Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity

Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity | 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 Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity Tatsuya Ishiguro, Haruka Ueda, Yutaro Mori, Kaoru Yamawaki, Takayuki Enomoto, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3547380/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 11 Oct, 2024 Read the published version in Cell Death Discovery → Version 1 posted You are reading this latest preprint version Abstract Cancer stem cells are associated with aggressive phenotypes of malignant tumors. A prominent feature of uterine endometrial cancer is the activation of the PI3K-Akt-mTOR pathway. Herein, we present variations in sensitivities to a PI3K-Akt-mTORC1 inhibitor among in vitro endometrial cancer stem cell-enriched spheroid cells from clinical specimens. Intriguingly, the in vitro sensitivity mirrored effects observed in in vivo spheroid-derived xenograft tumor models. Our findings reveal a complementary suppressive effect on endometrial cancer spheroid cell growth when aldehyde dehydrogenase (ALDH) and PI3K-Akt inhibitors are combined. In the PI3K-Akt-mTORC1 signaling cascade, the influence of ALDH on mTORC1 is partially channeled through retinoic acid-induced lactate dehydrogenase A (LDHA) activation. LDHA inhibition was found to reduce endometrial cancer cell growth, paralleling the effects of mTORC1 inhibition. Building upon our prior findings highlighting ALDH-driven glycolysis through GLUT1 in uterine endometrial cancer spheroid cells, curbing mTORC1 bolstered glucose transport via GLUT1 activation. Notably, elevated LDHA expression correlated with adverse clinical survival and escalated tumor grade, especially in advanced stages. Collectively, our findings emphasize the pivotal role of ALDH-LDHA-mTORC1 cascade in the proliferation of endometrial cancer. Targeting the intricate interplay between mTORC1 and ALDH-influenced glycolysis could pave the way for novel strategies to combat this aggressive cancer. Biological sciences/Cancer/Cancer stem cells Biological sciences/Cancer/Gynaecological cancer/Endometrial cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Uterine endometrial cancer is a significant gynecological ailment. While early-stage and low-grade uterine endometrial cancers present relatively mild behaviors, standard chemotherapy regimens fail to adequately address high-grade or metastatic tumors ( 1 ). A common molecular characteristic of both type I and type II endometrial cancers is the dysregulation of the PI3K-Akt-mTORC1 signaling pathway ( 2 ). Positioned as a downstream target, mTORC1 is influenced by numerous oncogenic pathways in cancer, such as the PI3K/Akt and MAPK pathways ( 3 ). Furthermore, the mTORC1 complex is modulated by growth factors, amino acids, energy levels, and stress, and it plays a role in cell growth through protein, lipid, and nucleotide synthesis as well as autophagy ( 3 ). Similar to many cancers, most endometrial cancers exhibit genomic aberrations in the PI3K-Akt-mTORC1 signaling pathway, which often lead to mTORC1 hyperactivation ( 4 ). Thus, therapies targeting the PI3K-Akt-mTORC1 signaling could potentially enhance patient outcomes in endometrial cancer ( 3 , 5 ). However, results from a recent phase I/II trial involving an mTOR inhibitor combined with an aromatase inhibitor, anastrozole, showed limited improvements in hormone receptor-positive recurrent or metastatic endometrial cancer patients, with an overall response rate of 24.5% ( 6 ). Another phase II clinical trial demonstrated that dual PI3K/mTOR inhibitors offered modest clinical benefits (overall response rate: 16%; duration of response: 4.2 months) with manageable side effects ( 7 ). Delving deeper into the mechanism of mTORC1 signaling and formulating methods to identify benefiting patients remains of paramount importance. Cancer stem cells, identified as primary contributors to cancer origination, when eliminated, significantly retard cancer progression ( 8 ). Aldehyde dehydrogenase (ALDH) serves as a specific marker for many types of cancer stem cells ( 9 ). ALDH, pivotal in retinoid metabolism and toxic aldehyde removal, influences cancer progression, stemness, and chemotherapy resistance across various cancers. Notably, among the 19 ALDH isoforms, ALDH1A1, ALDH1A3, and ALDH3A1 are tied to cancer stem cells ( 10 ). We previously identified ALDH as a functional marker for both ovarian and uterine endometrial cancer stem cells ( 11 , 12 ). In particular, we highlighted ALDH1A1 as the specific isoform marking uterine endometrial cancer stem cells and uncovering the role of the ALDH-GLUT cascade in paclitaxel resistance among these cells ( 12 ). However, comprehending the ALDH mechanism in endometrial cancer proliferation remains an open question. Patient-derived tridimensional cells, encompassing spheroid and organoid cells from clinical specimens, retain several clinical traits. They are invaluable platforms for assessing drug sensitivity in numerous solid cancers, including ovarian and uterine endometrial cancers ( 13 , 14 ). Our earlier research indicated that gynecological cancer patient-derived spheroid cells exhibit cellular diversity and potent tumorigenic capabilities in vivo , which are hallmark characteristics of cancer stem cells ( 11 , 12 , 15 ). Moreover, our spheroid cells have proven effective in drug sensitivity determination ( 16 ), echoing the utility of organoid cells that are universally recognized for drug sensitivity assays ( 17 ). In this study, we delved into the sensitivity of inhibitors targeting the PI3K-Akt-mTORC1 signaling, employing uterine endometrial cancer patient-derived spheroid cells. We juxtaposed protein and genomic mutational profiles of these spheroid cells. A drug sensitivity test elucidated the interplay between ALDH and mTOR, uncovering the novel function of ALDH in cancer and its contribution to mTORC1 activation via LDHA. Furthermore, we identified the reciprocal relationship between glycolysis and mTORC1 in promoting the proliferation of endometrial cancer cells. These findings underscore that targeting ALDH-LDHA-mTORC1 signaling is a novel treatment strategy for aggressive uterine endometrial cancer. Materials and Methods Tumor-derived Spheroid Culture Endometrial cancer spheroid cells isolated from clinical cancer specimens were cultured using ultra-low-attachment culture dishes (Corning, Corning, NY, USA). The culture medium was STEMPRO hESC SFM (Gibco, Grand Island, NY, USA) with a supplementation of 8 ng/ml basic fibroblast growth factor (Invitrogen, Carlsbad, CA, USA) and penicillin/streptomycin. Cells were maintained under specific conditions at 37°C with 5% CO 2 concentration ( 15 ). Spheroid cells were dissociated using Accumax (Innovative Cell Technologies, San Diego, CA, USA) for serial passaging. All the culture and handling protocols strictly adhered to the guidelines of the Ethics Committee of Niigata University and the National Cancer Center. Informed consent was duly acquired from all involved patients. Animal Experiments The drug efficacy experiments followed a randomized selection of mice into distinct groups. Accumax (Innovative Cell Technologies) was used to dissociate spheroid cells into single-cell structures. These cells were then suspended in a medium, comprising 50% Matrigel (BD Biosciences, San Jose, CA, USA; 3564234), before being subcutaneously injected using a 27-G needle into NOG (NOD/Shi-SCID-IL-2Rγnull) mice. These mice were sourced from the Central Institute for Experimental Animals, Kawasaki, Japan. The drug was administered every 2–3 days from 10–14 days after spheroid cell injection. The control group was exposed to dimethyl sulfoxide (DMSO) only. Mice were monitored every 3–4 days for a span of 4–6 weeks post-cell transplantation. The Animal Care and Use Committee of Niigata University approved all mouse procedures, ensuring they align with institutional policies. Lentivirus-mediated Transduction Both pCMV3-ALDH1A1 plasmid (HG11388-UT) and pCMV3 control vector were procured from Sino Biological (Beijing, China). The process of creating virus-containing supernatants and the subsequent viral infections were carried out as elaborated in a previously established protocol ( 11 ). Post-infection, cells underwent selection in a milieu containing 100 µg/ml hygromycin. Western Blot Analyses The cell lysis procedure was facilitated using radioimmunoprecipitation assay buffer. This buffer contained specific concentrations of several compounds, including 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM ethylenediaminetetraacetic acid. To this mixture, protease and phosphatase inhibitors (Roche, Basel, Switzerland) were added. The resultant samples were then subjected to western blot analysis as detailed in a previous protocol ( 11 ), with the use of specific primary antibodies (Details provided in Supplementary Table 1). Statistical Analyses The statistical evaluation of both in vitro and in vivo spheroid cell experiments utilized Welch’s t -tests or Student’s t -tests, contingent on the outcomes of F tests. A threshold of p < 0.05 was set for significance. Clinical sample statistics were carried out using the EZR software ( 18 ). The Kaplan–Meier method was used for univariate survival analysis, whereas the significance of variances between groups was determined through log-rank tests. Data Availability Statement The microarray data analyzed in this study were obtained from the Gene Expression Omnibus database (accession number: GSE123530). Results PI3K-Akt-mTOR signaling patterns in human uterine endometrial cancer spheroid cells First, we evaluated the protein expression and gene mutation profile of PI3K-Akt-mTOR signaling to delineate the signaling status in endometrial cancer spheroid cells. Through western blot analysis of seven distinct endometrial cancer spheroid cells, we observed variations in the expression levels of signaling factors, including phospho-Akt, phospho-PTEN, and phospho-p70S6K. Intriguingly, their expression did not correlate with the ALDH1A1 expression within the spheroid cell types (Fig. 1 A). Subsequently, an immunohistochemical evaluation of spheroid-derived xenograft tumors was undertaken alongside the original clinical cancer tumor specimens. This highlighted that the expression patterns of phospho-Akt, phospho-p70S6K, and phospho-PTEN echoed the results observed in the aforementioned western blot analysis of spheroid cells. Notably, although a heterogeneous expression was discerned across tumors, a considerable number of cancer cells exhibited elevated phospho-Akt expression in tumors originating from or derivatives of EMN24 and EMN144 cells. Furthermore, the tumors from EMN24 cells displayed pronounced phospho-p70S6K expression, and the tumors from EMN108 cells showed evident phospho-PTEN expression (Supplementary Fig. S1 A). In a broader context, mutation profiles across 22 endometrial cancer spheroid cells mirrored those witnessed in the original clinical tumor specimens (Fig. 1 B) ( 12 ). Among these PI3K-Akt-mTORC1-related signaling factors, a previous study showed that phospho-p70S6K is a predictive tool for the outcomes of patients with type II endometrial cancer when used as an immunohistochemistry (IHC)-based marker ( 2 ). This was particularly significant among markers related to the activation of the PI3K-Akt-mTORC1 signaling pathway ( 2 ). Furthermore, the phosphorylation observed at T389 showcased a connection with malignancy-related p70S6K activity ( 19 ). Merging this insight with the observed expression results of phospho-p70S6K in both spheroid cells and spheroid-derived xenograft tumors, we sought to confirm the expression of phospho-p70S6K in 35 clinically advanced endometrial cancer tissue specimens through immunohistochemical staining (Fig. 1 C). Kaplan–Meier survival analyses showed that an elevated phospho-p70S6K expression may be correlated with overall survival (Fig. 1 D, p < 0.05), not with progression-free survival (Supplementary Fig. S2A). The expression was not correlated with histological grade or clinical stage in these advanced-stage cases (Supplementary Fig. S2B and S2C, Fisher’s exact test). Though conclusions drawn from a limited case pool remain preliminary, our findings accentuate that phospho-p70S6K expression might indeed be intertwined with an adverse prognosis in advanced-stage uterine endometrial cancer. PI3K Inhibitors curbing the proliferation of endometrial cancer spheroid cells With the aforementioned results of phospho-p70S6K expression being related to the activation of the PI3K-Akt-mTORC1 signaling pathway ( 2 ), we then explored the effects of PI3K-Akt-mTORC1 signaling in endometrial cancer spheroid cells. Our in vitro cancer spheroid model has displayed superior efficacy in comparison to the cancer stem cell model ( 11 , 12 ) and existing in vitro drug sensitivity evaluations ( 16 ). Riding on this fundamental advantage, we first assessed the sensitivity of endometrial cancer cells toward PI3K inhibitors. The particular focus was on Alpelisib, a prominent PI3K inhibitor deployed in clinical trials to treat breast and several other cancers ( 20 , 21 ). Upon assessment, we categorized the endometrial cancer cells into three distinct sensitivity groups with respect to Alpelisib: High-sensitivity group: EMN18 and EMN21 cells. Low-sensitivity group: EMN103 and EMN144 cells. Intermediate-sensitivity group: Comprising the remaining cell types (Fig. 2 A and 2 B). Our examination of other PI3K inhibitors, namely Taselisib and Copanlisib, showed sensitivity patterns similar to those observed for Alpelisib (Supplementary Fig. S3A and S3B). Alpelisib treatment enhanced caspase levels when the spheroid cellular growth was inhibited (Fig. 2 B and 2 C). To juxtapose in vivo sensitivity against its in vitro counterpart, we administered Alpelisib to NOG mice thrice weekly. The outcomes were illuminating: the xenograft tumors stemming from EMN18 and EMN21 spheroid cells (from the in vitro high-sensitivity group) exhibited a decline post-Alpelisib treatment (Fig. 2 D). Moreover, tumors from EMN108 and EMN81 spheroid cells (intermediate-sensitive) also exhibited growth suppression (Supplementary Fig. S3C). Nonetheless, tumors originating from EMN144 cells (low-sensitivity group) remained unaffected by Alpelisib treatment (Supplementary Fig. S3D). Collectively, these findings highlight that the Alpelisib sensitivity observed in our in vitro spheroid model mirrors the outcomes witnessed during in vivo applications. Additive effects of PI3K and ALDH inhibitors on endometrial cancer spheroid cells' growth Next, we elucidated the interactions between PI3K-Akt-mTORC1 signaling and cancer stemness using ALDH, which was found to be a functional marker of uterine endometrial cancer stem cells in our previous study ( 12 ). Intriguingly, a targeted sequencing analysis revealed identical mutational profiles between ALDH-high and ALDH-low spheroid cells (Fig. 3 A). Moreover, despite the suppression of phospho-Akt and phospho-p70S6K expression post-Alpelisib treatment in sensitive spheroid cells (Fig. 3 B), we observed no discernable differences in the in vitro sensitivity to Alpelisib between ALDH-high and ALDH-low cells (data not shown). This observation was consistent with the changes in ALDH activity or ALDH1A1 expression (Fig. 3 B and Supplementary Fig. 4A). Further, Alpelisib treatment diminished phospho-p70S6K expression in both ALDH-high and ALDH-low cells (Supplementary Fig. S4B). Given the above, we postulated that ALDH activity might be independent of PI3K sensitivity. To put this theory to the test, we co-treated spheroid cells with disulfiram (an ALDH inhibitor) and Alpelisib. Remarkably, this combination treatment led to a substantial inhibition of spheroid growth (Fig. 3 C). Validating these in vitro findings, we introduced this co-treatment to spheroid cell-transplanted mice. Tumors post-co-treatment were roughly half the size compared with those treated solely with Alpelisib. This was consistent for both high-sensitivity (EMN21, Fig. 3 D) and intermediate-sensitivity (EMN24, Supplementary Fig. S4C) cell groups. Additionally, consistent with in vitro results, Alpelisib alone did not alter the ALDH activity in xenograft tumors (Supplementary Fig. S4D). Conclusively, while ALDH activity appears to have no direct impact on PI3K inhibition, a combination of ALDH and PI3K inhibitors results in an additive inhibition of endometrial cancer progression. Additive inhibition of endometrial cancer spheroid cells by combining Akt and ALDH inhibitors Pivoting from our work with PI3K inhibitors, we turned our attention to the effects of Akt inhibitors on endometrial cancer spheroid cells. Notably, cells that exhibited low-to-intermediate sensitivity to PI3K inhibitors—specifically, the EMN81, EMN103, and EMN144 cell lines (Fig. 2 A and Supplementary Fig. S3A and S3B)—responded to low-dose treatment with Akt inhibitors, Ipatasertib and Caplivasertib (Fig. 4 A– 4 C). This sensitivity was marked by an uptick in activated caspases post-Ipatasertib treatment, implying cytotoxic effects (Fig. 4 D). However, it is worth noting that while EMN24 cell-derived xenograft tumors demonstrated resistance to Ipatasertib in vitro , they remained unresponsive to the drug even in vivo (Supplementary Fig. S5). Such findings underscore the potential of baseline Akt activity, especially linked to PTEN mutations, as a valuable predictor for Akt inhibitor sensitivity (Fig. 1 A and 1 B) ( 22 ). While we observed a reduction in phospho-p70S6K expression after treatment with the Akt inhibitor, there was no noticeable shift in ALDH activity or expression (Fig. 4 E). Such observations led us to hypothesize that ALDH activity remained unaffected by Akt inhibition. However, dual inhibition of both Akt and ALDH might collectively hinder endometrial cancer cell growth. To test this, we investigated the combined impact of ALDH and Akt inhibitors, both in vitro and in vivo . The resulting data was as follows: combination treatment not only triggered apoptosis in endometrial cancer cells but also hindered their proliferation (Fig. 4 F– 4 I). Mirroring our findings with PI3K inhibitors, this co-treatment approach further validated the additive repression of endometrial cancer progression, independent of any influence of ALDH activity on Akt inhibition. mTOR inhibitor reduces the proliferation of endometrial cancer spheroid cells with ALDH activity Drawing insights from our experiments with PI3K and Akt inhibitors, it became evident that PI3K/Akt activity was independent of ALDH activity. Our exploration then shifted toward understanding the influence of mTOR inhibition on endometrial cancer spheroid cell proliferation. Upon conducting in vitro sensitivity assays, we observed varying responses to mTOR inhibitors, everolimus, and Torin1. Notably, the range of sensitivities was more consistent than what was observed for PI3K and Akt inhibitors (Fig. 5 A and Supplementary Fig. S6A). Following mTOR inhibitor administration, there was a decline in the levels of both phospho-p70S6K and phospho-4EBP1 across the spheroid cell population (Fig. 5 B). Intriguingly, this decline in phospho-p70S6K was more accentuated in ALDH-high cells compared with ALDH-low cells (Fig. 5 C and Supplementary Fig. S6B). Moreover, the ALDH-high cells exhibited heightened sensitivity to everolimus in comparison to their ALDH-low counterparts (Fig. 5 D). It was discerned that the exogenous overexpression of ALDH1A1 further augmented this sensitivity to everolimus (Fig. 5 E). Collectively, these findings bolster the notion that cells with higher ALDH activity inherently possess increased mTOR activation relative to cells with low ALDH activity. Alternatively, combination treatment with disulfiram and everolimus also hindered the spheroid cell proliferation (Fig. 5 F). In conclusion, while ALDH activity appears to be linked to mTORC1 activity, a combination of ALDH and mTORC1 inhibitors additively impedes endometrial cancer progression. Endometrial cancer spheroid cells exhibiting high ALDH activity show enhanced sensitivity to mTOR inhibitors Based on the aforementioned results, we speculated that heightened ALDH activity might partially enhance mTOR sensitivity in endometrial cancer. To reveal the relationship between ALDH and mTOR, we examined changes in the expression of the PI3K-Akt-mTOR signaling pathway after manipulating ALDH activity. Disulfiram, which reduced ALDH activity, led to a decrease in phospho-p70S6K levels, whereas phospho-PI3K and phospho-Akt levels remained unchanged (Fig. 6 A and Supplementary Fig. S7A). Additionally, gene set enrichment analysis (GSEA) revealed the ALDH-high cells preferentially expressed genes included in the gene set of hallmark of mTORC1 signaling (false discovery rate [FDR] q-value < 0.01, normalized enrichment score [NES] 1.59, p -value < 0.01) ( 23 ), genes upregulated after ectopically expressing eIF4E, and genes upregulated in control cells compared with eIF4GI-silenced cells ( 24 , 25 ) (Fig. 6 B and Supplementary Fig. S7B). Moreover, ALDH-high cells clearly expressed more phospho-p70S6K than ALDH-low cells (Fig. 6 C). The exogenous overexpression of ALDH1A1 that led to ALDH activation ( 12 ) ultimately increased phospho-p70S6K levels (Fig. 6 D). These results suggested that ALDH activity primarily affected mTORC1 activation rather than PI3K or Akt. To further confirm the relationship between ALDH and mTORC1 signaling, we determined whether mTOR activation could rescue the inhibitory effect of ALDH inhibitor on cancer cells. As expected, mTOR activator MHY1485 partially mitigated disulfiram-induced cytotoxicity in endometrial cancer spheroid cells (Fig. 6 E, 6 F, and Supplementary Fig. S7C–S7F). Moreover, western blot analysis indicated that MHY1485 could partially revert the disulfiram-mediated reduction in phospho-p70S6K expression (Fig. 6 G and Supplementary Fig. S7G). LDHA bridges ALDH activity and mTORC1 activation in endometrial cancer spheroid cells To understand how ALDH influences mTOR signaling in endometrial cancer cells, we characterized its functional isoforms. Notably, of the 19 ALDH isoforms with analogous catalytic functions, ALDH1A1 predominantly dictates its activity ( 23 ). Our findings further cemented this observation, as endometrial cancer spheroid cells majorly expressed ALDH1A1, sidelining other ALDH isoforms ( 12 ). A pivotal function of ALDH1A1 includes the conversion of retinol to retinoic acids, driving cancer proliferation through multifaceted mechanisms ( 24 , 25 ). Corroborating our premise that ALDH-mediated retinoic acid modulates endometrial cancer development in vitro , we discerned that additional retinoic acid rescued the cell mortality caused by disulfiram (Supplementary Fig. S8A–S8E). This mirrored the outcome when mTORC1 was activated with MHY1485 (Fig. 6 E and 6 F and Supplementary Fig. S7C–S7F). Experiments conducted on cells overexpressing ALDH1A1 further supported this observation (Supplementary Fig. S8F). Moreover, western blot outcomes indicated that retinoic acid could partially revert the disulfiram-mediated decrease in phopho-p70S6K expression (Supplementary Fig. S8G and S8H). This modulation paralleled the effects of the mTORC1 activator MHY1485 (Fig. 6 G and Supplementary Fig. S7G). To assess the influence of RA on mTOR activation, we combined GSEA results from microarray data and published RARA binding data from the ChIP-Atlas ( http://chip-atlas.org/ ). Of 184 hallmark mTORC1 signaling genes (Fig. 6 B) ( 26 ), 85 were core enrichment genes associated with ALDH-high endometrial cancer cells. Merging this with RARA-regulated genes from ChIP-Atlas, specifically those linked to malignancies, yielded eight candidate genes (Fig. 7 A). Significantly, LDHA was predominantly expressed in ALDH-high cells (Fig. 7 B). Delving deeper, ALDH1A1 overexpression augmented LDHA levels, whereas ALDH inhibition, using disulfiram, attenuated its expression (Fig. 7 C, 7 D and Supplementary Fig. S9A). Additionally, ALDH-high cells and exogenous ALDH1A1-overexpressing cells had higher LDHA activity than control bulk spheroid cells (Fig. 7 E). Upon investigating LDHA inhibition in cancer cells, we observed that using LDHA inhibitor AZ-33 considerably hampered endometrial cancer cell viability, especially in cells with ALDH activity (Fig. 7 F). Interestingly, while the LDHA inhibitor diminished LDH activity and phospho-p70S6K expression, ALDH expression and its functional activity remained unaltered (Fig. 7 G). Furthermore, treatment with AZ-33 led to a marked reduction in stemness indicators such as Nanog, Oct-4, and c-Myc (Fig. 7 G). Additionally, activating mTORC1 with MHY1485 could partially counteract the inhibitory effects of AZ-33 on cancer cell proliferation, although MHY1485 in isolation did not make a significant difference (Fig. 7 H and Supplementary Fig. S9B and S9C). Taken together, our findings highlight that ALDH influences mTORC1 through LDHA, promoting the proliferation of endometrial cancer spheroid cells. Notably, while fluctuations in mTORC1 activation remained agnostic to LDHA levels, inhibiting mTOR with everolimus led to a surge in GLUT1. Conversely, stimulating mTOR with MHY1485 reduced GLUT1 levels (Fig. 7 I and Supplementary Fig. S9D), influencing glucose transport (Fig. 7 J and Supplementary Fig. S9E and S9F). These intricate interplays underscore a reciprocal relationship between glycolysis and mTORC1 in ALDH-active endometrial cancer progression (Supplementary Fig. S10). LDHA expression is associated with adverse prognosis in endometrial cancer patients Our study suggests that glycolysis and mTORC1 signaling play a pivotal role in regulating ALDH-high endometrial cancer cells. Seeking to delineate the distribution of ALDH and LDHA in a clinical framework, we performed immunostaining analyses on endometrial cancer samples. Intriguingly, ALDH-positive cells exhibited more pronounced LDHA expression than their ALDH-negative counterparts across both early and advanced cancer stages (Fig. 8 A and Supplementary Fig. S11A). To further elucidate the clinical implications of LDHA, serum LDH levels were quantified in 244 patients with endometrial cancer from our institution. Notably, LDH levels increased in patients with high-grade cancer relative to their low-grade counterparts across all stages, a trend particularly evident in stage I (Fig. 8 B and 8 C). Moreover, advanced-stage patients consistently exhibited elevated LDH titers in contrast to those in the early stages (Fig. 8 D). This underscores a direct correlation between serum LDH levels, tumor grade, and clinical cancer progression. Delving deeper into the clinical ramifications of LDHA expression, we utilized The Cancer Genome Atlas (TCGA) database ( 4 ) to investigate its correlation with the prognosis of patients with endometrial cancer. LDHA mRNA levels were discernibly elevated in high-grade tumors (Fig. 8 E). Alarmingly, patients manifesting elevated LDHA expression experienced a significantly reduced overall survival (Fig. 8 F, p = 0.02). Moreover, progression-free survival showed a concerning trend, with LDHA -high-expressing cases displaying a tendency toward shorter survival compared with their low-expression counterparts (Supplementary Fig. S11B, p = 0.09). A significant correlation was also evident between LDHA and RPS6KB1 expression in endometrial cancer tissue (Supplementary Fig. S8B, p = 0.01). In summary, our findings highlight LDHA as a crucial biomarker, revealing its strong association with tumor grade and suggesting its potential role as an indicator of unfavorable prognosis in endometrial cancer. Discussion The utilization of the patient-derived cell model offers promising avenues for understanding therapeutic responses to cytotoxic chemotherapeutic agents on an individual patient basis ( 27 ). Recent findings suggest organoid cells can effectively serve as models for gauging responses to therapies that target specific genetic mutations ( 28 , 29 ). In alignment with these studies, our research highlights the correlation between in vitro and in vivo tests concerning the sensitivity to PI3K-Akt-mTOR signaling inhibitors in endometrial cancer patient-derived spheroid cells. Particularly, the response to PI3K and Akt inhibitors showed distinct differentiation based on genomic mutation profiles. These observations remark on the clinical potential of spheroid cells. Endometrial, breast, and colon cancers often present activation in the mutant-dependent PI3K-Akt-mTOR signaling pathway ( 2 , 4 , 30 ). While PI3K-Akt-mTOR signaling inhibitors have theoretical potential to significantly suppress mutated cancer cells, challenges arise from negative feedback release and detrimental impacts on non-tumor cells. Furthermore, elements such as growth factors, energy, and stress play roles in mTOR activation ( 3 ). This drove our investigation into the non-genetic mechanisms underlying PI3K-Akt-mTOR pathway activation during endometrial cancer proliferation. The PI3K-Akt-mTOR pathway has been implicated in enhancing stemness, a trait linked to aggressive cancer manifestations ( 31 ). Numerous studies have pinpointed the role of PI3K signaling in cancer stemness, affecting markers such as Nanog, SOX2, and CD133 ( 32 – 34 ). For instance, mTORC1 activation has been noted to boost colon cancer stem cell proliferation ( 35 ). mTOR pathway is the downstream mediator of ALDH1A3 in gastric cancer ( 38 ). Our studies on uterine endometrial cancer emphasized the influence of both genomic and ALDH-mediated non-genomic aberrations on mTOR activation. Spheroid cells, enriched with cancer stem cells, proved instrumental in elucidating the nexus between cancer stemness and PI3K-Akt-mTORC1 signaling ( 36 ). Lactate dehydrogenase (LDH), an intracellular enzyme, can manifest as either a homotetramer or heterotetramer, encompassing LDHA and LDHB subunits, giving rise to five isoforms. Each isoform is competent in transforming pyruvate to lactate ( 37 ). Echoing prior reports on cancers such as breast and pancreatic ( 38 – 40 ), our analysis attests to the association between LDHA expression and advanced-stage endometrial cancer prognosis. Furthermore, elevated LDHA expression and serum LDH levels were more prevalent in high-grade cancer patients. Past research has drawn parallels between elevated serum LDH levels and reduced survival rates in solid tumors such as melanoma and prostate carcinomas, highlighting its potential as a prognostic biomarker for metastatic carcinomas ( 41 ). Collectively, these insights underline the role of LDHA in exacerbating endometrial cancer progression and its clinical malignancy. The interplay between glycolysis and cancer proliferation, especially processes such as epithelial-to-mesenchymal transition and cancer stemness, is the subject of extensive research ( 42 ). Our prior work revealed the centrality of the glucose transporter GLUT1 in dictating the stemness and chemoresistance traits of ALDH-active endometrial cancer stem cells ( 12 ). We later discovered the regulatory role of ALDH-mediated retinoic acid over glycolytic functional factor LDHA. LDHA is crucial for sustaining breast cancer stemness and triggering metastasis ( 39 ). Few studies have analyzed the role of LDHA in governing mTOR activation. In the context of pancreatic adenocarcinoma, LDHA-induced proliferation was found to be mediated by AMPK-mTOR signaling due to L-lactate ( 38 ). Other findings emphasize the role of LDHA in activating glycolysis, subsequently stimulating mTORC1 and thus fostering cellular proliferation. This phenomenon is particularly observed in K-Ras activated colon and pancreatic cancer cells ( 43 ). Furthermore, LDHA is required for the activation of mTOR in gastric cancer ( 44 ). Corroborating these findings, our research establishes that inhibiting LDHA impedes mTOR activation and curtails cancer cell proliferation, especially in ALDH-active endometrial cancer spheroids, regardless of K-Ras genomic aberrations (Fig. 7 ). Moreover, our findings reveal the role of mTOR in modulating glycolysis. Curbing Akt-mTORC1 signaling diminishes GLUT1 expression, thereby stalling glycolysis and cancer cell survival in diseases such as leukemia ( 45 , 46 ). The intricate dynamics between mTOR and glycolysis in cancer encompass both direct and indirect mechanisms. A standout feature of our study underscores the mutual regulatory relationship between glycolysis and mTOR activation. This feedback mechanism orchestrates the proliferation and survival of ALDH-active endometrial cancer stem cells (Fig. S10). Targeting this interplay could potentially enhance endometrial cancer treatment outcomes, even when mTORC1 activation is dictated by PI3K-Akt-mTOR genomic aberrations. In summary, we disclosed the ALDH-LDHA-mTORC1 cascade to be a novel facet of the ALDH-related signaling that governs the proliferation of uterine endometrial cancer stem cells. Future studies should delve deeper into the cascade and interplay between glycolysis and mTORC1 to develop clinical treatment strategies to tackle the outcome of aggressive uterine endometrial cancer. Additional materials and methods can be retrieved in Supplemental Experimental Procedures. Declarations Author Contributions Conception and design: H.U., T. I. Development of methodology: H.U., T.I. Acquisition of data: H.U., T.I., Y.M. Analysis and interpretation of data: H.U., T.I., Y.M., K.Yamawaki Writing, review, and/or revision of the manuscript: H.U., T.I., Y. M., K.Yamawaki, K. Yoshihara, K. O., and T.E. Study supervision: T. E., K.Y. Acknowledgments We thank Ryo Tamura, Kazuaki Suda, Nozomi Yachida, Manako Yamaguchi, Kentaro Sugino, Tomoyuki Sekizuka, Kyota Saito, Anna Ishida (Department of Obstetrics and Gynecology, Niigata University Medical School) for scientific advice and technical assistance. This research was supported by Grants-in-Aid for Scientific Research (C) (Grant No. 18K09250, 21K09490) to T.I. from the Japan Society for the Promotion of Science. This study was partially supported by the National Cancer Center Research and Development Fund (29-A-2). References de Boer SM, Powell ME, Mileshkin L, Katsaros D, Bessette P, Haie-Meder C, et al. 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Cui B, Luo Y, Tian P, Peng F, Lu J, Yang Y, et al. Stress-induced epinephrine enhances lactate dehydrogenase A and promotes breast cancer stem-like cells. J Clin Invest. 2019; 129:1030–1046. Petrelli F, Cabiddu M, Coinu A, Borgonovo K, Ghilardi M, Lonati V, et al. Prognostic role of lactate dehydrogenase in solid tumors: a systematic review and meta-analysis of 76 studies. Acta Oncol. 2015; 54:961–970. Guo Q, Qiu Y, Liu Y, He Y, Zhang G, Du Y, et al. Cell adhesion molecule CD44v10 promotes stem-like properties in triple-negative breast cancer cells via glucose transporter GLUT1-mediated glycolysis. J Biol Chem. 2022; 298:102588. Byun JK, Park M, Yun JW, Lee J, Kim JS, Cho SJ, et al. Oncogenic KRAS signaling activates mTORC1 through COUP-TFII-mediated lactate production. EMBO Rep. 2019; 20:e47451. Zhao Z, Han F, Yang S, Wu J, Zhan W. Oxamate-mediated inhibition of lactate dehydrogenase induces protective autophagy in gastric cancer cells: involvement of the Akt-mTOR signaling pathway. Cancer Lett. 2015; 358:17–26. Chen S, Tao Y, Wang Q, Ren J, Jing Y, Huang J, et al. Glucose induced-AKT/mTOR activation accelerates glycolysis and promotes cell survival in acute myeloid leukemia. Leuk Res. 2023; 128:107059. Artico LL, Ruas JS, Teixeira Junior JR, Migita NA, Seguchi G, Shi X, et al. IGFBP7 fuels the glycolytic metabolism in B-cell precursor acute lymphoblastic Leukemia by sustaining activation of the IGF1R-Akt-GLUT1 axis. Int J Mol Sci. 2023; 24:9679. Additional Declarations There is NO conflict of interest to disclose. Supplementary Files SupplementaryData20merge.pdf Cite Share Download PDF Status: Published Journal Publication published 11 Oct, 2024 Read the published version in Cell Death Discovery → Version 1 posted 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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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3547380","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":246970586,"identity":"78c05631-3f30-4679-ac46-0d2cc668d7ea","order_by":0,"name":"Tatsuya Ishiguro","email":"data:image/png;base64,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","orcid":"https://orcid.org/0000-0002-6445-4066","institution":"Department of Obstetrics and Gynecology, Niigata University Graduate School of Medical and Dental Sciences","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Tatsuya","middleName":"","lastName":"Ishiguro","suffix":""},{"id":246970587,"identity":"5dc4ac66-18fe-4360-a967-b22a222d3420","order_by":1,"name":"Haruka Ueda","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Haruka","middleName":"","lastName":"Ueda","suffix":""},{"id":246970588,"identity":"934fe3f9-fe99-48c7-8b40-b17b0b977c39","order_by":2,"name":"Yutaro Mori","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yutaro","middleName":"","lastName":"Mori","suffix":""},{"id":246970589,"identity":"3c2e464e-f6c3-42f1-8350-e62922a9bbee","order_by":3,"name":"Kaoru Yamawaki","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kaoru","middleName":"","lastName":"Yamawaki","suffix":""},{"id":246970590,"identity":"d8472312-6c6f-4bc9-9200-74cb1e5c5459","order_by":4,"name":"Takayuki Enomoto","email":"","orcid":"","institution":"","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Takayuki","middleName":"","lastName":"Enomoto","suffix":""},{"id":246970591,"identity":"ba4299f9-552f-4f78-bc31-8920d2355969","order_by":5,"name":"Kosuke Yoshihara","email":"","orcid":"https://orcid.org/0000-0002-2254-3378","institution":"Niigata University Graduate School of Medical and Dental Sciences","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Kosuke","middleName":"","lastName":"Yoshihara","suffix":""},{"id":246970592,"identity":"1da74301-e13f-4c93-b7cb-cb20e7746da0","order_by":6,"name":"Koji Okamoto","email":"","orcid":"https://orcid.org/0000-0001-7587-0164","institution":"Teikyo University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Koji","middleName":"","lastName":"Okamoto","suffix":""}],"badges":[],"createdAt":"2023-11-02 22:25:17","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3547380/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3547380/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41420-024-02204-y","type":"published","date":"2024-10-11T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":47655899,"identity":"a9cf2435-945c-4b6f-83fe-31f8aa4363bf","added_by":"auto","created_at":"2023-12-05 17:42:16","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":341875,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ePI3K-Akt-mTOR signaling expression in endometrial cancer. See also Supplementary Fig. S1 and S2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot of endometrial cancer spheroid cells. (B) Targeted sequencing analysis of primary tumors and spheroid cells. (C) Representative immunostaining images for phospho-p70S6K-negative (left) and phospho-p70S6K-positive (right). Scale bars: 50 µm. (D) Kaplan–Meier plots of overall survival in patients with advanced-stage endometrial cancer stratified by phospho-p70S6K positivity (red, \u003cem\u003en =\u003c/em\u003e 13) and negativity (black, \u003cem\u003en =\u003c/em\u003e 22). Notably, most patients received first-line taxane and platinum-based chemotherapy. Refer to Supplementary Fig. S1 and S2 for supplemental data.\u003c/p\u003e","description":"","filename":"Slide1.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/1e3ee7b82054014285d38da5.png"},{"id":47654796,"identity":"4fa00091-85e6-4354-bb6c-0c8d28d58dda","added_by":"auto","created_at":"2023-12-05 17:26:16","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":328538,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of PI3K inhibitors on spheroid cell formation and growth. See also Supplementary Fig. S3.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Spheroid cell responses to varied Alpelisib concentrations over 4 days. (B) Bright-phase images of EMN18 spheroids after 4 days of 5 nM Alpelisib treatment. Scale bars: 100 µm. (C) Caspase activity in EMN18 cells pre- and post-Alpelisib exposure. (D) Xenograft tumor volumes (mean ± SEM) post-subcutaneous injection of 5 × 10\u003csup\u003e5\u003c/sup\u003e EMN18 spheroids. Mice in the Alpelisib group were intraperitoneally administered Alpelisib (15mg/kg), while the control group received DMSO. Data from \u003cem\u003en =\u003c/em\u003e 6 independent experiments, \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, Student’s \u003cem\u003et\u003c/em\u003e-test. Scale bars: 10 mm. Supplemental data available in Supplementary Fig. S3.\u003c/p\u003e","description":"","filename":"Slide2.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/7bd0ee80f36903d76a724908.png"},{"id":47655703,"identity":"de596d14-8791-4000-8ec3-3d792c7cbb9b","added_by":"auto","created_at":"2023-12-05 17:34:16","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":221472,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDual therapy of Alpelisib and ALDH inhibitor obstructs endometrial cancer cell progression. See also Supplementary Fig. S4.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Target sequencing of ALDH-high and ALDH-low spheroid cells. (B) Western blot of spheroid cells treated with or without Alpelisib. (C) Relative EMN21 spheroid cell viability under specified treatments over 4 days. (D) Xenograft tumor volumes (mean ± SEM) from 1 × 10\u003csup\u003e6\u003c/sup\u003e EMN21 spheroids, treated \u003cem\u003ein vivo\u003c/em\u003e with combinations of Alpelisib (15 mg/kg) and/or disulfiram (40 mg/kg). \u003cem\u003en =\u003c/em\u003e 8, Student’s \u003cem\u003et\u003c/em\u003e-test. Xenograft tumor images post-excision on day 42 are on the right. Scale bar: 10 mm. Refer to Supplementary Fig. S4 for additional data.\u003c/p\u003e","description":"","filename":"Slide3.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/f16ad7150c5bdf6319e472b8.png"},{"id":47654800,"identity":"ad9b9a82-fcef-4fb9-82d6-1d04cba7c75d","added_by":"auto","created_at":"2023-12-05 17:26:16","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":823364,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eIpatasertib and ALDH inhibitor combination impacts endometrial cancer cell progression. See also Supplementary Fig. S5.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A-B), Responses of spheroid cells to different concentrations of (A) Ipatasertib and (B) Capivasertib after incubation for 4 days. (C) Bright-phase images of EMN103 and EMN144 spheroid cells (4 days after Ipatasertib treatment). Scale bars, 100 µm. (D) Caspase activity before and after 1 nM Ipatasertib treatment to EMN144 cells. (E) Western blot analyses of spheroid cells in the presence or absence of Ipatasertib treatment. (F) Relative cell viability in the presence or absence of 100 nM Ipatasertib and 0.5 µM disulfiram treatment for 4 days. (G) Volumes (mean ± SEM) of xenograft tumors from 1 × 10\u003csup\u003e6 \u003c/sup\u003eEMN144 spheroid cells with the presence or absence of Ipatasertib (15 mg/kg) and/or disulfiram (40 mg/kg) after \u003cem\u003ein vivo\u003c/em\u003e treatment. \u003cem\u003en =\u003c/em\u003e 8, Student’s \u003cem\u003et\u003c/em\u003e-tests. (H) and (I) immunostaining of xenograft tumors of xenograft tumors. Scale bars:100 µm. [(H) Ki67 and (I) cleaved-caspase].\u003c/p\u003e","description":"","filename":"Slide4.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/9eb84b5cb1818177831282a7.png"},{"id":47654802,"identity":"c2f7e6d9-725d-46a5-a023-e558791c9fb9","added_by":"auto","created_at":"2023-12-05 17:26:16","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":282444,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emTOR inhibitor blocks the proliferation of endometrial cancer spheroid cells. See also Supplementary Fig. S6.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Responses of spheroid cells to different concentrations of everolimus after incubation for 4 days. (B) Western blot analyses of spheroid cells in the presence or absence of everolimus treatment. (C) Western blot analyses of ALDH-low and ALDH-high spheroid cells in the presence or absence of everolimus treatment. (D) Relative ALDH-high and ALDH-low spheroid cell viability with the indicated everolimus \u003cem\u003ein vitro\u003c/em\u003e treatment for 4 days. (E) Relative infected spheroid cell viability with the indicated everolimus \u003cem\u003ein vitro\u003c/em\u003etreatment for 4 days. (F) Relative cell viability in the presence or absence of 5 µM everolimus and 10 µM disulfiram treatment for 4 days.\u003c/p\u003e","description":"","filename":"Slide5.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/9cc13e63206ffaffd7b021c3.png"},{"id":47654799,"identity":"e6c1a8e6-5e4e-47d4-b69a-11fa30325405","added_by":"auto","created_at":"2023-12-05 17:26:16","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":378933,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eALDH inhibitor blocks the proliferation of endometrial cancer cells via mTOR activity (EMN24 cells). See also Supplementary Fig. S7 and S8.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Western blot analyses of spheroids cells after disulfiram treatment for 24 h. (B) Gene set enrichment analyses of gene expression profiles between ALDH-high and ALDH-low cells (HALLMARK_MTORC1_SIGNALING, genes upregulated through activation of mTORC1 complex). (C) Western blot analyses of ALDH-high and ALDH-low spheroids cells. (D) Western blot analyses of the infected spheroids cells. (E) Relative spheroid cell viability in the presence or absence of 10 µM disulfiram and/or 5 µM MHY1485 \u003cem\u003ein vitro\u003c/em\u003etreatment for 4 days. (F) Bright-phase images of spheroid cells in the presence or absence of disulfiram and/or MHY1485 \u003cem\u003ein vitro\u003c/em\u003etreatment for 4 days. Scale bars: 100 µm. (G) Western blot analyses of spheroids cells after disulfiram and/or MHY1485 treatment for 24 h.\u003c/p\u003e","description":"","filename":"Slide6.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/1e92aa405e02b072867756d6.png"},{"id":47655701,"identity":"55cc38c9-5f8e-4ebe-83ba-a39c8eba3e98","added_by":"auto","created_at":"2023-12-05 17:34:16","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":228812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInteraction between glycolysis and mTOR controls the proliferation of ALDH-high endometrial cancer cells. See also Supplementary Fig. S9.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) \u003cem\u003eIn silico\u003c/em\u003e screening of RARA target genes connected to mTORC1 signaling using a combination of public chromatin immunoprecipitation (ChIP) database and gene set enrichment analysis based on microarray analysis of endometrial cancer spheroid cells. Combined with the 85 genes from 184 genes in the gene set of hallmark of mTORC1 signaling and putative RARA-regulated genes retrieved from ChIP-Atlas (http://chip-atlas.org/), eight genes were identified as the candidate genes correlated to ALDH-RARA-mTOR axis. (B-D), Western blot analyses of (B) ALDH-high and ALDH-low spheroids cells, (C) the infected spheroids cells, and (D) disulfiram treatment. (E) Relative LDHA activity of control bulk, ALDH-high, and exogenous ALDH1A1 overexpressing spheroid cells. (F) Relative ALDH-high and ALDH-low spheroid cell viability with the indicated \u003cem\u003ein vitro\u003c/em\u003e AZ-33 treatment for 4 days. (G) Western blot analyses of spheroids cells after AZ-33 treatment for 24 h. (H) Relative spheroid cell viability in the presence or absence of 100 µM AZ-33 and/or 1 µM MHY1485 \u003cem\u003ein vitro\u003c/em\u003e treatment for 4 days. (I) Western blot analyses of spheroids cells after everolimus or MHY1485 treatment for 24 h. (J) Relative glucose uptake of spheroid cells with 80 µM everolimus \u003cem\u003ein vitro\u003c/em\u003e treatment.\u003c/p\u003e","description":"","filename":"Slide7.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/929d554b7d354fea7badc28f.png"},{"id":47655900,"identity":"97e8e6d9-f2bb-44be-9845-467ea5a47975","added_by":"auto","created_at":"2023-12-05 17:42:16","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":214018,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLDH is associated with clinical advanced stage and poor prognosis in endometrial cancer. See also Supplementary Fig. S11.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Combination staining of ALDH1A1 (green), LDHA (red), and DAPI (blue) in human endometrial cancer tumor samples. (B–D) Serum LDH titer of the endometrial cancer patients before primary treatment (B) in different grades of all-clinical stage patients, (C) of advanced-stage patients, and (D) in different clinical stages of endometrial cancer. (E) \u003cem\u003eLDHA\u003c/em\u003e mRNA level of endometrial cancer patients in different grades (TCGA database). (F) Overall survival of advanced-stage high-grade endometrial cancer patients with a high or low level of \u003cem\u003eLDHA\u003c/em\u003eexpression (TCGA database, black; \u003cem\u003eLDHA\u003c/em\u003e-high, \u003cem\u003en =\u003c/em\u003e 29. Red; \u003cem\u003eLDHA\u003c/em\u003e-low, \u003cem\u003en =\u003c/em\u003e37; \u003cem\u003ep\u003c/em\u003e = 0.021).\u003c/p\u003e","description":"","filename":"Slide8.png","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/2f9c79e5f47c0bed52d5c145.png"},{"id":66450558,"identity":"aa7d9edd-3e72-4061-a878-48348c61f077","added_by":"auto","created_at":"2024-10-12 07:10:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3798012,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/38620449-2f5e-46bc-b1ba-46760f8358fc.pdf"},{"id":47654804,"identity":"083391a5-7e28-4fff-b71f-0a65de188a59","added_by":"auto","created_at":"2023-12-05 17:26:16","extension":"pdf","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":1980853,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryData20merge.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3547380/v1/0f9ab8df63aa6ea2be7730b7.pdf"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose.","formattedTitle":"Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity","fulltext":[{"header":"Introduction","content":"\u003cp\u003eUterine endometrial cancer is a significant gynecological ailment. While early-stage and low-grade uterine endometrial cancers present relatively mild behaviors, standard chemotherapy regimens fail to adequately address high-grade or metastatic tumors (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). A common molecular characteristic of both type I and type II endometrial cancers is the dysregulation of the PI3K-Akt-mTORC1 signaling pathway (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Positioned as a downstream target, mTORC1 is influenced by numerous oncogenic pathways in cancer, such as the PI3K/Akt and MAPK pathways (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Furthermore, the mTORC1 complex is modulated by growth factors, amino acids, energy levels, and stress, and it plays a role in cell growth through protein, lipid, and nucleotide synthesis as well as autophagy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Similar to many cancers, most endometrial cancers exhibit genomic aberrations in the PI3K-Akt-mTORC1 signaling pathway, which often lead to mTORC1 hyperactivation (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Thus, therapies targeting the PI3K-Akt-mTORC1 signaling could potentially enhance patient outcomes in endometrial cancer (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). However, results from a recent phase I/II trial involving an mTOR inhibitor combined with an aromatase inhibitor, anastrozole, showed limited improvements in hormone receptor-positive recurrent or metastatic endometrial cancer patients, with an overall response rate of 24.5% (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Another phase II clinical trial demonstrated that dual PI3K/mTOR inhibitors offered modest clinical benefits (overall response rate: 16%; duration of response: 4.2 months) with manageable side effects (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Delving deeper into the mechanism of mTORC1 signaling and formulating methods to identify benefiting patients remains of paramount importance.\u003c/p\u003e \u003cp\u003eCancer stem cells, identified as primary contributors to cancer origination, when eliminated, significantly retard cancer progression (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). Aldehyde dehydrogenase (ALDH) serves as a specific marker for many types of cancer stem cells (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). ALDH, pivotal in retinoid metabolism and toxic aldehyde removal, influences cancer progression, stemness, and chemotherapy resistance across various cancers. Notably, among the 19 ALDH isoforms, ALDH1A1, ALDH1A3, and ALDH3A1 are tied to cancer stem cells (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). We previously identified ALDH as a functional marker for both ovarian and uterine endometrial cancer stem cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). In particular, we highlighted ALDH1A1 as the specific isoform marking uterine endometrial cancer stem cells and uncovering the role of the ALDH-GLUT cascade in paclitaxel resistance among these cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). However, comprehending the ALDH mechanism in endometrial cancer proliferation remains an open question.\u003c/p\u003e \u003cp\u003ePatient-derived tridimensional cells, encompassing spheroid and organoid cells from clinical specimens, retain several clinical traits. They are invaluable platforms for assessing drug sensitivity in numerous solid cancers, including ovarian and uterine endometrial cancers (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Our earlier research indicated that gynecological cancer patient-derived spheroid cells exhibit cellular diversity and potent tumorigenic capabilities \u003cem\u003ein vivo\u003c/em\u003e, which are hallmark characteristics of cancer stem cells (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Moreover, our spheroid cells have proven effective in drug sensitivity determination (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), echoing the utility of organoid cells that are universally recognized for drug sensitivity assays (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn this study, we delved into the sensitivity of inhibitors targeting the PI3K-Akt-mTORC1 signaling, employing uterine endometrial cancer patient-derived spheroid cells. We juxtaposed protein and genomic mutational profiles of these spheroid cells. A drug sensitivity test elucidated the interplay between ALDH and mTOR, uncovering the novel function of ALDH in cancer and its contribution to mTORC1 activation via LDHA. Furthermore, we identified the reciprocal relationship between glycolysis and mTORC1 in promoting the proliferation of endometrial cancer cells. These findings underscore that targeting ALDH-LDHA-mTORC1 signaling is a novel treatment strategy for aggressive uterine endometrial cancer.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eTumor-derived Spheroid Culture\u003c/h2\u003e \u003cp\u003eEndometrial cancer spheroid cells isolated from clinical cancer specimens were cultured using ultra-low-attachment culture dishes (Corning, Corning, NY, USA). The culture medium was STEMPRO hESC SFM (Gibco, Grand Island, NY, USA) with a supplementation of 8 ng/ml basic fibroblast growth factor (Invitrogen, Carlsbad, CA, USA) and penicillin/streptomycin. Cells were maintained under specific conditions at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e concentration (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). Spheroid cells were dissociated using Accumax (Innovative Cell Technologies, San Diego, CA, USA) for serial passaging. All the culture and handling protocols strictly adhered to the guidelines of the Ethics Committee of Niigata University and the National Cancer Center. Informed consent was duly acquired from all involved patients.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eAnimal Experiments\u003c/h2\u003e \u003cp\u003eThe drug efficacy experiments followed a randomized selection of mice into distinct groups. Accumax (Innovative Cell Technologies) was used to dissociate spheroid cells into single-cell structures. These cells were then suspended in a medium, comprising 50% Matrigel (BD Biosciences, San Jose, CA, USA; 3564234), before being subcutaneously injected using a 27-G needle into NOG (NOD/Shi-SCID-IL-2Rγnull) mice. These mice were sourced from the Central Institute for Experimental Animals, Kawasaki, Japan. The drug was administered every 2\u0026ndash;3 days from 10\u0026ndash;14 days after spheroid cell injection. The control group was exposed to dimethyl sulfoxide (DMSO) only. Mice were monitored every 3\u0026ndash;4 days for a span of 4\u0026ndash;6 weeks post-cell transplantation. The Animal Care and Use Committee of Niigata University approved all mouse procedures, ensuring they align with institutional policies.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eLentivirus-mediated Transduction\u003c/h2\u003e \u003cp\u003eBoth pCMV3-ALDH1A1 plasmid (HG11388-UT) and pCMV3 control vector were procured from Sino Biological (Beijing, China). The process of creating virus-containing supernatants and the subsequent viral infections were carried out as elaborated in a previously established protocol (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Post-infection, cells underwent selection in a milieu containing 100 \u0026micro;g/ml hygromycin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analyses\u003c/h2\u003e \u003cp\u003eThe cell lysis procedure was facilitated using radioimmunoprecipitation assay buffer. This buffer contained specific concentrations of several compounds, including 50 mM Tris (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1 mM ethylenediaminetetraacetic acid. To this mixture, protease and phosphatase inhibitors (Roche, Basel, Switzerland) were added. The resultant samples were then subjected to western blot analysis as detailed in a previous protocol (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e), with the use of specific primary antibodies (Details provided in Supplementary Table\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analyses\u003c/h2\u003e \u003cp\u003eThe statistical evaluation of both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e spheroid cell experiments utilized Welch\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests or Student\u0026rsquo;s \u003cem\u003et\u003c/em\u003e-tests, contingent on the outcomes of F tests. A threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was set for significance. Clinical sample statistics were carried out using the EZR software (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). The Kaplan\u0026ndash;Meier method was used for univariate survival analysis, whereas the significance of variances between groups was determined through log-rank tests.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eData Availability Statement\u003c/h2\u003e \u003cp\u003eThe microarray data analyzed in this study were obtained from the Gene Expression Omnibus database (accession number: GSE123530).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003ePI3K-Akt-mTOR signaling patterns in human uterine endometrial cancer spheroid cells\u003c/h2\u003e \u003cp\u003eFirst, we evaluated the protein expression and gene mutation profile of PI3K-Akt-mTOR signaling to delineate the signaling status in endometrial cancer spheroid cells. Through western blot analysis of seven distinct endometrial cancer spheroid cells, we observed variations in the expression levels of signaling factors, including phospho-Akt, phospho-PTEN, and phospho-p70S6K. Intriguingly, their expression did not correlate with the ALDH1A1 expression within the spheroid cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, an immunohistochemical evaluation of spheroid-derived xenograft tumors was undertaken alongside the original clinical cancer tumor specimens. This highlighted that the expression patterns of phospho-Akt, phospho-p70S6K, and phospho-PTEN echoed the results observed in the aforementioned western blot analysis of spheroid cells. Notably, although a heterogeneous expression was discerned across tumors, a considerable number of cancer cells exhibited elevated phospho-Akt expression in tumors originating from or derivatives of EMN24 and EMN144 cells. Furthermore, the tumors from EMN24 cells displayed pronounced phospho-p70S6K expression, and the tumors from EMN108 cells showed evident phospho-PTEN expression (Supplementary Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA). In a broader context, mutation profiles across 22 endometrial cancer spheroid cells mirrored those witnessed in the original clinical tumor specimens (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong these PI3K-Akt-mTORC1-related signaling factors, a previous study showed that phospho-p70S6K is a predictive tool for the outcomes of patients with type II endometrial cancer when used as an immunohistochemistry (IHC)-based marker (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This was particularly significant among markers related to the activation of the PI3K-Akt-mTORC1 signaling pathway (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Furthermore, the phosphorylation observed at T389 showcased a connection with malignancy-related p70S6K activity (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e). Merging this insight with the observed expression results of phospho-p70S6K in both spheroid cells and spheroid-derived xenograft tumors, we sought to confirm the expression of phospho-p70S6K in 35 clinically advanced endometrial cancer tissue specimens through immunohistochemical staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Kaplan\u0026ndash;Meier survival analyses showed that an elevated phospho-p70S6K expression may be correlated with overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), not with progression-free survival (Supplementary Fig. S2A). The expression was not correlated with histological grade or clinical stage in these advanced-stage cases (Supplementary Fig. S2B and S2C, Fisher\u0026rsquo;s exact test). Though conclusions drawn from a limited case pool remain preliminary, our findings accentuate that phospho-p70S6K expression might indeed be intertwined with an adverse prognosis in advanced-stage uterine endometrial cancer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003ePI3K Inhibitors curbing the proliferation of endometrial cancer spheroid cells\u003c/h2\u003e \u003cp\u003eWith the aforementioned results of phospho-p70S6K expression being related to the activation of the PI3K-Akt-mTORC1 signaling pathway (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), we then explored the effects of PI3K-Akt-mTORC1 signaling in endometrial cancer spheroid cells. Our \u003cem\u003ein vitro\u003c/em\u003e cancer spheroid model has displayed superior efficacy in comparison to the cancer stem cell model (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) and existing \u003cem\u003ein vitro\u003c/em\u003e drug sensitivity evaluations (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e). Riding on this fundamental advantage, we first assessed the sensitivity of endometrial cancer cells toward PI3K inhibitors. The particular focus was on Alpelisib, a prominent PI3K inhibitor deployed in clinical trials to treat breast and several other cancers (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUpon assessment, we categorized the endometrial cancer cells into three distinct sensitivity groups with respect to Alpelisib:\u003c/p\u003e \u003cp\u003e \u003col\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eHigh-sensitivity group: EMN18 and EMN21 cells.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eLow-sensitivity group: EMN103 and EMN144 cells.\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003cspan\u003e \u003cli\u003e \u003cp\u003eIntermediate-sensitivity group: Comprising the remaining cell types (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB).\u003c/p\u003e \u003c/li\u003e \u003c/span\u003e \u003c/ol\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eOur examination of other PI3K inhibitors, namely Taselisib and Copanlisib, showed sensitivity patterns similar to those observed for Alpelisib (Supplementary Fig. S3A and S3B). Alpelisib treatment enhanced caspase levels when the spheroid cellular growth was inhibited (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). To juxtapose \u003cem\u003ein vivo\u003c/em\u003e sensitivity against its \u003cem\u003ein vitro\u003c/em\u003e counterpart, we administered Alpelisib to NOG mice thrice weekly. The outcomes were illuminating: the xenograft tumors stemming from EMN18 and EMN21 spheroid cells (from the \u003cem\u003ein vitro\u003c/em\u003e high-sensitivity group) exhibited a decline post-Alpelisib treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Moreover, tumors from EMN108 and EMN81 spheroid cells (intermediate-sensitive) also exhibited growth suppression (Supplementary Fig. S3C). Nonetheless, tumors originating from EMN144 cells (low-sensitivity group) remained unaffected by Alpelisib treatment (Supplementary Fig. S3D).\u003c/p\u003e \u003cp\u003eCollectively, these findings highlight that the Alpelisib sensitivity observed in our \u003cem\u003ein vitro\u003c/em\u003e spheroid model mirrors the outcomes witnessed during \u003cem\u003ein vivo\u003c/em\u003e applications.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAdditive effects of PI3K and ALDH inhibitors on endometrial cancer spheroid cells' growth\u003c/h2\u003e \u003cp\u003eNext, we elucidated the interactions between PI3K-Akt-mTORC1 signaling and cancer stemness using ALDH, which was found to be a functional marker of uterine endometrial cancer stem cells in our previous study (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Intriguingly, a targeted sequencing analysis revealed identical mutational profiles between ALDH-high and ALDH-low spheroid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Moreover, despite the suppression of phospho-Akt and phospho-p70S6K expression post-Alpelisib treatment in sensitive spheroid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), we observed no discernable differences in the \u003cem\u003ein vitro\u003c/em\u003e sensitivity to Alpelisib between ALDH-high and ALDH-low cells (data not shown). This observation was consistent with the changes in ALDH activity or ALDH1A1 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and Supplementary Fig.\u0026nbsp;4A). Further, Alpelisib treatment diminished phospho-p70S6K expression in both ALDH-high and ALDH-low cells (Supplementary Fig. S4B).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eGiven the above, we postulated that ALDH activity might be independent of PI3K sensitivity. To put this theory to the test, we co-treated spheroid cells with disulfiram (an ALDH inhibitor) and Alpelisib. Remarkably, this combination treatment led to a substantial inhibition of spheroid growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eValidating these \u003cem\u003ein vitro\u003c/em\u003e findings, we introduced this co-treatment to spheroid cell-transplanted mice. Tumors post-co-treatment were roughly half the size compared with those treated solely with Alpelisib. This was consistent for both high-sensitivity (EMN21, Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) and intermediate-sensitivity (EMN24, Supplementary Fig. S4C) cell groups. Additionally, consistent with \u003cem\u003ein vitro\u003c/em\u003e results, Alpelisib alone did not alter the ALDH activity in xenograft tumors (Supplementary Fig. S4D).\u003c/p\u003e \u003cp\u003eConclusively, while ALDH activity appears to have no direct impact on PI3K inhibition, a combination of ALDH and PI3K inhibitors results in an additive inhibition of endometrial cancer progression.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAdditive inhibition of endometrial cancer spheroid cells by combining Akt and ALDH inhibitors\u003c/h2\u003e \u003cp\u003ePivoting from our work with PI3K inhibitors, we turned our attention to the effects of Akt inhibitors on endometrial cancer spheroid cells. Notably, cells that exhibited low-to-intermediate sensitivity to PI3K inhibitors\u0026mdash;specifically, the EMN81, EMN103, and EMN144 cell lines (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and Supplementary Fig. S3A and S3B)\u0026mdash;responded to low-dose treatment with Akt inhibitors, Ipatasertib and Caplivasertib (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). This sensitivity was marked by an uptick in activated caspases post-Ipatasertib treatment, implying cytotoxic effects (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHowever, it is worth noting that while EMN24 cell-derived xenograft tumors demonstrated resistance to Ipatasertib \u003cem\u003ein vitro\u003c/em\u003e, they remained unresponsive to the drug even \u003cem\u003ein vivo\u003c/em\u003e (Supplementary Fig. S5). Such findings underscore the potential of baseline Akt activity, especially linked to PTEN mutations, as a valuable predictor for Akt inhibitor sensitivity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB) (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile we observed a reduction in phospho-p70S6K expression after treatment with the Akt inhibitor, there was no noticeable shift in ALDH activity or expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). Such observations led us to hypothesize that ALDH activity remained unaffected by Akt inhibition. However, dual inhibition of both Akt and ALDH might collectively hinder endometrial cancer cell growth.\u003c/p\u003e \u003cp\u003eTo test this, we investigated the combined impact of ALDH and Akt inhibitors, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The resulting data was as follows: combination treatment not only triggered apoptosis in endometrial cancer cells but also hindered their proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF\u0026ndash;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eI). Mirroring our findings with PI3K inhibitors, this co-treatment approach further validated the additive repression of endometrial cancer progression, independent of any influence of ALDH activity on Akt inhibition.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003emTOR inhibitor reduces the proliferation of endometrial cancer spheroid cells with ALDH activity\u003c/h2\u003e \u003cp\u003eDrawing insights from our experiments with PI3K and Akt inhibitors, it became evident that PI3K/Akt activity was independent of ALDH activity. Our exploration then shifted toward understanding the influence of mTOR inhibition on endometrial cancer spheroid cell proliferation. Upon conducting \u003cem\u003ein vitro\u003c/em\u003e sensitivity assays, we observed varying responses to mTOR inhibitors, everolimus, and Torin1. Notably, the range of sensitivities was more consistent than what was observed for PI3K and Akt inhibitors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and Supplementary Fig. S6A). Following mTOR inhibitor administration, there was a decline in the levels of both phospho-p70S6K and phospho-4EBP1 across the spheroid cell population (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Intriguingly, this decline in phospho-p70S6K was more accentuated in ALDH-high cells compared with ALDH-low cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and Supplementary Fig. S6B). Moreover, the ALDH-high cells exhibited heightened sensitivity to everolimus in comparison to their ALDH-low counterparts (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). It was discerned that the exogenous overexpression of ALDH1A1 further augmented this sensitivity to everolimus (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). Collectively, these findings bolster the notion that cells with higher ALDH activity inherently possess increased mTOR activation relative to cells with low ALDH activity. Alternatively, combination treatment with disulfiram and everolimus also hindered the spheroid cell proliferation (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF). In conclusion, while ALDH activity appears to be linked to mTORC1 activity, a combination of ALDH and mTORC1 inhibitors additively impedes endometrial cancer progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eEndometrial cancer spheroid cells exhibiting high ALDH activity show enhanced sensitivity to mTOR inhibitors\u003c/h2\u003e \u003cp\u003eBased on the aforementioned results, we speculated that heightened ALDH activity might partially enhance mTOR sensitivity in endometrial cancer. To reveal the relationship between ALDH and mTOR, we examined changes in the expression of the PI3K-Akt-mTOR signaling pathway after manipulating ALDH activity. Disulfiram, which reduced ALDH activity, led to a decrease in phospho-p70S6K levels, whereas phospho-PI3K and phospho-Akt levels remained unchanged (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA and Supplementary Fig. S7A). Additionally, gene set enrichment analysis (GSEA) revealed the ALDH-high cells preferentially expressed genes included in the gene set of hallmark of mTORC1 signaling (false discovery rate [FDR] q-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01, normalized enrichment score [NES] 1.59, \u003cem\u003ep\u003c/em\u003e-value\u0026thinsp;\u0026lt;\u0026thinsp;0.01) (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e), genes upregulated after ectopically expressing eIF4E, and genes upregulated in control cells compared with eIF4GI-silenced cells (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB and Supplementary Fig. S7B). Moreover, ALDH-high cells clearly expressed more phospho-p70S6K than ALDH-low cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eC). The exogenous overexpression of ALDH1A1 that led to ALDH activation (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) ultimately increased phospho-p70S6K levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eD). These results suggested that ALDH activity primarily affected mTORC1 activation rather than PI3K or Akt. To further confirm the relationship between ALDH and mTORC1 signaling, we determined whether mTOR activation could rescue the inhibitory effect of ALDH inhibitor on cancer cells. As expected, mTOR activator MHY1485 partially mitigated disulfiram-induced cytotoxicity in endometrial cancer spheroid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE, \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF, and Supplementary Fig. S7C\u0026ndash;S7F). Moreover, western blot analysis indicated that MHY1485 could partially revert the disulfiram-mediated reduction in phospho-p70S6K expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and Supplementary Fig. S7G).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eLDHA bridges ALDH activity and mTORC1 activation in endometrial cancer spheroid cells\u003c/h2\u003e \u003cp\u003eTo understand how ALDH influences mTOR signaling in endometrial cancer cells, we characterized its functional isoforms. Notably, of the 19 ALDH isoforms with analogous catalytic functions, ALDH1A1 predominantly dictates its activity (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e). Our findings further cemented this observation, as endometrial cancer spheroid cells majorly expressed ALDH1A1, sidelining other ALDH isoforms (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). A pivotal function of ALDH1A1 includes the conversion of retinol to retinoic acids, driving cancer proliferation through multifaceted mechanisms (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Corroborating our premise that ALDH-mediated retinoic acid modulates endometrial cancer development \u003cem\u003ein vitro\u003c/em\u003e, we discerned that additional retinoic acid rescued the cell mortality caused by disulfiram (Supplementary Fig. S8A\u0026ndash;S8E). This mirrored the outcome when mTORC1 was activated with MHY1485 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eE and \u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF and Supplementary Fig. S7C\u0026ndash;S7F). Experiments conducted on cells overexpressing ALDH1A1 further supported this observation (Supplementary Fig. S8F). Moreover, western blot outcomes indicated that retinoic acid could partially revert the disulfiram-mediated decrease in phopho-p70S6K expression (Supplementary Fig. S8G and S8H). This modulation paralleled the effects of the mTORC1 activator MHY1485 (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eG and Supplementary Fig. S7G).\u003c/p\u003e \u003cp\u003eTo assess the influence of RA on mTOR activation, we combined GSEA results from microarray data and published RARA binding data from the ChIP-Atlas (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://chip-atlas.org/\u003c/span\u003e\u003cspan address=\"http://chip-atlas.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Of 184 hallmark mTORC1 signaling genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eB) (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), 85 were core enrichment genes associated with ALDH-high endometrial cancer cells. Merging this with RARA-regulated genes from ChIP-Atlas, specifically those linked to malignancies, yielded eight candidate genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). Significantly, LDHA was predominantly expressed in ALDH-high cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). Delving deeper, ALDH1A1 overexpression augmented LDHA levels, whereas ALDH inhibition, using disulfiram, attenuated its expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eD and Supplementary Fig. S9A). Additionally, ALDH-high cells and exogenous ALDH1A1-overexpressing cells had higher LDHA activity than control bulk spheroid cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eE).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUpon investigating LDHA inhibition in cancer cells, we observed that using LDHA inhibitor AZ-33 considerably hampered endometrial cancer cell viability, especially in cells with ALDH activity (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eF). Interestingly, while the LDHA inhibitor diminished LDH activity and phospho-p70S6K expression, ALDH expression and its functional activity remained unaltered (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Furthermore, treatment with AZ-33 led to a marked reduction in stemness indicators such as Nanog, Oct-4, and c-Myc (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eG). Additionally, activating mTORC1 with MHY1485 could partially counteract the inhibitory effects of AZ-33 on cancer cell proliferation, although MHY1485 in isolation did not make a significant difference (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eH and Supplementary Fig. S9B and S9C).\u003c/p\u003e \u003cp\u003eTaken together, our findings highlight that ALDH influences mTORC1 through LDHA, promoting the proliferation of endometrial cancer spheroid cells. Notably, while fluctuations in mTORC1 activation remained agnostic to LDHA levels, inhibiting mTOR with everolimus led to a surge in GLUT1. Conversely, stimulating mTOR with MHY1485 reduced GLUT1 levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eI and Supplementary Fig. S9D), influencing glucose transport (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eJ and Supplementary Fig. S9E and S9F). These intricate interplays underscore a reciprocal relationship between glycolysis and mTORC1 in ALDH-active endometrial cancer progression (Supplementary Fig. S10).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eLDHA expression is associated with adverse prognosis in endometrial cancer patients\u003c/h2\u003e \u003cp\u003eOur study suggests that glycolysis and mTORC1 signaling play a pivotal role in regulating ALDH-high endometrial cancer cells. Seeking to delineate the distribution of ALDH and LDHA in a clinical framework, we performed immunostaining analyses on endometrial cancer samples. Intriguingly, ALDH-positive cells exhibited more pronounced LDHA expression than their ALDH-negative counterparts across both early and advanced cancer stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA and Supplementary Fig. S11A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further elucidate the clinical implications of LDHA, serum LDH levels were quantified in 244 patients with endometrial cancer from our institution. Notably, LDH levels increased in patients with high-grade cancer relative to their low-grade counterparts across all stages, a trend particularly evident in stage I (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB and \u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). Moreover, advanced-stage patients consistently exhibited elevated LDH titers in contrast to those in the early stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eD). This underscores a direct correlation between serum LDH levels, tumor grade, and clinical cancer progression.\u003c/p\u003e \u003cp\u003eDelving deeper into the clinical ramifications of LDHA expression, we utilized The Cancer Genome Atlas (TCGA) database (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e) to investigate its correlation with the prognosis of patients with endometrial cancer. \u003cem\u003eLDHA\u003c/em\u003e mRNA levels were discernibly elevated in high-grade tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE). Alarmingly, patients manifesting elevated \u003cem\u003eLDHA\u003c/em\u003e expression experienced a significantly reduced overall survival (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eF, p\u0026thinsp;=\u0026thinsp;0.02). Moreover, progression-free survival showed a concerning trend, with \u003cem\u003eLDHA\u003c/em\u003e-high-expressing cases displaying a tendency toward shorter survival compared with their low-expression counterparts (Supplementary Fig. S11B, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.09). A significant correlation was also evident between \u003cem\u003eLDHA\u003c/em\u003e and RPS6KB1 expression in endometrial cancer tissue (Supplementary Fig. S8B, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.01).\u003c/p\u003e \u003cp\u003eIn summary, our findings highlight \u003cem\u003eLDHA\u003c/em\u003e as a crucial biomarker, revealing its strong association with tumor grade and suggesting its potential role as an indicator of unfavorable prognosis in endometrial cancer.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe utilization of the patient-derived cell model offers promising avenues for understanding therapeutic responses to cytotoxic chemotherapeutic agents on an individual patient basis (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e). Recent findings suggest organoid cells can effectively serve as models for gauging responses to therapies that target specific genetic mutations (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e). In alignment with these studies, our research highlights the correlation between \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e tests concerning the sensitivity to PI3K-Akt-mTOR signaling inhibitors in endometrial cancer patient-derived spheroid cells. Particularly, the response to PI3K and Akt inhibitors showed distinct differentiation based on genomic mutation profiles. These observations remark on the clinical potential of spheroid cells.\u003c/p\u003e \u003cp\u003eEndometrial, breast, and colon cancers often present activation in the mutant-dependent PI3K-Akt-mTOR signaling pathway (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). While PI3K-Akt-mTOR signaling inhibitors have theoretical potential to significantly suppress mutated cancer cells, challenges arise from negative feedback release and detrimental impacts on non-tumor cells. Furthermore, elements such as growth factors, energy, and stress play roles in mTOR activation (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). This drove our investigation into the non-genetic mechanisms underlying PI3K-Akt-mTOR pathway activation during endometrial cancer proliferation. The PI3K-Akt-mTOR pathway has been implicated in enhancing stemness, a trait linked to aggressive cancer manifestations (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Numerous studies have pinpointed the role of PI3K signaling in cancer stemness, affecting markers such as Nanog, SOX2, and CD133 (\u003cspan additionalcitationids=\"CR33\" citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). For instance, mTORC1 activation has been noted to boost colon cancer stem cell proliferation (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). mTOR pathway is the downstream mediator of ALDH1A3 in gastric cancer (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Our studies on uterine endometrial cancer emphasized the influence of both genomic and ALDH-mediated non-genomic aberrations on mTOR activation. Spheroid cells, enriched with cancer stem cells, proved instrumental in elucidating the nexus between cancer stemness and PI3K-Akt-mTORC1 signaling (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLactate dehydrogenase (LDH), an intracellular enzyme, can manifest as either a homotetramer or heterotetramer, encompassing LDHA and LDHB subunits, giving rise to five isoforms. Each isoform is competent in transforming pyruvate to lactate (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Echoing prior reports on cancers such as breast and pancreatic (\u003cspan additionalcitationids=\"CR39\" citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e), our analysis attests to the association between LDHA expression and advanced-stage endometrial cancer prognosis. Furthermore, elevated LDHA expression and serum LDH levels were more prevalent in high-grade cancer patients. Past research has drawn parallels between elevated serum LDH levels and reduced survival rates in solid tumors such as melanoma and prostate carcinomas, highlighting its potential as a prognostic biomarker for metastatic carcinomas (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Collectively, these insights underline the role of LDHA in exacerbating endometrial cancer progression and its clinical malignancy.\u003c/p\u003e \u003cp\u003eThe interplay between glycolysis and cancer proliferation, especially processes such as epithelial-to-mesenchymal transition and cancer stemness, is the subject of extensive research (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Our prior work revealed the centrality of the glucose transporter GLUT1 in dictating the stemness and chemoresistance traits of ALDH-active endometrial cancer stem cells (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). We later discovered the regulatory role of ALDH-mediated retinoic acid over glycolytic functional factor LDHA. LDHA is crucial for sustaining breast cancer stemness and triggering metastasis (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Few studies have analyzed the role of LDHA in governing mTOR activation. In the context of pancreatic adenocarcinoma, LDHA-induced proliferation was found to be mediated by AMPK-mTOR signaling due to L-lactate (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Other findings emphasize the role of LDHA in activating glycolysis, subsequently stimulating mTORC1 and thus fostering cellular proliferation. This phenomenon is particularly observed in K-Ras activated colon and pancreatic cancer cells (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e). Furthermore, LDHA is required for the activation of mTOR in gastric cancer (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e). Corroborating these findings, our research establishes that inhibiting LDHA impedes mTOR activation and curtails cancer cell proliferation, especially in ALDH-active endometrial cancer spheroids, regardless of K-Ras genomic aberrations (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eMoreover, our findings reveal the role of mTOR in modulating glycolysis. Curbing Akt-mTORC1 signaling diminishes GLUT1 expression, thereby stalling glycolysis and cancer cell survival in diseases such as leukemia (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). The intricate dynamics between mTOR and glycolysis in cancer encompass both direct and indirect mechanisms. A standout feature of our study underscores the mutual regulatory relationship between glycolysis and mTOR activation. This feedback mechanism orchestrates the proliferation and survival of ALDH-active endometrial cancer stem cells (Fig. S10). Targeting this interplay could potentially enhance endometrial cancer treatment outcomes, even when mTORC1 activation is dictated by PI3K-Akt-mTOR genomic aberrations.\u003c/p\u003e \u003cp\u003eIn summary, we disclosed the ALDH-LDHA-mTORC1 cascade to be a novel facet of the ALDH-related signaling that governs the proliferation of uterine endometrial cancer stem cells. Future studies should delve deeper into the cascade and interplay between glycolysis and mTORC1 to develop clinical treatment strategies to tackle the outcome of aggressive uterine endometrial cancer.\u003c/p\u003e \u003cp\u003eAdditional materials and methods can be retrieved in Supplemental Experimental Procedures.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConception and design: H.U., T. I.\u003c/p\u003e\n\u003cp\u003eDevelopment of methodology: H.U., T.I.\u003c/p\u003e\n\u003cp\u003eAcquisition of data: H.U., T.I., Y.M.\u003c/p\u003e\n\u003cp\u003eAnalysis and interpretation of data: H.U., T.I., Y.M., K.Yamawaki\u003c/p\u003e\n\u003cp\u003eWriting, review, and/or revision of the manuscript: H.U., T.I., Y. M., K.Yamawaki, K. Yoshihara, K. O., and T.E.\u003c/p\u003e\n\u003cp\u003eStudy supervision: T. E., K.Y.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Ryo Tamura, Kazuaki Suda, Nozomi Yachida, Manako Yamaguchi, Kentaro Sugino, Tomoyuki Sekizuka, Kyota Saito, Anna Ishida (Department of Obstetrics and Gynecology, Niigata University Medical School) for scientific advice and technical assistance. This research was supported by Grants-in-Aid for Scientific Research (C) (Grant No. 18K09250, 21K09490) to T.I. from the Japan Society for the Promotion of Science. This study was partially supported by the National Cancer Center Research and Development Fund (29-A-2).\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ede Boer SM, Powell ME, Mileshkin L, Katsaros D, Bessette P, Haie-Meder C, et al. Adjuvant chemoradiotherapy versus radiotherapy alone for women with high-risk endometrial cancer (PORTEC-3): final results of an international, open-label, multicentre, randomised, phase 3 trial. Lancet Oncol. 2018; 19:295\u0026ndash;309.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWild PJ, Ikenberg K, Fuchs TJ, Rechsteiner M, Georgiev S, Fankhauser N, et al. p53 suppresses type II endometrial carcinomas in mice and governs endometrial tumour aggressiveness in humans. EMBO Mol Med. 2012; 4:808\u0026ndash;824.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaxton RA, Sabatini DM. mTOR signaling in growth, metabolism, and disease. 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Int J Mol Sci. 2023; 24:9679.\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-3547380/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3547380/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCancer stem cells are associated with aggressive phenotypes of malignant tumors. A prominent feature of uterine endometrial cancer is the activation of the PI3K-Akt-mTOR pathway. Herein, we present variations in sensitivities to a PI3K-Akt-mTORC1 inhibitor among \u003cem\u003ein vitro\u003c/em\u003e endometrial cancer stem cell-enriched spheroid cells from clinical specimens. Intriguingly, the \u003cem\u003ein vitro\u003c/em\u003e sensitivity mirrored effects observed in \u003cem\u003ein vivo\u003c/em\u003e spheroid-derived xenograft tumor models. Our findings reveal a complementary suppressive effect on endometrial cancer spheroid cell growth when aldehyde dehydrogenase (ALDH) and PI3K-Akt inhibitors are combined. In the PI3K-Akt-mTORC1 signaling cascade, the influence of ALDH on mTORC1 is partially channeled through retinoic acid-induced lactate dehydrogenase A (LDHA) activation. LDHA inhibition was found to reduce endometrial cancer cell growth, paralleling the effects of mTORC1 inhibition. Building upon our prior findings highlighting ALDH-driven glycolysis through GLUT1 in uterine endometrial cancer spheroid cells, curbing mTORC1 bolstered glucose transport via GLUT1 activation. Notably, elevated LDHA expression correlated with adverse clinical survival and escalated tumor grade, especially in advanced stages. Collectively, our findings emphasize the pivotal role of ALDH-LDHA-mTORC1 cascade in the proliferation of endometrial cancer. Targeting the intricate interplay between mTORC1 and ALDH-influenced glycolysis could pave the way for novel strategies to combat this aggressive cancer.\u003c/p\u003e","manuscriptTitle":"Glycolysis-mTORC1 crosstalk drives rapid proliferation in patient-derived endometrial cancer spheroids with ALDH activity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-12-05 17:26:11","doi":"10.21203/rs.3.rs-3547380/v1","editorialEvents":[{"type":"communityComments","content":0}],"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":"6169ecaf-8bb2-4ee8-a4b2-8c4d51d55c0d","owner":[],"postedDate":"December 5th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":26073985,"name":"Biological sciences/Cancer/Cancer stem cells"},{"id":26073986,"name":"Biological sciences/Cancer/Gynaecological cancer/Endometrial cancer"}],"tags":[],"updatedAt":"2024-10-12T07:10:45+00:00","versionOfRecord":{"articleIdentity":"rs-3547380","link":"https://doi.org/10.1038/s41420-024-02204-y","journal":{"identity":"cell-death-discovery","isVorOnly":false,"title":"Cell Death Discovery"},"publishedOn":"2024-10-11 04:00:00","publishedOnDateReadable":"October 11th, 2024"},"versionCreatedAt":"2023-12-05 17:26:11","video":"","vorDoi":"10.1038/s41420-024-02204-y","vorDoiUrl":"https://doi.org/10.1038/s41420-024-02204-y","workflowStages":[]},"version":"v1","identity":"rs-3547380","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3547380","identity":"rs-3547380","version":["v1"]},"buildId":"WrCJVZZCHTDjtuVLN7oU0","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}