CRYβB2 is a biomarker for poor prognosis and response to CDK4/6 inhibitors in breast cancer

CRYβB2 is a biomarker for poor prognosis and response to CDK4/6 inhibitors in breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article CRYβB2 is a biomarker for poor prognosis and response to CDK4/6 inhibitors in breast cancer Yu Yan, Athira Narayan, Marzieh Mazinani, Harumi Saeki, Tao Huang, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6431382/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Breast Cancer Research → Version 1 posted 12 You are reading this latest preprint version Abstract Background Hazard rates of breast cancer death are significantly higher for women of African American (AA) origin compared with European American (EA) and the molecular mechanisms underlying this difference in outcome are understudied. Previously, we showed that β-crystallin B2 (CRYβB2) expression is up-regulated in breast cancer of AA women, activates nucleolin (NCL), and mediates oncogenesis in triple negative breast cancer (TNBC). Presently no biomarkers, other than estrogen receptor (ER)/ progesterone receptor (PR) expression, are used to prescribe cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) for women with hormone receptor positive (HR + ) metastatic breast cancer. Methods Western blot and flow cytometry was used to determine the activation of CDK4/6 pathway and cell cycle of cells isolated from CRYβB2 overexpressing tumors, respectively. Response to CDK4/6i was determined following treatment of mice containing control and CRYβB2- overexpressing tumors and TNBC and ER + cells. The correlation of CRYβB2 expression with CDK4/6 activation and survival was determined by Western blot, immunohistochemistry, and Kaplan Meier curves using TNBC and ER + tumors from AA and EA women. Results Here, we report that tumors overexpressing CRYβB2 showed an increase in cell cycle progression and activation of the CDK4/6 pathway in models of premalignant- and ductal carcinoma in situ (DCIS) lesions, and TNBC and ER + breast cancer cells. Targeting CRYβB2 and NCL resulted in lower levels of CDK4, cell division cycle 25 A (Cdc25a), and phosphorylated retinoblastoma (ppRb). Only tumors expressing CRYβB2 showed growth inhibition by the CDK4/6i, palbociclib. The expression of CRYβB2 protein inversely correlated with the IC50 of palbociclib in both TNBC and ER + cells. In accord with this, CRYβB2 knockdown in TNBC and ER + cells conferred greater resistance to inhibition with palbociclib. Further, the NCL aptamer AS-1411 sensitized TNBC and ER + cells to palbociclib. Higher levels of CRYβB2 expression in TNBC and ER + tumors of AA patients correlated with higher CDK4/pRb activation. Expression of both CRYβB2 and ppRb correlated with decreased survival in AA women with TNBC. Conclusions CRYβB2 expression correlated with CDK4/6 activation and response to CDK4/6i, and may be useful as a biomarker of prognosis and response to palbociclib therapy. European American African American CRYβB2 nucleolin CDK4 pRb palbociclib Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The addition of cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) to anti-hormonal therapy has become a first-line option for patients with the hormone receptor (HR) + / human epidermal growth factor receptor 2 negative (HER2 − ) phenotype in the metastatic setting [ 1 , 2 ]. The cyclin D–CDK4/6 complex phosphorylates the cell cycle repressor protein retinoblastoma 1 (RB1), reducing the inhibitory effects of RB1 on E2F-mediated transcription of cell cycle genes, leading to cell cycle progression [ 2 ]. The clinical use of CDK4/6i is confounded by the high individual variability in clinical response [ 3 ]. As treatment paradigms become more complex, substantial interest has emerged in the prospective identification of patients who are most likely to derive maximum benefit from CDK4/6i, and those whose tumors might be intrinsically resistant to therapy [2] . Presently, no clinically available biomarkers, other than ER/PR expression, are used to prescribe CDK4/6i. It has been demonstrated that fully functional RB is required for the effective use of CDK4/6i in the clinic [ 4 , 5 ]. However, not all RB + /ER + patients benefit from CDK4/6i therapy. Due to frequent RB loss [ 6 ], TNBC patients are considered to be poor candidates for CDK inhibition [ 6 ]. However, cell lines and mouse xenograft models reflecting the luminal androgen receptor (LAR) subtype of TNBC have been shown to respond to palbociclib [ 7 ]. Additionally, from 180 TNBC patient samples, 51% were found to be RB- positive; thus RB represents a relevant target for therapy [8] . TNBC has a notoriously poor prognosis and paucity of targeted therapies. As a result, even today, chemotherapy is the mainstay of treatment [ 9 ]. We showed that β-crystallin B2 (CRYβB2) induced the growth of xenografts of MCF10A breast cancer cells with a single hit mutation in the MAPK pathway [ 10 ]. CRYβB2 interacted with several proteins that regulate cell proliferation and invasion, including nucleolin (NCL), zinc finger protein 495 (ZNF495), acrosin binding protein (ACRBP), Growth factor receptor-bound protein 2 (GRB2), annexin A1 and A2 (ANXA1 and 2), drebrin-like (DBNL), and erythrocyte membrane protein band 4.1 like 2 (EPB41L2) [ 10 ]. NCL is a shuttling nucleolar protein, which induces malignancy by regulation of the cell cycle [ 11 ], apoptosis [ 12 ], cell proliferation [ 13 ], metastasis [ 11 ], and stem cell maintenance [ 14 – 16 ]. We observed that it mediates the tumorigenic effects of CRYβB2 [ 10 ]. Notably, CRYβB2 sensitizes TNBC cells to NCL inhibition with the specific aptamer AS-1411 [ 10 ]. Anti-NCL drugs have being evaluated as anticancer agents in phase II clinical trials [ 17 ]. NCL was also shown to be an independent marker of prognosis in breast cancer [ 18 ]. Here we show that CRYβB2-interaction with NCL results in activation of the CDK4/6 pathway and predisposes breast tumors to management with CDK4/6i. NCL inhibition with the aptamer AS-1411 synergized with CDK4/6i. Finally, higher levels of CRYβB2 correlate with 1) higher activation of the CDK4/6 pathway in TNBC and ER + tumors of AA women, and 2) decreased survival of AA women with TNBC. Methods Patient samples, Cell Lines and Reagents . Primary tumors from women undergoing treatment were provided by the Johns Hopkins Surgical Pathology Department, under protocols approved by the institutional review board. Tissue microarrays (TMAs) were provided by Dr. Naab and Dr. Kanaan from Howard University. Briefly, the TMAs were constructed by Pantomics (Fairfield, CA) using FFPE tumor blocks from primary TNBC (87) and axillary lymph nodes (15) from AA women [ 10 ]. Breast cancer cells were obtained from the American Type Culture Collection. MCF10A premalignant cells were obtained from Ben H. Park. MCF10AT1 and DCIS.COM cells were obtained from the Barbara Ann Karmanos Cancer Institute. Cells were authenticated using short tandem repeat (STR) profiling and tested for mycoplasma using MycoAlert PLUS mycoplasma detection kit (Lonza). Palbociclib was purchased from Selleck Chemicals. The nucleolin aptamer AS-1411 (5′-GGTGGTGGTGGTTGTGGTGGTGGTGG) and CRO control (5′-CCTCCTCCTCCTTCTCCTCCTCCTCC) were purchased from Integrated DNA technologies. Constructs . CRYβB2 coding sequence was cloned into a lentivirus vector (Addgene) using the Gateway Technology System (Thermo Fisher). MCF10A, MCF10AT1 and DCIS.COM cells overexpressing CRYβB2 were generated as previous described [ 10 ]. For immunofluorescence, MCF10AT1 cells were infected with lentivirus containing the CRYβB2 sequence tagged with the myc-DDK (flag) sequence (Origene). For CRISPR knockout, nucleolin and CRYβB2 guide RNAs were designed using sgRNA online web page from Broad Institute and cloned into Lenticrispr V2 (Addgene) [ 10 ]. 293T cells were transfected with the lentivirus constructs using Lipofectamine and virus were used to infect cancer cells [ 19 ]. Xenograft . All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the Johns Hopkins School of Medicine. Xenografts of MCF10AT1 cells expressing vector control and CRYβB2 constructs were established in 6–8 weeks NOD-SCID IL2Rgnull (NSG) mice (from an in-house colony at Hopkins) by injecting 5x10 6 tumor cells into the fourth mammary gland. Mice bearing MCF10AT1 tumors were treated for 2 weeks, receiving palbociclib (50 mg/kg) or saline (pH 4.0) as vehicle for 5 days/ week orally. Western blot, Immunohistochemistry and Immunofluorescence . Western blot, immunohistochemistry (IHC) [ 19 ] and immunofluorescence (IF) [ 20 ] were performed as previously described using antibodies against CRYβB2 and cell cycle proteins (Supplementary Methods). For IF, the slides were probed with the following primary antibodies: CRYβB2 (Thermo Fisher), CRYBB2-myc DDK flag (Cell Signaling Technology), and nuclear staining (Hoechst; Fisher Scientific) [ 20 ]. ImageJ was used for quantification. Cell cycle and proliferation analysis For cell cycle determinations tumors were digested with collagenase/ hyaluronidase [ 19 ]. Tumor-derived cells were permeabilized with cold 70% ethanol and stained with propidium iodide (Sigma). Samples were run on the BD FACSCalibur system (Becton Dickinson) [ 21 ]. MTT (thiazolyl blue tetrazolium bromide, Amresco, #0793) solution (0.8 µg/µL) was used to measure cell proliferation after 48h of drug treatment [ 19 ]. Values are expressed as percent survival of the vehicle treated control (given as 100%). Statistical Analysis . Two-tailed Mann Whitney Test and Student’s T-tests were performed on pairwise combinations of data to determine statistical significance defined as P < 0.05. Statistical analyses were performed using GraphPad Prism version 8.3. Results CRYβB2 induces cell cycle progression and regulates the CDK4/6 pathway in premalignant and ductal carcinoma in situ (DCIS) models We showed that CRYβB2 induced tumorigenesis of breast cells with low malignant potential, and its knockdown (KD) decreased TNBC growth in immunodeficient mice[ 10 ]. CRYβB2 interacted with, and induced NCL, leading to activation of AKT and EGFR with silencing of p53 [ 10 ]. Decrease of NCL expression was shown to reduce proliferation of glioblastoma cells, and induced cell cycle arrest [ 22 ]. NCL is a substrate for CDKs [ 23 , 24 ]. Extensive NCL phosphorylation occurs during the cell cycle, regulating its functions and localization [ 25 ]. NCL associates with two major cellular tumor suppressors, pRb [ 26 ] and p53 [ 27 ], and is involved in post-transcriptional inhibition of p53 [ 27 ]. The p53 pathway has been shown to mediate cellular stress responses, initiate DNA repair, cell-cycle arrest, senescence and apoptosis [ 28 ]. The MCF10 model is a series of cell lines that originated from the human breast epithelial cells, MCF10A [ 29 ]. MCF10AT1 is a premalignant cell line produced by transfection of MCF10A with constitutively active HRAS [ 29 ]. Approximately 25% of the MCF10AT1 cells transplanted into immunodeficient mice progressed to invasive ductal carcinoma (IDC) after a long latency, which indicated low tumorigenic potential of MCF10AT1 with slow progression [ 29 ]. MCF10DCIS.com is a cell line cloned from cell culture of an MCF10AT1 xenograft lesion. DCIS.com cells reproducibly form DCIS-like comedo lesions that spontaneously progress over time to IDC when grown as xenografts in immunodeficient mice [ 30 ]. Previously, we showed that CRYβB2 hastened the growth of MCF10AT1 and DCIS.com tumors [ 10 ]. Highly proliferative MCF10AT1-CRYβB2 tumors and associated metastases showed an increase in cell cycle progression in comparison to MCF10AT1-vec controls (Fig. 1 A). In line with this observation, MCF10AT1 and DCIS.COM tumors overexpressing CRYβB2 showed an increase in cell cycle mediators, including CDK4, cell division cycle 25 A (Cdc25a), and phosphorylated pRb (ppRb) proteins (Fig. 1 B). CRYβB2 overexpression resulted in upregulation of p53 expression in MCF10AT1 tumors. But p53 was downregulated in DCIS.com tumors (Fig. 1 B)[ 10 ] and in premalignant MCF10A-BRCA1-185delAG knockin (KI) mutant cells [ 31 ] (Fig. 1 C and Supplementary Fig. 1A ). The expression of cell cycle proteins were also increased by overexpression of CRYβB2 in MCF10A-BRCA1-KI [ 31 ] and MCF10A-p53 knockout (KO) cells [ 32 ] (Fig. 1 C and Supplementary Fig. 1A ). A lower effect of CRYβB2 induction of cell cycle proteins was observed in MCF10A-p53-R248W KI cells [ 33 ], which have higher endogenous levels of p53 (Fig. 1 C and Supplementary Fig. 1A ). These data suggest that p53 may restrict CRYβB2-dependent activation of cell cycle progression. Further analysis of the effects of knockout of NCL in MCF10AT1 cells showed that reduced NCL expression impaired the CRYβB2-dependent activation of the CDK4/6 pathway (Fig. 1 D and Supplementary Fig. 1B ). No effect on the expression of cell cycle proteins, except a slight decrease of ppRb, was observed by NCL deficiency in MCF10AT1- vector cells, which lack CRYβB2 expression (Fig. 1 D and Supplementary Fig. 1B ). We found that CRYβB2 co-localized with ppRb ( Supplementary Fig. 1C ) and is mutually exclusively expressed with p53 ( Supplementary Fig. 1D ) in the nucleus of MCF10AT1 cells. Collectively, these results suggest that CRYβB2 may recruit NCL to activate the CDK4/pRb pathway, and induce cell cycle progression in premalignant and early-stage breast cancer. CRYβB2 regulates CDK4/6 pathway in ER positive (ER) and TNBC cells Since we observed CRYβB2-mediated regulation of CDK4/pRb in early breast cancer models, we investigated its effect on regulation of cell cycle proteins in models of established tumors. We found that, similar to the premalignant models, CRYβB2 and NCL expression correlated with CDK4/pRb activation in ER + (Fig. 2 A) and TNBC (Fig. 2 B) cells. We next evaluated the effect of downregulation of CRYβB2 and NCL expression in breast cancer cells on activation of the CDK4/6 pathway. Knockdown of CRYβB2 and NCL in AA (MDA-MB-157) and EA (SUM-159 and HCC-1806) TNBC cells resulted in decreased levels of proteins of the CDK4/pRb pathway (Fig. 2 C). We observed a decrease of total pRb in AA ER + (MDAMB-175) CRYβB2- KD cells (Fig. 2 D). Collectively, these results suggest that, in established breast cancer cell lines, CRYβB2 may recruit NCL to activate CDK4/pRb pathway and induce cell cycle progression. CRYβB2 expression correlates to the response of tumor xenografts to CDK4/6 inhibitors We observed CRYβB2-dependent regulation of CDK4/pRb pathway in both premalignant and malignant ER + and TNBC breast cancer models. We next evaluated the effect of induction and downregulation of CRYβB2 and NCL in breast cancer cells on tumor response to CDK4/6i. We observed that the growth of MCF10AT1-CRYβB2 tumors was significantly decreased by treatment of tumor-bearing mice with palbociclib compared to vehicle (Fig. 3 A). Strikingly, MCF10AT1-vector tumors, which lacked detectable CRYβB2 expression did not respond significantly to palbociclib (Fig. 3 A). As shown in Fig. 3 B, expression of CRYβB2 protein correlated inversely with the reported IC50 of palbociclib [ 34 ] in both TNBC and ER + cells. Knockdown of CRYβB2 in TNBC and ER + cells decreased response to palbociclib (Fig. 3 C). These data suggest that CRYβB2 expression in breast tumor cells enhances their sensitivity to CDK4/6i. Since NCL mediates CRYβB2-activation of CDK4/pRb pathway (Fig. 1 D and Fig. 2 C ) , we determined if genetic and pharmacological decrease of NCL expression alters response to CDK4/6i. TNBC NCL-KO cells are more sensitive to palbociclib in comparison to vector control cells (Fig. 3 D). ER + cell lines were described to be most sensitive to growth inhibition by CDK4/6i while TNBC cells were most resistant [ 34 ]. A combination of palbociclib with the NCL aptamer AS-1411[ 35 ] significantly decreased TNBC and ER + tumor cell viability in comparison to single agents (Fig. 3 E). Collectively, these results suggest that CRYβB2 is a biomarker of response to CDK4/6i. A clinically available aptamer [ 35 ], targeting NCL synergizes with the cytotoxic effect of CDK4/6i. Expression of the CRYβB2 and CDK4/pRb pathways correlates in ER − and ER + tumors of AA women We next investigated if the preclinical findings could be corroborated with clinical samples. We confirmed that CRYβB2 expression is higher in ER − tumors of AA women in comparison to EA women [ 10 ] (Fig. 4 A and Fig. 4 C). CDK4 expression was also higher in ER − tumors of AA women and its expression correlated with CRYβB2 expression in tumors of AA women (Fig. 4 A, Fig. 4 C and Supplementary Fig. 2A ). The expression of ppRb (upper band) tended to be higher in AA ER − tumors ( Supplementary Fig. 2B and Supplementary Fig. 2C ). CRYβB2 expression tended to be higher and ppRb is significantly higher in ER + tumors of AA women in comparison to EA women (Fig. 4 B and Fig. 4 C). These data showed that CRYβB2 is a potential biomarker of CDK4/ pRb pathway activation in TNBC of AA women. The increase of ppRb in ER + tumors of AA women may stratify this population as likely responders to CDK4/6i. CRYβB2 and ppRb expression are associated with poor TNBC outcome in AA women CRYβB2 is overexpressed in ER-negative tumors from AA patients in comparison to EA women [ 10 ] (Fig. 4 A and Fig. 4 C), promoted growth of TNBC xenografts, and its nucleolar expression is associated with poor outcome of AA women with TNBC [ 10 ]. Since we observed a correlation of CRYβB2 expression and CDK4 expression in primary TNBC tumors (Fig. 4 A and Fig. 4 C), we sought to address its relationship with ppRb expression and survival in a larger cohort of TNBC patients. The RB gene is mutated or lost in 20% of basal-like tumors [ 6 ]. Accordingly, we observed expression of ppRb in 85% (87/102) of TNBC cases from AA women. ppRb protein expression correlated with nucleolar CRYβB2 expression in TNBC patients (Fig. 5 A and Supplementary Fig. 3A ). Our analysis showed that 85% (95% CI: 77% − 92%) of TNBC from AA women were ppRb + (n = 102), with 46% having ppRb high and 39% ppRb low expression levels (Fig. 5 B). We detected ppRb expression in 94% (95% CI; 78% − 99%) of TNBC from AA patients that were never disease-free (n = 33), with 61% having a ppRb high and 33% ppRb low expression (Fig. 5 B). The expression of ppRb in AA-TNBC was associated with a significant decrease in disease-free (n = 87, P = 0.0002) and overall survival (n = 87, P = 0.0049) (Fig. 5 C and Supplementary Fig. 3B ). Most importantly, patients with TNBC tumors expressing both CRYβB2 and ppRb (double positive) showed a more significant decrease in disease-free ( P < 0.0001) and overall survival ( P = 0.0456) than patients with ppRb + /CRYβB2 − tumors (Fig. 5 D). Collectively, these data show that the CDK4/pRb pathway is active and associated with CRYβB2 and poor prognosis in AA-TNBC patients. Discussion The approval of CDK4/6i for women with HR + metastatic breast cancer has permanently changed the treatment paradigm of this disease [ 36 ], and their effect on TNBC is under investigation [ 37 ]. However, in reality, only a subset of treated patients respond [ 3 ]. That highlights the need for complementary or companion diagnostics to pinpoint potential responders. Presently, no clinically available biomarkers, other than ER- and PR-expression, are used to prescribe CDK4/6i for women with HR + metastatic breast cancer [ 2 ]. However, some patients with ER/PR positive tumors may still develop resistance to CDK4/6i, and research is ongoing to identify additional biomarkers that could further refine patient selection [ 2 ]. We showed that CRYβB2 is up-regulated in breast cancer of AA women, activates NCL, and mediates oncogenesis in TNBC [ 10 ]. In addition to regulation of protein synthesis, NCL induces malignancy by regulation of the cell cycle [ 22 , 38 ]. NCL expression has an impact on both pRb [ 26 ] and p53 [ 27 ]. It is involved in post-transcriptional inhibition of p53 [ 27 ]. Inactivation of NCL was shown to induce cell cycle arrest [ 22 , 38 ]. We observed a CRYβB2-based decrease of p53 protein and activation of NCL and the CDK4/pRb pathway, resulting in an expansion of tumor cells in the proliferative S phase of the cell cycle. These findings are in agreement with previous findings that CRYβB2 regulates CDK4 and cyclin D2 in ovarian cells [ 39 ]. CRYβB2- tumors were sensitive to inhibition of CDK4/6 by palbociclib. Importantly, palbociclib was ineffective in control tumors, which lacked detectable CRYβB2 expression, demonstrating the ability of CRYβB2 to sensitize breast tumor cells to CDK4/6i. In addition to activation of the CDK4/pRb pathway, we observed that CRYβB2 expression also correlated with response to palbociclib in TNBC and ER + cell lines. Similarly, ER-negative tumors from AA women showed higher expression and a correlation of CRYβB2 with CDK4 and ppRb expression. ER + tumors from AA women showed higher ppRb expression. Collectively, our data suggests that CRYβB2 can define a subgroup of patients with TNBC and ER + tumors which are more likely to activate the CDK4/pRb pathway and respond to CDK4/6i. We have previously shown that nucleolar, and to a lesser extent nuclear, CRYβB2 expression most effectively identifies the AA-TNBC patients who are less likely to survive [ 10 ]. Here, we show that high ppRb protein levels correlate with a worse TNBC outcome in AA women compared to ppRb-negative tumors, as its presence indicates increased cell proliferation due to the inactivation of the tumor suppressor function of pRb. In accord with the role of CRYβB2 in activation of CDK4/pRb, we observed that only ppRb + tumors with nucleolar CRYβB2 expression have a significant decrease in disease-free and overall survival. Conclusions CRYβB2 induces cell cycle progression in breast tumors through regulation of NCL and the CDK4/pRb pathway. CRYβB2 is overexpressed in ER-negative tumors of AA patients and can be used as a biomarker of disease outcome and response to CDK4/6i. Higher expression of ppRb in ER + tumors of AA women stratify this population as likely responders to CDK4/6i. Declarations Ethical Approval and Consent to participate : Primary tumors from women undergoing treatment were provided by the Johns Hopkins Surgical Pathology Department, under protocols approved by the institutional review board. All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the Johns Hopkins School of Medicine. Consent for publication : Not applicable. Availability of supporting data : Not applicable. Competing interests : The authors declare no competing interests. Funding : This work was funded by the DOD BCRP Center of Excellence Grant W81XWH-04-1-0595 to S.S, DOD BCRP, W81XWH-15-1-0017 to V.M and the Division of Nuclear Medicine and Molecular Imaging. Authors' contributions : Conception and experimental design: Y.Y., V.M., and S.S. Performed the experiments: Y.Y., A.N., V.M., M.M., H.S., G.L. Acquisition of data: V.M., Y.Y., Y.K., A.A., T.N., H.S., T.H., E.G., L.C., and S.S. Analysis and interpretation of data: V.M., M.P., and S.S. Writing and/or revision of the manuscript: V.M., M.P., and S.S. Study supervision: V.M., M.P., and S.S. Acknowledgements : The authors would like to acknowledge the contribution to this study from the Sidney Kimmel Cancer Center flow cytometry and confocal core facilities at Johns Hopkins. References Pernas S, Tolaney SM, Winer EP, Goel S. CDK4/6 inhibition in breast cancer: current practice and future directions. Therapeutic Adv Med Oncol. 2018;10:1758835918786451. Morrison L, Loibl S, Turner NC. The CDK4/6 inhibitor revolution - a game-changing era for breast cancer treatment. Nat Rev Clin Oncol. 2024;21(2):89–105. Schoninger SF, Blain SW. The Ongoing Search for Biomarkers of CDK4/6 Inhibitor Responsiveness in Breast Cancer. Mol Cancer Ther. 2020;19(1):3–12. Dean JL, McClendon AK, Hickey TE, Butler LM, Tilley WD, Witkiewicz AK, Knudsen ES. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors. Cell Cycle. 2012;11(14):2756–61. Karakas C, Biernacka A, Bui T, Sahin AA, Yi M, Akli S, Schafer J, Alexander A, Adjapong O, Hunt KK, et al. Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer. Am J Pathol. 2016;186(7):1900–12. Cancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490(7418):61–70. Asghar US, Barr AR, Cutts R, Beaney M, Babina I, Sampath D, Giltnane J, Lacap JA, Crocker L, Young A, et al. Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer. Clin Cancer Res. 2017;23(18):5561–72. Patel JM, Goss A, Garber JE, Torous V, Richardson ET, Haviland MJ, Hacker MR, Freeman GJ, Nalven T, Alexander B, et al. Retinoblastoma protein expression and its predictors in triple-negative breast cancer. NPJ Breast Cancer. 2020;6:19. Andreopoulou E, Kelly CM, McDaid HM. Therapeutic Advances and New Directions for Triple-Negative Breast Cancer. Breast care. 2017;12(1):21–8. Yan Y, Narayan A, Cho S, Cheng Z, Liu JO, Zhu H, Wang G, Wharram B, Lisok A, Brummet M et al. CRYbetaB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer. Oncogene 2021. Chen Z, Xu X. Roles of nucleolin. Focus on cancer and anti-cancer therapy. Saudi Med J. 2016;37(12):1312–8. Soundararajan S, Chen W, Spicer EK, Courtenay-Luck N, Fernandes DJ. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells. Cancer Res. 2008;68(7):2358–65. Bugler B, Caizergues-Ferrer M, Bouche G, Bourbon H, Amalric F. Detection and localization of a class of proteins immunologically related to a 100-kDa nucleolar protein. Eur J Biochem. 1982;128(2–3):475–80. Yang A, Shi G, Zhou C, Lu R, Li H, Sun L, Jin Y. Nucleolin maintains embryonic stem cell self-renewal by suppression of p53 protein-dependent pathway. J Biol Chem. 2011;286(50):43370–82. Mahotka C, Bhatia S, Kollet J, Grinstein E. Nucleolin promotes execution of the hematopoietic stem cell gene expression program. Leukemia. 2018;32(8):1865–8. Fonseca NA, Rodrigues AS, Rodrigues-Santos P, Alves V, Gregorio AC, Valerio-Fernandes A, Gomes-da-Silva LC, Rosa MS, Moura V, Ramalho-Santos J, et al. Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination. Biomaterials. 2015;69:76–88. Rosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN Jr., Harzstark AL, Wagle N, Figlin RA, Smith GW, Garraway LA, et al. A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma. Investig New Drugs. 2014;32(1):178–87. Van Nguyen F, Lardy-Cleaud A, Bray S, Chabaud S, Dubois T, Diot A, Thompson AM, Bourdon JC, Perol D, Bouvet P et al. Druggable Nucleolin Identifies Breast Tumours Associated with Poor Prognosis That Exhibit Different Biological Processes. Cancers 2018, 10(10). Merino VF, Nguyen N, Jin K, Sadik H, Cho S, Korangath P, Han L, Foster YMN, Zhou XC, Zhang Z, et al. Combined Treatment with Epigenetic, Differentiating, and Chemotherapeutic Agents Cooperatively Targets Tumor-Initiating Cells in Triple-Negative Breast Cancer. Cancer Res. 2016;76(7):2013–24. Foss CA, Kulik L, Ordonez AA, Jain SK, Michael Holers V, Thurman JM, Pomper MG. SPECT/CT Imaging of Mycobacterium tuberculosis Infection with [(125)I]anti-C3d mAb. Mol imaging biology. 2019;21(3):473–81. Merino VF, Cho S, Nguyen N, Sadik H, Narayan A, Talbot C Jr., Cope L, Zhou XC, Zhang Z, Gyorffy B, et al. Induction of cell cycle arrest and inflammatory genes by combined treatment with epigenetic, differentiating, and chemotherapeutic agents in triple-negative breast cancer. Breast cancer research: BCR. 2018;20(1):145. Xu Z, Joshi N, Agarwal A, Dahiya S, Bittner P, Smith E, Taylor S, Piwnica-Worms D, Weber J, Leonard JR. Knocking down nucleolin expression in gliomas inhibits tumor growth and induces cell cycle arrest. J Neurooncol. 2012;108(1):59–67. Sarcevic B, Lilischkis R, Sutherland RL. Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes. J Biol Chem. 1997;272(52):33327–37. Huang F, Wu Y, Tan H, Guo T, Zhang K, Li D, Tong Z. Phosphorylation of nucleolin is indispensable to its involvement in the proliferation and migration of non-small cell lung cancer cells. Oncol Rep. 2019;41(1):590–8. Belenguer P, Baldin V, Mathieu C, Prats H, Bensaid M, Bouche G, Amalric F. Protein kinase NII and the regulation of rDNA transcription in mammalian cells. Nucleic Acids Res. 1989;17(16):6625–36. Grinstein E, Shan Y, Karawajew L, Snijders PJ, Meijer CJ, Royer HD, Wernet P. Cell cycle-controlled interaction of nucleolin with the retinoblastoma protein and cancerous cell transformation. J Biol Chem. 2006;281(31):22223–35. Takagi M, Absalon MJ, McLure KG, Kastan MB. Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell. 2005;123(1):49–63. Mantovani F, Collavin L, Del Sal G. Mutant p53 as a guardian of the cancer cell. Cell Death Differ. 2019;26(2):199–212. Dawson PJ, Wolman SR, Tait L, Heppner GH, Miller FR. MCF10AT: a model for the evolution of cancer from proliferative breast disease. Am J Pathol. 1996;148(1):313–9. Miller FR, Santner SJ, Tait L, Dawson PJ. MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ. J Natl Cancer Inst. 2000;92(14):1185–6. Konishi H, Mohseni M, Tamaki A, Garay JP, Croessmann S, Karnan S, Ota A, Wong HY, Konishi Y, Karakas B, et al. Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells. Proc Natl Acad Sci USA. 2011;108(43):17773–8. Weiss MB, Vitolo MI, Mohseni M, Rosen DM, Denmeade SR, Park BH, Weber DJ, Bachman KE. Deletion of p53 in human mammary epithelial cells causes chromosomal instability and altered therapeutic response. Oncogene. 2010;29(33):4715–24. Croessmann S, Wong HY, Zabransky DJ, Chu D, Mendonca J, Sharma A, Mohseni M, Rosen DM, Scharpf RB, Cidado J, et al. NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability. Proc Natl Acad Sci USA. 2015;112(37):11583–8. Finn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, Ginther C, Atefi M, Chen I, Fowst C, et al. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast cancer research: BCR. 2009;11(5):R77. Bates PJ, Reyes-Reyes EM, Malik MT, Murphy EM, O'Toole MG, Trent JO. G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochim et Biophys acta Gen Subj. 2017;1861(5 Pt B):1414–28. Cristofanilli M, Turner NC, Bondarenko I, Ro J, Im SA, Masuda N, Colleoni M, DeMichele A, Loi S, Verma S, et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol. 2016;17(4):425–39. Matutino A, Amaro C, Verma S. CDK4/6 inhibitors in breast cancer: beyond hormone receptor-positive HER2-negative disease. Therapeutic Adv Med Oncol. 2018;10:1758835918818346. Ugrinova I, Monier K, Ivaldi C, Thiry M, Storck S, Mongelard F, Bouvet P. Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication. BMC Mol Biol. 2007;8:66. Gao Q, Sun LL, Xiang FF, Gao L, Jia Y, Zhang JR, Tao HB, Zhang JJ, Li WJ. Crybb2 deficiency impairs fertility in female mice. Biochem Biophys Res Commun. 2014;453(1):37–42. Additional Declarations No competing interests reported. Supplementary Files YanetalSupplementsBCR.docx YanBCRoriginalgelimagesSupplements.pdf Cite Share Download PDF Status: Published Journal Publication published 14 Jan, 2026 Read the published version in Breast Cancer Research → Version 1 posted Editorial decision: Revision requested 26 Sep, 2025 Reviews received at journal 20 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviews received at journal 18 Jul, 2025 Reviewers agreed at journal 03 Jul, 2025 Reviewers agreed at journal 01 Jul, 2025 Reviewers agreed at journal 30 Jun, 2025 Reviewers agreed at journal 02 May, 2025 Reviewers invited by journal 30 Apr, 2025 Editor assigned by journal 24 Apr, 2025 Submission checks completed at journal 24 Apr, 2025 First submitted to journal 11 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-6431382","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451284847,"identity":"1ab0164f-bef4-481e-aba3-8217cf520297","order_by":0,"name":"Yu Yan","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Yan","suffix":""},{"id":451284848,"identity":"989cd993-70bd-4100-ab67-f1868f32ba8f","order_by":1,"name":"Athira Narayan","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Athira","middleName":"","lastName":"Narayan","suffix":""},{"id":451284850,"identity":"e60d367b-13c2-4d99-b15f-961840f89d02","order_by":2,"name":"Marzieh Mazinani","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Marzieh","middleName":"","lastName":"Mazinani","suffix":""},{"id":451284852,"identity":"61d9d73e-abcf-43fc-9dc9-38ca519177a4","order_by":3,"name":"Harumi Saeki","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Harumi","middleName":"","lastName":"Saeki","suffix":""},{"id":451284853,"identity":"c89287ea-4704-4eec-ad1a-1dd91113b8a8","order_by":4,"name":"Tao Huang","email":"","orcid":"","institution":"Huazhong University of Science and Technology","correspondingAuthor":false,"prefix":"","firstName":"Tao","middleName":"","lastName":"Huang","suffix":""},{"id":451284854,"identity":"4efa795e-b4ba-4f8f-9ab0-7b68ab5ee48e","order_by":5,"name":"Gabi Lofland","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Gabi","middleName":"","lastName":"Lofland","suffix":""},{"id":451284855,"identity":"9005360c-1696-4d3f-b30c-13ff4810eacf","order_by":6,"name":"Edward Gabrielson","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Edward","middleName":"","lastName":"Gabrielson","suffix":""},{"id":451284856,"identity":"781a2c91-280c-428f-96a4-582381ce9552","order_by":7,"name":"Leslie Cope","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Leslie","middleName":"","lastName":"Cope","suffix":""},{"id":451284858,"identity":"4f061615-77f2-4a44-8833-2c01f21fa9bd","order_by":8,"name":"Yasmine Kanaan","email":"","orcid":"","institution":"Howard University","correspondingAuthor":false,"prefix":"","firstName":"Yasmine","middleName":"","lastName":"Kanaan","suffix":""},{"id":451284860,"identity":"702a5257-8bea-436b-bfca-6eb208a3f394","order_by":9,"name":"Ali Afsari","email":"","orcid":"","institution":"Howard University","correspondingAuthor":false,"prefix":"","firstName":"Ali","middleName":"","lastName":"Afsari","suffix":""},{"id":451284862,"identity":"bdfd833f-577b-4b68-9652-3086d9ed3ddc","order_by":10,"name":"Tammey Naab","email":"","orcid":"","institution":"Athari BioSciences","correspondingAuthor":false,"prefix":"","firstName":"Tammey","middleName":"","lastName":"Naab","suffix":""},{"id":451284863,"identity":"35ead4f1-02b4-4f12-9154-24b77c42c7aa","order_by":11,"name":"Saraswati Sukumar","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Saraswati","middleName":"","lastName":"Sukumar","suffix":""},{"id":451284864,"identity":"5dd1ed33-d1df-4a1c-af0d-137b37af16bc","order_by":12,"name":"Martin Pomper","email":"","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":false,"prefix":"","firstName":"Martin","middleName":"","lastName":"Pomper","suffix":""},{"id":451284866,"identity":"c595adb5-4fdd-4a43-bca0-2fea55cb8053","order_by":13,"name":"Vanessa Merino","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAvklEQVRIiWNgGAWjYLCCBAYGHn6GM2AGYwOxWmQkG0jSAgQ2Bgd4wAzCWgyuHT724eEOGx7jg2ePbnjAYCO74QAhLbfTkmcknknjMTtwLu1GAkOaMUEtkrNzjBkS2w4DtZwxA2o5nEiElvzPYC3GDWAt/wlr4ZfOYQZrMWAAazlAjJY0kMPSeCTADjNINp5JSAubdPJjxp9tNvb8M86Y3fxRYSfbR0gLAkiAlBoQrRzsxAaSlI+CUTAKRsEIAgANAkba2oWoaAAAAABJRU5ErkJggg==","orcid":"","institution":"Johns Hopkins University School of Medicine","correspondingAuthor":true,"prefix":"","firstName":"Vanessa","middleName":"","lastName":"Merino","suffix":""}],"badges":[],"createdAt":"2025-04-11 23:38:09","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6431382/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6431382/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13058-025-02188-2","type":"published","date":"2026-01-14T16:28:38+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":82177774,"identity":"197322fc-0f18-4bb1-9145-859bcdba607b","added_by":"auto","created_at":"2025-05-07 11:21:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":430117,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRYβB2 induces cell cycle progression and activation of CDK4/ pRb pathway in premalignant tumors and DCIS\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eFlow cytometry determination of cell cycle distribution in cells isolated from MCF10AT1- CRYβB2 and control xenografts and their distal mammary gland metastases. Western blot analysis and ImageJ quantification of proteins involved in cell cycle progression in: MCF10AT1 and DCIS.COM- CRYβB2 and control tumors (\u003cstrong\u003eB\u003c/strong\u003e); premalignant MCF10A-p53 null (KO) [32], p53-R248W knockin (KI) [33], and MCF10A-BRCA1-185delAG KI [31]cells (\u003cstrong\u003eC\u003c/strong\u003e); and MCF10AT1- CRYβB2 and vector cells, containing NCL wild-type or knockout (NCL-KO#1 and #2) (\u003cstrong\u003eD\u003c/strong\u003e). β-actin: loading control. * p \u0026lt; 0.05, ** p\u0026lt; 0.01 and *** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/2729207323665e5eb1b3a9d8.png"},{"id":82178886,"identity":"ea17e7b8-d762-4a3a-8ccf-3791a43f2c10","added_by":"auto","created_at":"2025-05-07 11:29:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":425003,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRYβB2 activates CDK4/ pRb pathway in ER\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e and TNBC breast tumors\u003c/strong\u003e. Western blot analysis of proteins involved in cell cycle progression in: Estrogen receptor (ER)\u003csup\u003e +\u003c/sup\u003e (\u003cstrong\u003eA\u003c/strong\u003e); triple negative breast cancer (TNBC) cells (\u003cstrong\u003eB\u003c/strong\u003e); and CRISPR engineered NCL knockout (NCL-KO#1 and #2), CRYβB2 knockdown (KD), and control (ctrl) TNBC (\u003cstrong\u003eC\u003c/strong\u003e) and ER\u003csup\u003e+\u003c/sup\u003e (\u003cstrong\u003eD\u003c/strong\u003e) from AA and EA women. Pearson correlation coefficient (r) and the p value (p) are shown. β-actin: loading control. * p \u0026lt; 0.05, ** p\u0026lt; 0.01 and *** p\u0026lt; 0.001.\u003cbr\u003e\n\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/6c97a3b6daa19da7d19f78a4.png"},{"id":82178887,"identity":"bd80ebae-95ec-4e07-95bb-e749215e1acd","added_by":"auto","created_at":"2025-05-07 11:29:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":477087,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRYβB2 induces sensitivity to CDK4/6 inhibitor\u003c/strong\u003e. \u003cstrong\u003eA\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eTumor volume and weight ± SEM of 5 mice per group containing MCF10AT1-CRYβB2 or vector control tumors., treated for 2 weeks (yellow bar) with vehicle or palbociclib (Palbo, 50 mg/kg, oral). \u003cstrong\u003eB\u003c/strong\u003e. Direct correlation of CRYβB2 protein expression and palbociclib IC50 nM [\u003ca href=\"#_ENREF_34\" title=\"Finn, 2009 #31\"\u003e34\u003c/a\u003e] in TNBC and ER\u003csup\u003e+\u003c/sup\u003e. Viability assay of TNBC and ER\u003csup\u003e+\u003c/sup\u003e control (ctrl) and CRYβB2 knockdown (KD) cells (\u003cstrong\u003eC\u003c/strong\u003e); and TNBC NCL knockout (KO) cells (\u003cstrong\u003eD\u003c/strong\u003e) treated for 48 hs with palbociclib (mM). \u003cstrong\u003eE\u003c/strong\u003e. Viability of TNBC (MDA-MB-231) and ER\u003csup\u003e+ \u003c/sup\u003e(MCF7) cells treated for 48hs with palbociclib (mM and nM, respectively) alone or in combination with the NCL aptamer AS-1411 (AS, mM). * p \u0026lt; 0.05, ** p\u0026lt; 0.01 and *** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/0216ff44d412e8ed5a675e21.png"},{"id":82178890,"identity":"3c0a927a-7504-4c5a-afe3-5278da674b41","added_by":"auto","created_at":"2025-05-07 11:29:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":399254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRYβB2 activates CDK4/ pRb pathway in ER\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e- \u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003eand ER\u003c/strong\u003e\u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003e\u003cstrong\u003e breast tumors\u003c/strong\u003e \u003cstrong\u003eof AA and EA women\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eRelative levels of CRYβB2 and cell cycle proteins in ER\u003csup\u003e-\u003c/sup\u003e tumors from AA and EA women (n=10 each) (\u003cstrong\u003eA\u003c/strong\u003e) and ER\u003csup\u003e+\u003c/sup\u003e tumors from AA and EA women (n=12 each) (\u003cstrong\u003eB\u003c/strong\u003e) and protein correlation (\u003cstrong\u003eC\u003c/strong\u003e). Arrows indicate hyper- (upper band) and hypo- (lower band) phosphorylated forms of pRb. * p \u0026lt; 0.05, ** p\u0026lt; 0.01 and *** p\u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/9910bba064b96cf9d9e6fa39.png"},{"id":82177779,"identity":"40f4fe03-c3a1-4af3-b3b8-1a1c1bcef641","added_by":"auto","created_at":"2025-05-07 11:21:55","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":436428,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCRYβB2 and ppRb associate with poor TNBC outcome in AA women\u003c/strong\u003e.\u003cstrong\u003e A\u003c/strong\u003e.\u003cstrong\u003e \u003c/strong\u003eCorrelation of nucleolar CRYβB2 and nuclear phosphorylated pRb (ppRb) protein IHC stain using a TNBC tissue microarray from AA women (n=102). The size of the dots represents the number of patients within each score. Pearson correlation coefficient (r) and the p value are shown. \u003cstrong\u003eB\u003c/strong\u003e. Distribution of ppRb negative, low and high tumors among AA-TNBC patients (n=102). DF: Disease-free, NDF: Never disease-free and Met: Metastatic. Kaplan Meier curves for disease-free and overall survival among AA- TNBC patients(n=86) according to ppRb (\u003cstrong\u003eC\u003c/strong\u003e) and both ppRb and nucleolar CRYβB2 staining intensity (positive: score 1-3; negative: score 0) (\u003cstrong\u003eD\u003c/strong\u003e).\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/4781fd062faa721e0725e9eb.jpeg"},{"id":100614352,"identity":"d7601880-822f-492d-a563-ce1bd7c9c71f","added_by":"auto","created_at":"2026-01-19 17:19:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3274895,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/b13a9989-6527-4ccd-837f-dbb013cb4fb5.pdf"},{"id":82177777,"identity":"e977d519-90aa-491f-82b3-afb08a2bcd1b","added_by":"auto","created_at":"2025-05-07 11:21:55","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":1898131,"visible":true,"origin":"","legend":"","description":"","filename":"YanetalSupplementsBCR.docx","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/c8844544c07b6080b58f2311.docx"},{"id":82178889,"identity":"d1fdbeb5-268b-476e-bcc7-689cdd31da10","added_by":"auto","created_at":"2025-05-07 11:29:55","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1267634,"visible":true,"origin":"","legend":"","description":"","filename":"YanBCRoriginalgelimagesSupplements.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6431382/v1/2e8cc111806e44c5ad9834cb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"CRYβB2 is a biomarker for poor prognosis and response to CDK4/6 inhibitors in breast cancer","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe addition of cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) to anti-hormonal therapy has become a first-line option for patients with the hormone receptor (HR)\u003csup\u003e+\u003c/sup\u003e/ human epidermal growth factor receptor 2 negative (HER2\u003csup\u003e\u0026minus;\u003c/sup\u003e) phenotype in the metastatic setting [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The cyclin D\u0026ndash;CDK4/6 complex phosphorylates the cell cycle repressor protein retinoblastoma 1 (RB1), reducing the inhibitory effects of RB1 on E2F-mediated transcription of cell cycle genes, leading to cell cycle progression [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The clinical use of CDK4/6i is confounded by the high individual variability in clinical response [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. As treatment paradigms become more complex, substantial interest has emerged in the prospective identification of patients who are most likely to derive maximum benefit from CDK4/6i, and those whose tumors might be intrinsically resistant to therapy \u003csup\u003e[2]\u003c/sup\u003e. Presently, no clinically available biomarkers, other than ER/PR expression, are used to prescribe CDK4/6i. It has been demonstrated that fully functional RB is required for the effective use of CDK4/6i in the clinic [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, not all RB\u003csup\u003e+\u003c/sup\u003e/ER\u003csup\u003e+\u003c/sup\u003e patients benefit from CDK4/6i therapy.\u003c/p\u003e \u003cp\u003eDue to frequent RB loss [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], TNBC patients are considered to be poor candidates for CDK inhibition [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. However, cell lines and mouse xenograft models reflecting the luminal androgen receptor (LAR) subtype of TNBC have been shown to respond to palbociclib [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, from 180 TNBC patient samples, 51% were found to be RB- positive; thus RB represents a relevant target for therapy \u003csup\u003e[8]\u003c/sup\u003e. TNBC has a notoriously poor prognosis and paucity of targeted therapies. As a result, even today, chemotherapy is the mainstay of treatment [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe showed that β-crystallin B2 (CRYβB2) induced the growth of xenografts of MCF10A breast cancer cells with a single hit mutation in the MAPK pathway [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CRYβB2 interacted with several proteins that regulate cell proliferation and invasion, including nucleolin (NCL), zinc finger protein 495 (ZNF495), acrosin binding protein (ACRBP), Growth factor receptor-bound protein 2 (GRB2), annexin A1 and A2 (ANXA1 and 2), drebrin-like (DBNL), and erythrocyte membrane protein band 4.1 like 2 (EPB41L2) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. NCL is a shuttling nucleolar protein, which induces malignancy by regulation of the cell cycle [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], apoptosis [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], cell proliferation [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], metastasis [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and stem cell maintenance [\u003cspan additionalcitationids=\"CR15\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. We observed that it mediates the tumorigenic effects of CRYβB2 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Notably, CRYβB2 sensitizes TNBC cells to NCL inhibition with the specific aptamer AS-1411 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Anti-NCL drugs have being evaluated as anticancer agents in phase II clinical trials [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. NCL was also shown to be an independent marker of prognosis in breast cancer [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHere we show that CRYβB2-interaction with NCL results in activation of the CDK4/6 pathway and predisposes breast tumors to management with CDK4/6i. NCL inhibition with the aptamer AS-1411 synergized with CDK4/6i. Finally, higher levels of CRYβB2 correlate with 1) higher activation of the CDK4/6 pathway in TNBC and ER\u003csup\u003e+\u003c/sup\u003e tumors of AA women, and 2) decreased survival of AA women with TNBC.\u003c/p\u003e"},{"header":"Methods","content":"\u003cp\u003e\u003cb\u003ePatient samples, Cell Lines and Reagents\u003c/b\u003e. Primary tumors from women undergoing treatment were provided by the Johns Hopkins Surgical Pathology Department, under protocols approved by the institutional review board. Tissue microarrays (TMAs) were provided by Dr. Naab and Dr. Kanaan from Howard University. Briefly, the TMAs were constructed by Pantomics (Fairfield, CA) using FFPE tumor blocks from primary TNBC (87) and axillary lymph nodes (15) from AA women [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Breast cancer cells were obtained from the American Type Culture Collection. MCF10A premalignant cells were obtained from Ben H. Park. MCF10AT1 and DCIS.COM cells were obtained from the Barbara Ann Karmanos Cancer Institute. Cells were authenticated using short tandem repeat (STR) profiling and tested for mycoplasma using MycoAlert PLUS mycoplasma detection kit (Lonza). Palbociclib was purchased from Selleck Chemicals. The nucleolin aptamer AS-1411 (5\u0026prime;-GGTGGTGGTGGTTGTGGTGGTGGTGG) and CRO control (5\u0026prime;-CCTCCTCCTCCTTCTCCTCCTCCTCC) were purchased from Integrated DNA technologies.\u003c/p\u003e \u003cp\u003e \u003cb\u003eConstructs\u003c/b\u003e. CRYβB2 coding sequence was cloned into a lentivirus vector (Addgene) using the Gateway Technology System (Thermo Fisher). MCF10A, MCF10AT1 and DCIS.COM cells overexpressing CRYβB2 were generated as previous described [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. For immunofluorescence, MCF10AT1 cells were infected with lentivirus containing the CRYβB2 sequence tagged with the myc-DDK (flag) sequence (Origene). For CRISPR knockout, nucleolin and CRYβB2 guide RNAs were designed using sgRNA online web page from Broad Institute and cloned into Lenticrispr V2 (Addgene) [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. 293T cells were transfected with the lentivirus constructs using Lipofectamine and virus were used to infect cancer cells [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e\u003cb\u003eXenograft\u003c/b\u003e. All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the Johns Hopkins School of Medicine. Xenografts of MCF10AT1 cells expressing vector control and CRYβB2 constructs were established in 6\u0026ndash;8 weeks NOD-SCID IL2Rgnull (NSG) mice (from an in-house colony at Hopkins) by injecting 5x10\u003csup\u003e6\u003c/sup\u003e tumor cells into the fourth mammary gland. Mice bearing MCF10AT1 tumors were treated for 2 weeks, receiving palbociclib (50 mg/kg) or saline (pH 4.0) as vehicle for 5 days/ week orally.\u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern blot, Immunohistochemistry and Immunofluorescence\u003c/b\u003e. Western blot, immunohistochemistry (IHC) [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e] and immunofluorescence (IF) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e] were performed as previously described using antibodies against CRYβB2 and cell cycle proteins (Supplementary Methods). For IF, the slides were probed with the following primary antibodies: CRYβB2 (Thermo Fisher), CRYBB2-myc DDK flag (Cell Signaling Technology), and nuclear staining (Hoechst; Fisher Scientific) [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. ImageJ was used for quantification.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eCell cycle and proliferation analysis\u003c/strong\u003e \u003cp\u003eFor cell cycle determinations tumors were digested with collagenase/ hyaluronidase [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Tumor-derived cells were permeabilized with cold 70% ethanol and stained with propidium iodide (Sigma). Samples were run on the BD FACSCalibur system (Becton Dickinson) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. MTT (thiazolyl blue tetrazolium bromide, Amresco, #0793) solution (0.8 \u0026micro;g/\u0026micro;L) was used to measure cell proliferation after 48h of drug treatment [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Values are expressed as percent survival of the vehicle treated control (given as 100%).\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eStatistical Analysis\u003c/b\u003e. Two-tailed Mann Whitney Test and Student\u0026rsquo;s T-tests were performed on pairwise combinations of data to determine statistical significance defined as P\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Statistical analyses were performed using GraphPad Prism version 8.3.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e \u003cb\u003eCRYβB2 induces cell cycle progression and regulates the CDK4/6 pathway in premalignant and ductal carcinoma in situ (DCIS) models\u003c/b\u003e \u003c/p\u003e \u003cp\u003eWe showed that CRYβB2 induced tumorigenesis of breast cells with low malignant potential, and its knockdown (KD) decreased TNBC growth in immunodeficient mice[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. CRYβB2 interacted with, and induced NCL, leading to activation of AKT and EGFR with silencing of p53 [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Decrease of NCL expression was shown to reduce proliferation of glioblastoma cells, and induced cell cycle arrest [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. NCL is a substrate for CDKs [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Extensive NCL phosphorylation occurs during the cell cycle, regulating its functions and localization [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. NCL associates with two major cellular tumor suppressors, pRb [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and p53 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], and is involved in post-transcriptional inhibition of p53 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The p53 pathway has been shown to mediate cellular stress responses, initiate DNA repair, cell-cycle arrest, senescence and apoptosis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe MCF10 model is a series of cell lines that originated from the human breast epithelial cells, MCF10A [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. MCF10AT1 is a premalignant cell line produced by transfection of MCF10A with constitutively active HRAS [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Approximately 25% of the MCF10AT1 cells transplanted into immunodeficient mice progressed to invasive ductal carcinoma (IDC) after a long latency, which indicated low tumorigenic potential of MCF10AT1 with slow progression [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. MCF10DCIS.com is a cell line cloned from cell culture of an MCF10AT1 xenograft lesion. DCIS.com cells reproducibly form DCIS-like comedo lesions that spontaneously progress over time to IDC when grown as xenografts in immunodeficient mice [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Previously, we showed that CRYβB2 hastened the growth of MCF10AT1 and DCIS.com tumors [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Highly proliferative MCF10AT1-CRYβB2 tumors and associated metastases showed an increase in cell cycle progression in comparison to MCF10AT1-vec controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In line with this observation, MCF10AT1 and DCIS.COM tumors overexpressing CRYβB2 showed an increase in cell cycle mediators, including CDK4, cell division cycle 25 A (Cdc25a), and phosphorylated pRb (ppRb) proteins (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). CRYβB2 overexpression resulted in upregulation of p53 expression in MCF10AT1 tumors. But p53 was downregulated in DCIS.com tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB)[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] and in premalignant MCF10A-BRCA1-185delAG knockin (KI) mutant cells [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). The expression of cell cycle proteins were also increased by overexpression of CRYβB2 in MCF10A-BRCA1-KI [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e] and MCF10A-p53 knockout (KO) cells [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). A lower effect of CRYβB2 induction of cell cycle proteins was observed in MCF10A-p53-R248W KI cells [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], which have higher endogenous levels of p53 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and \u003cb\u003eSupplementary Fig.\u0026nbsp;1A\u003c/b\u003e). These data suggest that p53 may restrict CRYβB2-dependent activation of cell cycle progression.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFurther analysis of the effects of knockout of NCL in MCF10AT1 cells showed that reduced NCL expression impaired the CRYβB2-dependent activation of the CDK4/6 pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). No effect on the expression of cell cycle proteins, except a slight decrease of ppRb, was observed by NCL deficiency in MCF10AT1- vector cells, which lack CRYβB2 expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and \u003cb\u003eSupplementary Fig.\u0026nbsp;1B\u003c/b\u003e). We found that CRYβB2 co-localized with ppRb (\u003cb\u003eSupplementary Fig.\u0026nbsp;1C\u003c/b\u003e) and is mutually exclusively expressed with p53 (\u003cb\u003eSupplementary Fig.\u0026nbsp;1D\u003c/b\u003e) in the nucleus of MCF10AT1 cells. Collectively, these results suggest that CRYβB2 may recruit NCL to activate the CDK4/pRb pathway, and induce cell cycle progression in premalignant and early-stage breast cancer.\u003c/p\u003e\n\u003ch3\u003eCRYβB2 regulates CDK4/6 pathway in ER positive (ER) and TNBC cells\u003c/h3\u003e\n\u003cp\u003eSince we observed CRYβB2-mediated regulation of CDK4/pRb in early breast cancer models, we investigated its effect on regulation of cell cycle proteins in models of established tumors. We found that, similar to the premalignant models, CRYβB2 and NCL expression correlated with CDK4/pRb activation in ER\u003csup\u003e+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA) and TNBC (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB) cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe next evaluated the effect of downregulation of CRYβB2 and NCL expression in breast cancer cells on activation of the CDK4/6 pathway. Knockdown of CRYβB2 and NCL in AA (MDA-MB-157) and EA (SUM-159 and HCC-1806) TNBC cells resulted in decreased levels of proteins of the CDK4/pRb pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). We observed a decrease of total pRb in AA ER\u003csup\u003e+\u003c/sup\u003e (MDAMB-175) CRYβB2- KD cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD). Collectively, these results suggest that, in established breast cancer cell lines, CRYβB2 may recruit NCL to activate CDK4/pRb pathway and induce cell cycle progression.\u003c/p\u003e\n\u003ch3\u003eCRYβB2 expression correlates to the response of tumor xenografts to CDK4/6 inhibitors\u003c/h3\u003e\n\u003cp\u003eWe observed CRYβB2-dependent regulation of CDK4/pRb pathway in both premalignant and malignant ER\u003csup\u003e+\u003c/sup\u003e and TNBC breast cancer models. We next evaluated the effect of induction and downregulation of CRYβB2 and NCL in breast cancer cells on tumor response to CDK4/6i.\u003c/p\u003e \u003cp\u003eWe observed that the growth of MCF10AT1-CRYβB2 tumors was significantly decreased by treatment of tumor-bearing mice with palbociclib compared to vehicle (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Strikingly, MCF10AT1-vector tumors, which lacked detectable CRYβB2 expression did not respond significantly to palbociclib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB, expression of CRYβB2 protein correlated inversely with the reported IC50 of palbociclib [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e] in both TNBC and ER\u003csup\u003e+\u003c/sup\u003e cells. Knockdown of CRYβB2 in TNBC and ER\u003csup\u003e+\u003c/sup\u003e cells decreased response to palbociclib (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). These data suggest that CRYβB2 expression in breast tumor cells enhances their sensitivity to CDK4/6i.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSince NCL mediates CRYβB2-activation of CDK4/pRb pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD and Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC\u003cb\u003e)\u003c/b\u003e, we determined if genetic and pharmacological decrease of NCL expression alters response to CDK4/6i. TNBC NCL-KO cells are more sensitive to palbociclib in comparison to vector control cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). ER\u003csup\u003e+\u003c/sup\u003e cell lines were described to be most sensitive to growth inhibition by CDK4/6i while TNBC cells were most resistant [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. A combination of palbociclib with the NCL aptamer AS-1411[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] significantly decreased TNBC and ER\u003csup\u003e+\u003c/sup\u003e tumor cell viability in comparison to single agents (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE). Collectively, these results suggest that CRYβB2 is a biomarker of response to CDK4/6i. A clinically available aptamer [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], targeting NCL synergizes with the cytotoxic effect of CDK4/6i.\u003c/p\u003e \u003cp\u003e \u003cb\u003eExpression of the CRYβB2 and CDK4/pRb pathways correlates in ER\u003c/b\u003e \u003csup\u003e \u003cb\u003e\u0026minus;\u003c/b\u003e \u003c/sup\u003e \u003cb\u003eand ER\u003c/b\u003e\u003csup\u003e\u003cb\u003e+\u003c/b\u003e\u003c/sup\u003e \u003cb\u003etumors of AA women\u003c/b\u003e\u003c/p\u003e \u003cp\u003eWe next investigated if the preclinical findings could be corroborated with clinical samples. We confirmed that CRYβB2 expression is higher in ER\u003csup\u003e\u0026minus;\u003c/sup\u003e tumors of AA women in comparison to EA women [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). CDK4 expression was also higher in ER\u003csup\u003e\u0026minus;\u003c/sup\u003e tumors of AA women and its expression correlated with CRYβB2 expression in tumors of AA women (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA, Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and \u003cb\u003eSupplementary Fig.\u0026nbsp;2A\u003c/b\u003e). The expression of ppRb (upper band) tended to be higher in AA ER\u003csup\u003e\u0026minus;\u003c/sup\u003e tumors (\u003cb\u003eSupplementary Fig.\u0026nbsp;2B\u003c/b\u003e and \u003cb\u003eSupplementary Fig.\u0026nbsp;2C\u003c/b\u003e). CRYβB2 expression tended to be higher and ppRb is significantly higher in ER\u003csup\u003e+\u003c/sup\u003e tumors of AA women in comparison to EA women (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). These data showed that CRYβB2 is a potential biomarker of CDK4/ pRb pathway activation in TNBC of AA women. The increase of ppRb in ER\u003csup\u003e+\u003c/sup\u003e tumors of AA women may stratify this population as likely responders to CDK4/6i.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eCRYβB2 and ppRb expression are associated with poor TNBC outcome in AA women\u003c/h3\u003e\n\u003cp\u003eCRYβB2 is overexpressed in ER-negative tumors from AA patients in comparison to EA women [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), promoted growth of TNBC xenografts, and its nucleolar expression is associated with poor outcome of AA women with TNBC [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Since we observed a correlation of CRYβB2 expression and CDK4 expression in primary TNBC tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC), we sought to address its relationship with ppRb expression and survival in a larger cohort of TNBC patients.\u003c/p\u003e \u003cp\u003eThe RB gene is mutated or lost in 20% of basal-like tumors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Accordingly, we observed expression of ppRb in 85% (87/102) of TNBC cases from AA women. ppRb protein expression correlated with nucleolar CRYβB2 expression in TNBC patients (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA and \u003cb\u003eSupplementary Fig.\u0026nbsp;3A\u003c/b\u003e). Our analysis showed that 85% (95% CI: 77% \u0026minus;\u0026thinsp;92%) of TNBC from AA women were ppRb\u003csup\u003e+\u003c/sup\u003e (n\u0026thinsp;=\u0026thinsp;102), with 46% having ppRb high and 39% ppRb low expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). We detected ppRb expression in 94% (95% CI; 78% \u0026minus;\u0026thinsp;99%) of TNBC from AA patients that were never disease-free (n\u0026thinsp;=\u0026thinsp;33), with 61% having a ppRb high and 33% ppRb low expression (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe expression of ppRb in AA-TNBC was associated with a significant decrease in disease-free (n\u0026thinsp;=\u0026thinsp;87, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0002) and overall survival (n\u0026thinsp;=\u0026thinsp;87, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0049) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cb\u003eSupplementary Fig.\u0026nbsp;3B\u003c/b\u003e). Most importantly, patients with TNBC tumors expressing both CRYβB2 and ppRb (double positive) showed a more significant decrease in disease-free (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001) and overall survival (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.0456) than patients with ppRb\u003csup\u003e+\u003c/sup\u003e/CRYβB2\u003csup\u003e\u0026minus;\u003c/sup\u003e tumors (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). Collectively, these data show that the CDK4/pRb pathway is active and associated with CRYβB2 and poor prognosis in AA-TNBC patients.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe approval of CDK4/6i for women with HR\u003csup\u003e+\u003c/sup\u003e metastatic breast cancer has permanently changed the treatment paradigm of this disease [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], and their effect on TNBC is under investigation [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. However, in reality, only a subset of treated patients respond [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. That highlights the need for complementary or companion diagnostics to pinpoint potential responders. Presently, no clinically available biomarkers, other than ER- and PR-expression, are used to prescribe CDK4/6i for women with HR\u003csup\u003e+\u003c/sup\u003e metastatic breast cancer [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. However, some patients with ER/PR positive tumors may still develop resistance to CDK4/6i, and research is ongoing to identify additional biomarkers that could further refine patient selection [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eWe showed that CRYβB2 is up-regulated in breast cancer of AA women, activates NCL, and mediates oncogenesis in TNBC [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. In addition to regulation of protein synthesis, NCL induces malignancy by regulation of the cell cycle [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. NCL expression has an impact on both pRb [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and p53 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. It is involved in post-transcriptional inhibition of p53 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Inactivation of NCL was shown to induce cell cycle arrest [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We observed a CRYβB2-based decrease of p53 protein and activation of NCL and the CDK4/pRb pathway, resulting in an expansion of tumor cells in the proliferative S phase of the cell cycle. These findings are in agreement with previous findings that CRYβB2 regulates CDK4 and cyclin D2 in ovarian cells [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. CRYβB2- tumors were sensitive to inhibition of CDK4/6 by palbociclib. Importantly, palbociclib was ineffective in control tumors, which lacked detectable CRYβB2 expression, demonstrating the ability of CRYβB2 to sensitize breast tumor cells to CDK4/6i. In addition to activation of the CDK4/pRb pathway, we observed that CRYβB2 expression also correlated with response to palbociclib in TNBC and ER\u003csup\u003e+\u003c/sup\u003e cell lines. Similarly, ER-negative tumors from AA women showed higher expression and a correlation of CRYβB2 with CDK4 and ppRb expression. ER\u003csup\u003e+\u003c/sup\u003e tumors from AA women showed higher ppRb expression. Collectively, our data suggests that CRYβB2 can define a subgroup of patients with TNBC and ER\u003csup\u003e+\u003c/sup\u003e tumors which are more likely to activate the CDK4/pRb pathway and respond to CDK4/6i.\u003c/p\u003e \u003cp\u003eWe have previously shown that nucleolar, and to a lesser extent nuclear, CRYβB2 expression most effectively identifies the AA-TNBC patients who are less likely to survive [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Here, we show that high ppRb protein levels correlate with a worse TNBC outcome in AA women compared to ppRb-negative tumors, as its presence indicates increased cell proliferation due to the inactivation of the tumor suppressor function of pRb. In accord with the role of CRYβB2 in activation of CDK4/pRb, we observed that only ppRb\u003csup\u003e+\u003c/sup\u003e tumors with nucleolar CRYβB2 expression have a significant decrease in disease-free and overall survival.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eCRYβB2 induces cell cycle progression in breast tumors through regulation of NCL and the CDK4/pRb pathway. CRYβB2 is overexpressed in ER-negative tumors of AA patients and can be used as a biomarker of disease outcome and response to CDK4/6i. Higher expression of ppRb in ER\u003csup\u003e+\u003c/sup\u003e tumors of AA women stratify this population as likely responders to CDK4/6i.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e:\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003ePrimary tumors from women undergoing treatment were provided by the Johns Hopkins Surgical Pathology Department, under protocols approved by the institutional review board. All animal studies were performed according to the guidelines and approval of the Animal Care Committee of the Johns Hopkins School of Medicine.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of supporting data\u003c/strong\u003e: Not applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e: The authors declare no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e: This work was funded by the DOD BCRP Center of Excellence Grant W81XWH-04-1-0595 to S.S, DOD BCRP, W81XWH-15-1-0017 to V.M and the Division of Nuclear Medicine and Molecular Imaging.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e:\u0026nbsp;Conception and experimental design: Y.Y., V.M., and S.S. Performed the experiments: Y.Y., A.N., V.M., M.M., H.S., G.L. Acquisition of data: V.M., Y.Y., Y.K., A.A., T.N., H.S., T.H., E.G., L.C., and S.S. Analysis and interpretation of data: V.M., M.P., and S.S. Writing and/or revision of the manuscript: V.M., M.P., and S.S. Study supervision: V.M., M.P., and S.S.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e: The authors would like to acknowledge the contribution to this study from the Sidney Kimmel Cancer Center flow cytometry and confocal core facilities at Johns Hopkins.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003ePernas S, Tolaney SM, Winer EP, Goel S. CDK4/6 inhibition in breast cancer: current practice and future directions. Therapeutic Adv Med Oncol. 2018;10:1758835918786451.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMorrison L, Loibl S, Turner NC. The CDK4/6 inhibitor revolution - a game-changing era for breast cancer treatment. Nat Rev Clin Oncol. 2024;21(2):89\u0026ndash;105.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchoninger SF, Blain SW. The Ongoing Search for Biomarkers of CDK4/6 Inhibitor Responsiveness in Breast Cancer. Mol Cancer Ther. 2020;19(1):3\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDean JL, McClendon AK, Hickey TE, Butler LM, Tilley WD, Witkiewicz AK, Knudsen ES. Therapeutic response to CDK4/6 inhibition in breast cancer defined by ex vivo analyses of human tumors. Cell Cycle. 2012;11(14):2756\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarakas C, Biernacka A, Bui T, Sahin AA, Yi M, Akli S, Schafer J, Alexander A, Adjapong O, Hunt KK, et al. Cytoplasmic Cyclin E and Phospho-Cyclin-Dependent Kinase 2 Are Biomarkers of Aggressive Breast Cancer. Am J Pathol. 2016;186(7):1900\u0026ndash;12.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCancer Genome Atlas N. Comprehensive molecular portraits of human breast tumours. Nature. 2012;490(7418):61\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAsghar US, Barr AR, Cutts R, Beaney M, Babina I, Sampath D, Giltnane J, Lacap JA, Crocker L, Young A, et al. Single-Cell Dynamics Determines Response to CDK4/6 Inhibition in Triple-Negative Breast Cancer. Clin Cancer Res. 2017;23(18):5561\u0026ndash;72.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePatel JM, Goss A, Garber JE, Torous V, Richardson ET, Haviland MJ, Hacker MR, Freeman GJ, Nalven T, Alexander B, et al. Retinoblastoma protein expression and its predictors in triple-negative breast cancer. NPJ Breast Cancer. 2020;6:19.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAndreopoulou E, Kelly CM, McDaid HM. Therapeutic Advances and New Directions for Triple-Negative Breast Cancer. Breast care. 2017;12(1):21\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYan Y, Narayan A, Cho S, Cheng Z, Liu JO, Zhu H, Wang G, Wharram B, Lisok A, Brummet M et al. CRYbetaB2 enhances tumorigenesis through upregulation of nucleolin in triple negative breast cancer. Oncogene 2021.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen Z, Xu X. Roles of nucleolin. Focus on cancer and anti-cancer therapy. Saudi Med J. 2016;37(12):1312\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSoundararajan S, Chen W, Spicer EK, Courtenay-Luck N, Fernandes DJ. The nucleolin targeting aptamer AS1411 destabilizes Bcl-2 messenger RNA in human breast cancer cells. Cancer Res. 2008;68(7):2358\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBugler B, Caizergues-Ferrer M, Bouche G, Bourbon H, Amalric F. Detection and localization of a class of proteins immunologically related to a 100-kDa nucleolar protein. Eur J Biochem. 1982;128(2\u0026ndash;3):475\u0026ndash;80.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang A, Shi G, Zhou C, Lu R, Li H, Sun L, Jin Y. Nucleolin maintains embryonic stem cell self-renewal by suppression of p53 protein-dependent pathway. J Biol Chem. 2011;286(50):43370\u0026ndash;82.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMahotka C, Bhatia S, Kollet J, Grinstein E. Nucleolin promotes execution of the hematopoietic stem cell gene expression program. Leukemia. 2018;32(8):1865\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFonseca NA, Rodrigues AS, Rodrigues-Santos P, Alves V, Gregorio AC, Valerio-Fernandes A, Gomes-da-Silva LC, Rosa MS, Moura V, Ramalho-Santos J, et al. Nucleolin overexpression in breast cancer cell sub-populations with different stem-like phenotype enables targeted intracellular delivery of synergistic drug combination. Biomaterials. 2015;69:76\u0026ndash;88.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRosenberg JE, Bambury RM, Van Allen EM, Drabkin HA, Lara PN Jr., Harzstark AL, Wagle N, Figlin RA, Smith GW, Garraway LA, et al. A phase II trial of AS1411 (a novel nucleolin-targeted DNA aptamer) in metastatic renal cell carcinoma. Investig New Drugs. 2014;32(1):178\u0026ndash;87.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVan Nguyen F, Lardy-Cleaud A, Bray S, Chabaud S, Dubois T, Diot A, Thompson AM, Bourdon JC, Perol D, Bouvet P et al. Druggable Nucleolin Identifies Breast Tumours Associated with Poor Prognosis That Exhibit Different Biological Processes. \u003cem\u003eCancers\u003c/em\u003e 2018, 10(10).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerino VF, Nguyen N, Jin K, Sadik H, Cho S, Korangath P, Han L, Foster YMN, Zhou XC, Zhang Z, et al. Combined Treatment with Epigenetic, Differentiating, and Chemotherapeutic Agents Cooperatively Targets Tumor-Initiating Cells in Triple-Negative Breast Cancer. Cancer Res. 2016;76(7):2013\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFoss CA, Kulik L, Ordonez AA, Jain SK, Michael Holers V, Thurman JM, Pomper MG. SPECT/CT Imaging of Mycobacterium tuberculosis Infection with [(125)I]anti-C3d mAb. Mol imaging biology. 2019;21(3):473\u0026ndash;81.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMerino VF, Cho S, Nguyen N, Sadik H, Narayan A, Talbot C Jr., Cope L, Zhou XC, Zhang Z, Gyorffy B, et al. Induction of cell cycle arrest and inflammatory genes by combined treatment with epigenetic, differentiating, and chemotherapeutic agents in triple-negative breast cancer. Breast cancer research: BCR. 2018;20(1):145.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu Z, Joshi N, Agarwal A, Dahiya S, Bittner P, Smith E, Taylor S, Piwnica-Worms D, Weber J, Leonard JR. Knocking down nucleolin expression in gliomas inhibits tumor growth and induces cell cycle arrest. J Neurooncol. 2012;108(1):59\u0026ndash;67.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSarcevic B, Lilischkis R, Sutherland RL. Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes. J Biol Chem. 1997;272(52):33327\u0026ndash;37.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHuang F, Wu Y, Tan H, Guo T, Zhang K, Li D, Tong Z. Phosphorylation of nucleolin is indispensable to its involvement in the proliferation and migration of non-small cell lung cancer cells. Oncol Rep. 2019;41(1):590\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBelenguer P, Baldin V, Mathieu C, Prats H, Bensaid M, Bouche G, Amalric F. Protein kinase NII and the regulation of rDNA transcription in mammalian cells. Nucleic Acids Res. 1989;17(16):6625\u0026ndash;36.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGrinstein E, Shan Y, Karawajew L, Snijders PJ, Meijer CJ, Royer HD, Wernet P. Cell cycle-controlled interaction of nucleolin with the retinoblastoma protein and cancerous cell transformation. J Biol Chem. 2006;281(31):22223\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTakagi M, Absalon MJ, McLure KG, Kastan MB. Regulation of p53 translation and induction after DNA damage by ribosomal protein L26 and nucleolin. Cell. 2005;123(1):49\u0026ndash;63.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMantovani F, Collavin L, Del Sal G. Mutant p53 as a guardian of the cancer cell. Cell Death Differ. 2019;26(2):199\u0026ndash;212.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDawson PJ, Wolman SR, Tait L, Heppner GH, Miller FR. MCF10AT: a model for the evolution of cancer from proliferative breast disease. Am J Pathol. 1996;148(1):313\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller FR, Santner SJ, Tait L, Dawson PJ. MCF10DCIS.com xenograft model of human comedo ductal carcinoma in situ. J Natl Cancer Inst. 2000;92(14):1185\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKonishi H, Mohseni M, Tamaki A, Garay JP, Croessmann S, Karnan S, Ota A, Wong HY, Konishi Y, Karakas B, et al. Mutation of a single allele of the cancer susceptibility gene BRCA1 leads to genomic instability in human breast epithelial cells. Proc Natl Acad Sci USA. 2011;108(43):17773\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWeiss MB, Vitolo MI, Mohseni M, Rosen DM, Denmeade SR, Park BH, Weber DJ, Bachman KE. Deletion of p53 in human mammary epithelial cells causes chromosomal instability and altered therapeutic response. Oncogene. 2010;29(33):4715\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCroessmann S, Wong HY, Zabransky DJ, Chu D, Mendonca J, Sharma A, Mohseni M, Rosen DM, Scharpf RB, Cidado J, et al. NDRG1 links p53 with proliferation-mediated centrosome homeostasis and genome stability. Proc Natl Acad Sci USA. 2015;112(37):11583\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFinn RS, Dering J, Conklin D, Kalous O, Cohen DJ, Desai AJ, Ginther C, Atefi M, Chen I, Fowst C, et al. PD 0332991, a selective cyclin D kinase 4/6 inhibitor, preferentially inhibits proliferation of luminal estrogen receptor-positive human breast cancer cell lines in vitro. Breast cancer research: BCR. 2009;11(5):R77.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBates PJ, Reyes-Reyes EM, Malik MT, Murphy EM, O'Toole MG, Trent JO. G-quadruplex oligonucleotide AS1411 as a cancer-targeting agent: Uses and mechanisms. Biochim et Biophys acta Gen Subj. 2017;1861(5 Pt B):1414\u0026ndash;28.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCristofanilli M, Turner NC, Bondarenko I, Ro J, Im SA, Masuda N, Colleoni M, DeMichele A, Loi S, Verma S, et al. Fulvestrant plus palbociclib versus fulvestrant plus placebo for treatment of hormone-receptor-positive, HER2-negative metastatic breast cancer that progressed on previous endocrine therapy (PALOMA-3): final analysis of the multicentre, double-blind, phase 3 randomised controlled trial. Lancet Oncol. 2016;17(4):425\u0026ndash;39.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMatutino A, Amaro C, Verma S. CDK4/6 inhibitors in breast cancer: beyond hormone receptor-positive HER2-negative disease. Therapeutic Adv Med Oncol. 2018;10:1758835918818346.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eUgrinova I, Monier K, Ivaldi C, Thiry M, Storck S, Mongelard F, Bouvet P. Inactivation of nucleolin leads to nucleolar disruption, cell cycle arrest and defects in centrosome duplication. BMC Mol Biol. 2007;8:66.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao Q, Sun LL, Xiang FF, Gao L, Jia Y, Zhang JR, Tao HB, Zhang JJ, Li WJ. Crybb2 deficiency impairs fertility in female mice. Biochem Biophys Res Commun. 2014;453(1):37\u0026ndash;42.\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":"breast-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brcr","sideBox":"Learn more about [Breast Cancer Research](http://breast-cancer-research.biomedcentral.com)","snPcode":"13058","submissionUrl":"https://submission.nature.com/new-submission/13058/3","title":"Breast Cancer Research","twitterHandle":"@BCRJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"European American, African American, CRYβB2, nucleolin, CDK4, pRb, palbociclib","lastPublishedDoi":"10.21203/rs.3.rs-6431382/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6431382/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eHazard rates of breast cancer death are significantly higher for women of African American (AA) origin compared with European American (EA) and the molecular mechanisms underlying this difference in outcome are understudied. Previously, we showed that β-crystallin B2 (CRYβB2) expression is up-regulated in breast cancer of AA women, activates nucleolin (NCL), and mediates oncogenesis in triple negative breast cancer (TNBC). Presently no biomarkers, other than estrogen receptor (ER)/ progesterone receptor (PR) expression, are used to prescribe cyclin-dependent kinases 4/6 inhibitors (CDK4/6i) for women with hormone receptor positive (HR\u003csup\u003e+\u003c/sup\u003e) metastatic breast cancer.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eWestern blot and flow cytometry was used to determine the activation of CDK4/6 pathway and cell cycle of cells isolated from CRYβB2 overexpressing tumors, respectively. Response to CDK4/6i was determined following treatment of mice containing control and CRYβB2- overexpressing tumors and TNBC and ER\u003csup\u003e+\u003c/sup\u003e cells. The correlation of CRYβB2 expression with CDK4/6 activation and survival was determined by Western blot, immunohistochemistry, and Kaplan Meier curves using TNBC and ER\u003csup\u003e+\u003c/sup\u003e tumors from AA and EA women.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eHere, we report that tumors overexpressing CRYβB2 showed an increase in cell cycle progression and activation of the CDK4/6 pathway in models of premalignant- and ductal carcinoma in situ (DCIS) lesions, and TNBC and ER\u003csup\u003e+\u003c/sup\u003e breast cancer cells. Targeting CRYβB2 and NCL resulted in lower levels of CDK4, cell division cycle 25 A (Cdc25a), and phosphorylated retinoblastoma (ppRb). Only tumors expressing CRYβB2 showed growth inhibition by the CDK4/6i, palbociclib. The expression of CRYβB2 protein inversely correlated with the IC50 of palbociclib in both TNBC and ER\u003csup\u003e+\u003c/sup\u003e cells. In accord with this, CRYβB2 knockdown in TNBC and ER\u003csup\u003e+\u003c/sup\u003e cells conferred greater resistance to inhibition with palbociclib. Further, the NCL aptamer AS-1411 sensitized TNBC and ER\u003csup\u003e+\u003c/sup\u003e cells to palbociclib. Higher levels of CRYβB2 expression in TNBC and ER\u003csup\u003e+\u003c/sup\u003e tumors of AA patients correlated with higher CDK4/pRb activation. Expression of both CRYβB2 and ppRb correlated with decreased survival in AA women with TNBC.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eCRYβB2 expression correlated with CDK4/6 activation and response to CDK4/6i, and may be useful as a biomarker of prognosis and response to palbociclib therapy.\u003c/p\u003e","manuscriptTitle":"CRYβB2 is a biomarker for poor prognosis and response to CDK4/6 inhibitors in breast cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-07 11:21:50","doi":"10.21203/rs.3.rs-6431382/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-09-26T16:50:16+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-20T18:03:30+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T23:12:31+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-07-18T19:01:37+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"251906353048577023489273767445259484244","date":"2025-07-03T12:45:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"329745724075870926941626366958021612603","date":"2025-07-01T21:16:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"273643089811817981810989933678381093470","date":"2025-07-01T02:45:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"86511143295864125883064563057616354766","date":"2025-05-02T15:30:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-30T15:16:29+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-04-24T14:29:38+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-24T13:34:24+00:00","index":"","fulltext":""},{"type":"submitted","content":"Breast Cancer Research","date":"2025-04-11T23:33:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"breast-cancer-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"brcr","sideBox":"Learn more about [Breast Cancer Research](http://breast-cancer-research.biomedcentral.com)","snPcode":"13058","submissionUrl":"https://submission.nature.com/new-submission/13058/3","title":"Breast Cancer Research","twitterHandle":"@BCRJournal","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b286abb-6683-4fce-bdc4-acf90bea906d","owner":[],"postedDate":"May 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T16:44:12+00:00","versionOfRecord":{"articleIdentity":"rs-6431382","link":"https://doi.org/10.1186/s13058-025-02188-2","journal":{"identity":"breast-cancer-research","isVorOnly":false,"title":"Breast Cancer Research"},"publishedOn":"2026-01-14 16:28:38","publishedOnDateReadable":"January 14th, 2026"},"versionCreatedAt":"2025-05-07 11:21:50","video":"","vorDoi":"10.1186/s13058-025-02188-2","vorDoiUrl":"https://doi.org/10.1186/s13058-025-02188-2","workflowStages":[]},"version":"v1","identity":"rs-6431382","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6431382","identity":"rs-6431382","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}