detected endogenous Bcl-xL but not Bcl-xS in CRC cells. These findings suggest that PBX1 increases the overall transcription of \u003cem\u003eBCL2L1\u003c/em\u003e, and in the presence of a cell-intrinsic splicing bias, promotes Bcl-xL expression, contributing to apoptosis resistance.\u003c/p\u003e\n\u003cp\u003eFunctional assays confirmed that \u003cem\u003ePBX1\u003c/em\u003e knockdown decreased Bcl-xL levels and increased apoptosis, while overexpression of \u003cem\u003ePBX1\u003c/em\u003e elevated Bcl-xL and reduced apoptotic cell death. These findings align with prior studies highlighting the oncogenic role of Bcl-xL in tumor survival and drug resistance [\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e]. Importantly, knocking down \u003cem\u003eBCL2L1\u003c/em\u003e in \u003cem\u003ePBX1\u003c/em\u003e-overexpressing cells reversed \u003cem\u003ePBX1\u003c/em\u003e\u0026rsquo;s anti-apoptotic effect, highlighting Bcl-xL as a key downstream effector of \u003cem\u003ePBX1\u003c/em\u003e. While \u003cem\u003ePBX1\u003c/em\u003e overexpression modestly reduced proliferation, it enhanced cell survival\u0026mdash;a phenotype that was abolished when \u003cem\u003eBCL2L1\u003c/em\u003e was knocked down. This finding suggests that \u003cem\u003ePBX1\u003c/em\u003e maintains a pool of non-proliferative, apoptosis-resistant tumor cells that may evade therapy and contribute to relapse.\u003c/p\u003e\n\u003cp\u003eFunctional assays further supported this regulatory preference: \u003cem\u003ePBX1\u003c/em\u003e knockdown decreased Bcl-xL and significantly increased apoptosis, while \u003cem\u003ePBX1\u003c/em\u003e overexpression elevated Bcl-xL expression and suppressed apoptotic activity. Importantly, functional interaction experiments in \u003cem\u003ePBX1\u003c/em\u003e-overexpressing cells demonstrated that \u003cem\u003eBCL2L1\u003c/em\u003e knockdown reversed the anti-apoptotic effect of \u003cem\u003ePBX1\u003c/em\u003e, confirming that Bcl-xL is a key functional effector downstream of \u003cem\u003ePBX1\u003c/em\u003e. In addition, while \u003cem\u003ePBX1\u003c/em\u003e overexpression modestly suppressed proliferation, it simultaneously enhanced cell survival\u0026mdash;an effect that was abrogated by \u003cem\u003eBCL2L1\u003c/em\u003e knockdown. This dual effect suggests that \u003cem\u003ePBX1\u003c/em\u003e may promote the persistence of non-proliferative, apoptosis-resistant tumor cells, which could contribute to treatment resistance or relapse if left unchecked.\u003c/p\u003e\n\u003cp\u003eTo assess the clinical relevance of this axis, we evaluated how \u003cem\u003ePBX1\u003c/em\u003e and \u003cem\u003eBCL2L1\u003c/em\u003e influence 5-FU chemotherapy response. \u003cem\u003ePBX1\u003c/em\u003e overexpression reduced cell viability under 5-FU exposure, and the combination of \u003cem\u003ePBX1\u003c/em\u003e overexpression with \u003cem\u003eBCL2L1\u003c/em\u003e knockdown further enhanced 5-FU cytotoxicity. These results suggest that targeting \u003cem\u003eBCL2L1\u003c/em\u003e can improve the therapeutic efficacy of \u003cem\u003ePBX1\u003c/em\u003e-based modulation. Similar patterns were observed in RKO cells, reinforcing the generalizability of the \u003cem\u003ePBX1\u0026ndash;BCL2L1\u003c/em\u003e axis in CRC.\u003c/p\u003e\n\u003cp\u003eIn vivo, xenograft experiments confirmed that \u003cem\u003ePBX1\u003c/em\u003e overexpression suppresses tumor growth, and this effect was significantly augmented by \u003cem\u003eBCL2L1\u003c/em\u003e knockdown. Tumor volume and weight were lowest in the \u003cem\u003ePBX1\u003c/em\u003e overexpression combined with \u003cem\u003eBCL2L1\u003c/em\u003e knockdown group, supporting a synergistic effect in vivo. These findings underscore the cooperative role of \u003cem\u003ePBX1\u003c/em\u003e and \u003cem\u003eBCL2L1\u003c/em\u003e in modulating tumorigenesis and therapeutic sensitivity.\u003c/p\u003e\n\u003cp\u003eBeyond \u003cem\u003eBCL2L1\u003c/em\u003e, we identified additional \u003cem\u003ePBX1\u003c/em\u003e-regulated targets such as \u003cem\u003eDUSP5\u003c/em\u003e and \u003cem\u003ePLK2\u003c/em\u003e, which are known to inhibit MAPK signaling and cell cycle progression, respectively [\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan class=\"CitationRef\"\u003e32\u003c/span\u003e]. These targets likely contribute to the anti-proliferative function of \u003cem\u003ePBX1\u003c/em\u003e and further establish its multifaceted role in CRC suppression.\u003c/p\u003e\n\u003cp\u003eIn summary, we propose a working model in which \u003cem\u003ePBX1\u003c/em\u003e simultaneously promotes anti-proliferative and anti-apoptotic pathways in CRC (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). \u003cem\u003ePBX1\u003c/em\u003e directly enhances \u003cem\u003eBCL2L1\u003c/em\u003e transcription, leading to Bcl-xL\u0026ndash;mediated apoptotic resistance. Concurrently, \u003cem\u003ePBX1\u003c/em\u003e activates tumor-suppressive genes like \u003cem\u003eDUSP5\u003c/em\u003e and \u003cem\u003ePLK2\u003c/em\u003e, inhibiting proliferation. This dual regulation creates a state in which tumor cells are growth-arrested yet resistant to apoptosis, potentially contributing to therapy resistance. Disrupting this balance by combining \u003cem\u003ePBX1\u003c/em\u003e overexpression with \u003cem\u003eBCL2L1\u003c/em\u003e knockdown effectively inhibits both proliferation and survival, thereby improving treatment efficacy. The \u003cem\u003ePBX1\u003c/em\u003e\u0026ndash;\u003cem\u003eBCL2L1\u003c/em\u003e axis thus emerges as a promising dual-action therapeutic target in colorectal cancer.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"4. Materials and Methods","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\n\u003ch2\u003e4.1 Patients and tissues\u003c/h2\u003e\nThis study examined 50 cases of colonic adenocarcinoma and corresponding adjacent normal colonic mucosa, collected from patients who underwent surgical treatment at Shantou Central Hospital (Guangdong, China) between 2020 and 2022. All tissues were paraffin-embedded and diagnosedindependently by two pathologists. Patients who had received radiotherapy or chemotherapy prior to surgery were excluded. Ethical approval was obtained from the local ethics committee (Shantou University Medical College, SUMC-2020-14), and informed consent was acquired.\u003c/div\u003e\n\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\n\u003ch2\u003e4.2 Cell Lines and Culture Conditions\u003c/h2\u003e\n\u003cp\u003eHuman colorectal cancer cell lines, including HCT116, HT-29, LoVo, RKO, SW480, and SW620, and the normal colon cell line CCD-18Co, were obtained from ATCC (Manassas, VA, USA) or the Shanghai Cell Bank, Chinese Academy of Sciences (LoVo). Cells were maintained in cell line-specific media as follows: McCoy\u0026rsquo;s 5A Medium (for HCT116 and HT-29; Gibco, Cat. No. 16600082); F-12K Nutrient Mixture (for LoVo; Gibco, Cat. No. 21127022); Minimum Essential Medium (MEM) supplemented with 2 mM L-glutamine and 1 mM sodium pyruvate (for RKO; Gibco, Cat. No. 11380037); Leibovitz\u0026rsquo;s L-15 Medium (for SW480 and SW620; Gibco, Cat. No. 11415064); MEM supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, and 1\u0026times; non-essential amino acids (NEAA) (for CCD-18Co; Gibco, Cat. No. 11140050). All media were supplemented with 10% fetal bovine serum (FBS) (Gibco, Cat. No. 10099141), and cells were incubated at 37\u0026deg;C in a humidified atmosphere containing 5% CO₂ (except for L-15, which was cultured in atmospheric air).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\n\u003ch2\u003e4.3 RNA Extraction, cDNA Synthesis, and qPCR\u003c/h2\u003e\n\u003cp\u003eTotal RNA was extracted using RNAiso Plus (Takara Bio Inc., Shiga, Japan; Cat. No. 9109). First-strand cDNA synthesis was performed using the HiScript II Q RT SuperMix for qPCR (Vazyme Biotech Co., Ltd., Nanjing, China; Cat. No. R223-01). Quantitative PCR was conducted with the ChamQ SYBR qPCR Master Mix (Vazyme Biotech Co., Ltd., Nanjing, China; Cat. No. Q331) on an ABI 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). \u0026beta;-actin was used as an internal control. Primer sequences are listed in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS1\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003e4.4 Western Blot Analysis\u003c/h2\u003e\n\u003cp\u003eProteins were resolved on 10% SDS-PAGE gels and transferred onto PVDF membranes (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. ISEQ00010). Membranes were incubated with primary antibodies followed by HRP-conjugated secondary antibodies. Signals were visualized using enhanced chemiluminescent detection reagents (Yeasen Biotechnology, Shanghai, China; Cat. No. 36208ES76). The primary antibodies used included: Rabbit anti-PBX1 (Proteintech Group, Wuhan, China; Cat. No. 18204-1-AP; dilution 1:500), Mouse anti-Bcl-xS/L (Santa Cruz Biotechnology, Dallas, TX, USA; Cat. No. sc-271121; dilution 1:200), Mouse anti-\u0026beta;-actin (Proteintech Group, Wuhan, China; Cat. No. 66009-1-Ig; dilution 1:10000). The secondary antibodies used were: HRP-conjugated goat anti-mouse IgG (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 31430; dilution 1:5000), HRP-conjugated goat anti-rabbit IgG (Thermo Fisher Scientific, Waltham, MA, USA; Cat. No. 31460; dilution 1:10000).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003e4.5 Database Analysis\u003c/h2\u003e\n\u003cp\u003e\u003cem\u003ePBX1\u003c/em\u003e expression in CRC was assessed using TCGA and Oncomine databases. TCGA RNA-seq data were analyzed using DESeq2 (v1.46.0), identifying significantly differentially expressed genes (adjusted p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, |fold change| \u0026ge; 2). Oncomine Database Analysis: We queried the database to analyze \u003cem\u003ePBX1\u003c/em\u003e mRNA expression in CRC patients. By comparing multiple datasets provided by Oncomine (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.oncomine.org\" target=\"_blank\"\u003ewww.oncomine.org\u003c/a\u003e\u003c/span\u003e\u003c/span\u003e, accessed on 1 March 2021), downloaded in March 2021, and data were analyzed with thresholds of p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 and fold change\u0026thinsp;\u0026gt;\u0026thinsp;2.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003e4.6 Cell Transfections\u003c/h2\u003e\n\u003cp\u003eFor stable gene modulation, lentiviral transduction was employed to achieve \u003cem\u003ePBX1\u003c/em\u003e overexpression and shRNA-mediated knockdown. Lentiviral particles were generated by co-transfecting packaging plasmids and expression vectors into HEK293T cells using standard calcium phosphate or lipid-based methods. Target CRC cells were infected with viral supernatants in the presence of 8 \u0026micro;g/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA; Cat. No. H9268) for 24 hours. Infected cells were selected using puromycin (1 \u0026micro;g/mL; Sangon Biotech, Shanghai, China; Cat. No. A610593) and/or blasticidin (8 \u0026micro;g/mL; Beyotime, Shanghai, China; Cat. No. ST018), or sorted by flow cytometry when applicable.\u003c/p\u003e\n\u003cp\u003eThe \u003cem\u003ePBX1\u003c/em\u003e overexpression plasmid was constructed by inserting the full-length coding sequence of \u003cem\u003ePBX1\u003c/em\u003e (NM_001204961.2) into the pCDH-CMV-EF1-copGFP lentiviral backbone (System Biosciences, Palo Alto, CA, USA). shRNA plasmids were obtained from GenePharma (Suzhou, China): shPBX1 was cloned into the LV3 (H1/GFP\u0026amp;Puro) backbone, targeting sequence: 5'-GTGGAGCATTCAGATTACA-3', and shBCL2L1 was cloned into the pLKO.1-blast backbone, targeting sequence: 5'-CCCTACCTGATTGGTGCAA-3'.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003e4.7 RNA Sequencing and Data Processing\u003c/h2\u003e\n\u003cp\u003eTotal RNA from \u003cem\u003ePBX1\u003c/em\u003e-knockdown and control HCT116 cells was sequenced by BGI Genomics (Shenzhen, China) using the BGISEQ platform. Reads were mapped to GRCh38 using RSEM, and differential expression analysis was conducted using DESeq2 (v1.46.0). Gene set enrichment analysis (GSEA, v4.3.3) was performed with the MSigDB collection c2.cp.wikipathways.v2024.1. NES and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.25 were considered significant.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\n\u003ch2\u003e4.8 CUT\u0026amp;Tag Assay and Data Processing\u003c/h2\u003e\n\u003cp\u003eCUT\u0026amp;Tag assays were conducted using the Hyperactive Universal CUT\u0026amp;Tag Assay Kit for Illumina Pro (Vazyme, TD904; Vazyme Biotech, Nanjing, China), with a mild formaldehyde crosslinking step added prior to cell permeabilization. Library construction was performed via Tn5 tagmentation followed by PCR amplification using the reagents provided in the kit. Final libraries were sequenced on the BGISEQ platform (BGI Genomics, Shenzhen, China). Raw reads were aligned to the human genome using Bowtie2 (v2.3.5.1), and peak calling was performed with SEACR (Sparse Enrichment Analysis for CUT\u0026amp;RUN). Peak annotation and motif enrichment analyses were carried out using HOMER (v4.11).\u003c/p\u003e\n\u003cp\u003eCUT\u0026amp;Tag experiments targeting PBX1 and H3K27ac were performed in-house. The following primary antibodies were used: anti-PBX1 (Abnova, H00005087-M01, mouse monoclonal, 1:50; Taipei, Taiwan), anti-H3K27ac (Invitrogen, 720096, rabbit polyclonal, 1:50; Carlsbad, CA, USA). Public ChIP-seq datasets for H3K4me3 (GEO: GSM1224663) and H3K4me1 (GEO: GSM1866700) were downloaded from the GEO database for comparative chromatin landscape profiling.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n\u003ch2\u003e4.9 Cell Proliferation Assay\u003c/h2\u003e\n\u003cp\u003eCell proliferation was assessed using the Cell Counting Kit-8 (CCK-8; MedChemExpress, HY-K0301; Monmouth Junction, NJ, USA) following the manufacturer\u0026rsquo;s instructions. Cells were seeded in 96-well plates at appropriate densities according to cell line characteristics. Absorbance at 450 nm was measured daily using a microplate reader to generate cell growth curves.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n\u003ch2\u003e4.10 Colony Formation Assay\u003c/h2\u003e\n\u003cp\u003eFor colony formation assays, cells were seeded in 6-well plates at a density of 300 cells per well and cultured for 2 weeks. Colonies were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, C0121; Shanghai, China) for 30 minutes at room temperature. The colony-covered area was quantified using ImageJ software (version 1.46r; NIH, Bethesda, MD, USA) and compared to the control group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n\u003ch2\u003e4.11 Transwell Migration and Invasion Assay\u003c/h2\u003e\n\u003cp\u003eFor cell migration and invasion assays, Transwell chambers (8-\u0026micro;m pore size; BD Falcon, 35309724; Franklin Lakes, NJ, USA) were used. For invasion assays, the upper chambers were pre-coated with Matrigel (BD Biosciences, 356234; San Jose, CA, USA). Cells were serum-starved for 12 hours, seeded into the upper chambers in serum-free medium, and allowed to migrate toward medium containing 20% fetal bovine serum in the lower chambers. After 24 hours of incubation, cells on the lower surface were fixed with 4% paraformaldehyde and stained with crystal violet (Beyotime, C0121; Shanghai, China) for 30 minutes at room temperature.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n\u003ch2\u003e4.12 Cell Apoptosis Assay\u003c/h2\u003e\n\u003cp\u003eApoptosis was evaluated using the APC Annexin V Apoptosis Detection Kit (Tonbo Biosciences, 20-6410-KIT; San Diego, CA, USA) according to the manufacturer\u0026rsquo;s instructions. Cells were stained with APC Annexin V and 7-AAD, and analyzed by flow cytometry using a Celula Sparrow2040 flow cytometer (Celula Inc., Shanghai, China). The percentages of early and late apoptotic cells were quantified.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\n\u003ch2\u003e4.13 Chromatin immunoprecipitation (ChIP)\u003c/h2\u003e\n\u003cp\u003eChromatin immunoprecipitation (ChIP) was performed using the EZ-ChIP\u0026trade; Chromatin Immunoprecipitation Kit (Sigma-Aldrich, 17\u0026ndash;611; St. Louis, MO, USA) according to the manufacturer\u0026rsquo;s instructions, as previously described [\u003cspan class=\"CitationRef\"\u003e43\u003c/span\u003e]. Immunoprecipitated DNA was analyzed by quantitative PCR, and results were normalized to input DNA. The following primary antibodies were used: anti-PBX1 (Abnova, H00005087-M01, mouse monoclonal, 1:50; Taipei, Taiwan), anti-H3K27ac (Invitrogen, 720096, rabbit polyclonal, 1:50; Carlsbad, CA, USA), mouse IgG control (Millipore, 12-371B, 1:50; Burlington, MA, USA), rabbit IgG control (Millipore, CS200581, 1:50; Burlington, MA, USA). Primer sequences used for qPCR analysis are listed in Supplementary Table \u003cspan class=\"InternalRef\"\u003eS2\u003c/span\u003e.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n\u003ch2\u003e4.14 Dual-luciferase reporter assay\u003c/h2\u003e\n\u003cp\u003eThe BCL2L1 promoter (~\u0026thinsp;1719 bp) and its truncated fragments (Fragment 1: 695 bp; Fragment 2: 638 bp; Fragment 3: 503 bp) were amplified using specific primers (sequences listed in Supplementary Table S3) and cloned into the pGL3-Basic vector (Promega, Madison, WI, USA) at the HindIII restriction site using the ClonExpress II One Step Cloning Kit (Vazyme, C116; Nanjing, China). For luciferase activity measurements, HCT116 and RKO cells were co-transfected with the constructed firefly luciferase plasmids and Renilla luciferase internal control plasmid (pRL-SV40, Promega, Madison, WI, USA) at a molar ratio of 100:1, using FuGENE\u0026reg; HD Transfection Reagent (Promega, E2311; Madison, WI, USA). After 48 hours, dual-luciferase activity was quantified using the Dual-Luciferase\u0026reg; Reporter Assay System (Vazyme, DD1205; Nanjing, China) according to the manufacturer\u0026rsquo;s protocol. Firefly luciferase activity was normalized to Renilla luciferase to control for transfection efficiency.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\n\u003ch2\u003e4.15 5-FU Treatment and Chemotherapy Sensitivity Assay\u003c/h2\u003e\n\u003cp\u003eTo evaluate chemotherapy response, HCT116 and RKO cells were treated with 0\u0026ndash;160 \u0026micro;M 5-fluorouracil (5-FU; MedChemExpress, HY-90006, Monmouth Junction, NJ, USA) for 24 hours. IC50 values were calculated using GraphPad Prism 8.0.2 (GraphPad Software, San Diego, CA, USA). Based on these values, a 10 \u0026micro;M concentration (approximately 1/4\u0026ndash;1/2 of the IC50) was selected for subsequent cell viability assays.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\n\u003ch2\u003e4.16 Xenograft Tumorigenesis in Nude Mice\u003c/h2\u003e\n\u003cp\u003eHCT116 cells (1\u0026times;10\u003csup\u003e7\u003c/sup\u003e) stably expressing Empty-pCDH\u0026thinsp;+\u0026thinsp;shNC, PBX1-pCDH\u0026thinsp;+\u0026thinsp;shNC, PBX1-pCDH\u0026thinsp;+\u0026thinsp;shBCL2L1 were injected subcutaneously into 5-week-old male BALB/c nude mice (n\u0026thinsp;=\u0026thinsp;5 per group; mice were randomly assigned to experimental groups using a random number table method; purchased from Guangdong Yaokang Biological Technology Co., Ltd., Guangdong, China). Tumor size and mouse weight were measured every 2\u0026ndash;3 days. Tumor volume was calculated as (length \u0026times; width\u003csup\u003e2\u003c/sup\u003e)/2. Mice were sacrificed at day 15, and tumors were photographed and weighed. Tumor measurements and outcome assessments were performed by investigators blinded to group allocation. All animal procedures were approved by the Institutional Animal Care and Use Committee of Shantou University Medical College ( SUMC-2022-289).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\n\u003ch2\u003e4.17 Statistical Analysis\u003c/h2\u003e\n\u003cp\u003eAll statistical analyses were performed using IBM SPSS Statistics version 26.0 (IBM Corp., Armonk, NY, USA). Each experiment with statistical comparison was conducted with at least three independent biological replicates. Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Homogeneity of variance was tested using Levene's test prior to t-test analysis. For comparisons with equal variance, standard Student\u0026rsquo;s t-test was applied; otherwise, Welch\u0026rsquo;s correction was used. One-way ANOVA followed by LSD post hoc test was used for multiple group comparisons. A P-value less than 0.05 was considered statistically significant. Detailed statistical methods and replicate numbers are provided in the corresponding figure legends.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec28\" class=\"Section2\"\u003e\n\u003ch2\u003e4.18 Code availability\u003c/h2\u003e\n\u003cp\u003eNo custom code was used in this study. All analyses were performed using standard bioinformatics packages and commercially available software.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003ePBX1\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003ePre-B-cell leukemia homeobox 1\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCRC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ecolorectal cancer\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003e\u003cem\u003eBCL2L1\u003c/em\u003e\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003e\u003cem\u003eBCL2 Like 1\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH3K27ac\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHistone H3 lysine 27 acetylation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH3K4me1\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHistone H3 lysine 4 monomethylation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eH3K4me3\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHistone H3 lysine 4 trimethylation\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBcl-xL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eB-cell lymphoma-extra large\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTALE\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThree amino acid loop extension\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBcl-xS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eB-cell lymphoma-extra small\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eATCC\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eAmerican Type Culture Collection\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTCGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThe Cancer Genome Atlas\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eBGI\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eBeijing Genomics Institute\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eRSEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eRNA-Seq by Expectation Maximization\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCUT\u0026amp;RUN\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eCleavage Under Targets and Release Using Nuclease\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSEACR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSparse Enrichment Analysis for CUT\u0026amp;RUN\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHOMER\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHypergeometric Optimization of Motif EnRichment\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eChIP\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eChromatin immunoprecipitation.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests Statement:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;The authors declare no competing interests. This research was supported by the Natural Science Foundation of Guangdong Province (Grant No. 2023A1515010434) and the National Natural Science Foundation of China (NSFC, Grant No. 32000456).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no specific acknowledgments for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors’ contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHao Lin and Ting Su contributed equally to this work and share first authorship. Hao Lin and Lingzhu Xie designed the study and supervised the project. Hao Lin and Xuanhao Lin collected and analyzed patient samples and clinical data. Ting Su, Rulan Deng, Jie Li, Ying Liu, Qiaoling Ke, Lele Meng, and Yijing Luo performed the experiments and collected data. Xuhong Song and Bin Liang conducted statistical analyses. Hao Lin, and Lingzhu Xie wrote the manuscript. Dongyang Huang and Lingzhu Xie provided overall supervision and revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Ethics Committee of Shantou University Medical College, China (Approval No. SUMC-2020-14). Written informed consent was obtained from all patients for tissue collection and analysis. All animal procedures were performed in accordance with institutional guidelines and approved by the Institutional Animal Care and Use Committee of Shantou University Medical College (Approval No. SUMC-2022-289).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the Natural Science Foundation of Guangdong Province (Grant No. 2023A1515010434) and the National Natural Science Foundation of China (NSFC, Grant No. 32000456).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analysed are available from the corresponding authors upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eSung H, Ferlay J, Siegel RL, Laversanne M, Soerjomataram I, Jemal A, Bray F. 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Aberrant activation of cyr61 enhancers in colorectal cancer development. \u003cem\u003eJ Exp Clin Cancer Res\u003c/em\u003e 38, 213\u0026ndash;28 (2019). \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/s13046-019-1217-9\u003c/span\u003e\u003cspan address=\"10.1186/s13046-019-1217-9\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7305360/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7305360/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003ePre-B-cell leukemia homeobox 1\u003c/em\u003e (\u003cem\u003ePBX1\u003c/em\u003e) is a transcription factor involved in diverse cellular processes, but its role in colorectal cancer (CRC) remains incompletely understood. In this study, we show that \u003cem\u003ePBX1\u003c/em\u003e is downregulated in CRC tissues and cell lines. Functional experiments revealed that \u003cem\u003ePBX1\u003c/em\u003e overexpression inhibits proliferation, migration, and invasion, but paradoxically suppresses apoptosis, suggesting a dual regulatory role. Transcriptome and CUT\u0026amp;Tag profiling identified \u003cem\u003eBCL2L1\u003c/em\u003e as a direct transcriptional target of PBX1. PBX1 binds the \u003cem\u003eBCL2L1\u003c/em\u003e promoter and enhances Bcl-xL expression, contributing to apoptotic resistance. \u003cem\u003eBCL2L1\u003c/em\u003e knockdown reversed the anti-apoptotic effects of \u003cem\u003ePBX1\u003c/em\u003e and restored apoptosis levels. Upon 5-fluorouracil (5-FU) treatment, \u003cem\u003ePBX1\u003c/em\u003e overexpression reduced cell viability, while concurrent \u003cem\u003eBCL2L1\u003c/em\u003e knockdown significantly enhanced drug sensitivity. In vivo, xenograft experiments demonstrated that \u003cem\u003ePBX1\u003c/em\u003e overexpression suppressed tumor growth, which was further augmented by \u003cem\u003eBCL2L1\u003c/em\u003e knockdown. These results underscore the dual role of \u003cem\u003ePBX1\u003c/em\u003e in simultaneously inhibiting tumor growth while promoting cell survival through the \u003cem\u003eBCL2L1\u003c/em\u003e\u0026ndash;Bcl-xL axis. Targeting this pathway could offer a novel therapeutic strategy for enhancing CRC treatment efficacy by simultaneously inhibiting proliferation and restoring apoptotic sensitivity.\u003c/p\u003e","manuscriptTitle":"Targeting the PBX1–BCL2L1 Axis as a Therapeutic Strategy in Colorectal Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-22 13:31:25","doi":"10.21203/rs.3.rs-7305360/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"transferred","content":"Cell Death Discovery","date":"2026-01-22T04:00:44+00:00","index":"","fulltext":""},{"type":"decision","content":"revise","date":"2025-09-11T14:15:53+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-09-03T09:43:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2025-08-29T08:03:39+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-21T17:37:04+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2025-08-21T16:28:57+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2025-08-15T01:43:45+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-06T11:09:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2025-08-06T03:52:27+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-06T03:52:27+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-discovery","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddiscovery","sideBox":"Learn more about [Cell Death Discovery](http://www.nature.com/cddiscovery/)","snPcode":"41420","submissionUrl":"https://mts-cddiscovery.nature.com/","title":"Cell Death Discovery","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"201242d0-bb16-4152-a912-276ea14c129a","owner":[],"postedDate":"August 22nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":53574733,"name":"Biological sciences/Cell biology/Mechanisms of disease"},{"id":53574734,"name":"Biological sciences/Cancer/Gastrointestinal cancer/Colorectal cancer"}],"tags":[],"updatedAt":"2026-02-20T16:50:26+00:00","versionOfRecord":[],"versionCreatedAt":"2025-08-22 13:31:25","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7305360","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7305360","identity":"rs-7305360","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}