Promotes Breast Cancer Cell Survival Research Articles

P21Waf1/Cip1 Is a Novel Downstream Target of 40S Ribosomal S6 Kinase 2

Background/Objectives: The ribosomal S6 kinase 2 (S6K2) acts downstream of the mechanistic target of rapamycin complex 1 and is a homolog of S6K1 but little is known about its downstream effectors. The objective of this study was to use an unbiased transcriptome profiling to uncover how S6K2 promotes breast cancer cell survival. Methods: RNA-Seq analysis was performed to identify novel S6K2 targets. Cells were transfected with siRNAs or plasmids containing genes of interest. Western blot analyses were performed to quantify total and phosphorylated proteins. Apoptosis was monitored by treating cells with different concentrations of doxorubicin. Results: Silencing of S6K2, but not S6K1, decreased p21 in MCF-7 and T47D breast cancer cells. Knockdown of Akt1 but not Akt2 decreased p21 in MCF-7 cells whereas both Akt1 and Akt2 knockdown attenuated p21 in T47D cells. While Akt1 overexpression enhanced p21 and partially reversed the effect of S6K2 deficiency on p21 downregulation in MCF-7 cells, it had little effect in T47D cells. S6K2 knockdown increased JUN mRNA and knockdown of cJun enhanced p21. Low concentrations of doxorubicin increased, and high concentrations decreased p21 levels in T47D cells. Silencing of S6K2 or p21 sensitized T47D cells to doxorubicin via c-Jun N-terminal kinase (JNK)-mediated downregulation of Mcl-1. Conclusions: S6K2 knockdown enhanced doxorubicin-induced apoptosis by downregulating the cell cycle inhibitor p21 and the anti-apoptotic protein Mcl-1 via Akt and/or JNK.

FAM3C in Cancer-Associated Adipocytes Promotes Breast Cancer Cell Survival and Metastasis.

High FAM3C levels in cancer-associated adipocytes contribute to tumor-supportive niches and are tightly associated with metastatic growth, indicating that FAM3C inhibition could be beneficial for treating patients with breast cancer.

Inhibition of Dipeptidyl Peptidase-4 Activates Autophagy to Promote Survival of Breast Cancer Cells via the mTOR/HIF-1α Pathway.

Autophagy plays a complex role in breast cancer cell survival, metastasis, and chemotherapeutic resistance. Dipeptidyl peptidase (DPP)-4, a therapeutic target for type 2 diabetes mellitus, is also involved in autophagic flux. The potential influence of DPP-4 suppression on cancer biology remains unknown. Here, we report that DPP-4 deficiency promotes breast cancer cell survival via the induction of autophagy by the C-X-C motif chemokine 12 (CXCL12)/C-X-C receptor 4 (CXCR4)/mammalian target of rapamycin (mTOR)/hypoxia inducible factor (HIF)-1α axis. DPP-4 knockdown and DPP-4 inhibitor KR62436 (KR) treatment both increased the levels of LC3II and HIF-1α in cultured human breast and mouse mammary cancer cells. The KR-induced autophagic phenotype in cancer cells was inhibited by treatment with the CXCR4 inhibitor AMD3100 and rapamycin. HIF-1α knockdown also suppressed breast cancer autophagy induced by KR. The autophagy inhibitor 3-methyladenine significantly blocked the KR-mediated suppression of cleaved caspase-3 levels and apoptosis in breast cancer cell lines. Finally, we found that the metformin-induced apoptosis of DPP-4-deficient 4T1 mammary cancer cells was associated with the suppression of autophagy. Our findings identify a novel role for DPP-4 inhibition in the promotion of breast cancer survival by inducing CXCL12/CXCR4/mTOR/HIF-1α axis-dependent autophagy. Metformin is a potential drug that counteracts the breast cancer cell survival system.

STAMBPL1 promotes breast cancer cell resistance to cisplatin partially by stabilizing MKP-1 expression.

Dual-specificity mitogen-activated protein kinase phosphatase-1 (MKP-1/DUSP1/CL-100) has been documented to promote breast cancer cell survival and chemoresistance. MKP-1 is an unstable protein that is ubiquitinated and degraded via the ubiquitin-proteasome system. However, it is not clear how MKP-1 protein stability is regulated in breast cancer. In this study, we performed a genome-wide siRNA library screen of deubiquitinases (DUBs) and identified STAMBPL1 as an MKP-1 DUB in breast cancer cells. STAMBPL1 interacts with MKP-1 and stabilizes MKP-1 via deubiquitination. Both STAMBPL1 and MKP-1 depletion sensitize breast cancer cells to cisplatin in vitro and in vivo, and ectopic overexpression of MKP-1 partially rescues STAMBPL1 depletion-induced cisplatin sensitivity. Furthermore, STAMBPL1 and MKP-1 depletion increased breast cancer sensitivity to cisplatin by increasing the phosphorylation and activation of c-Jun N-terminal protein kinase (JNK). Collectively, our findings not only identify STAMBPL1 as an MKP-1 DUB but also reveal a critical mechanism that regulates MKP-1 expression in breast cancer. Our findings indicate that the STAMBPL1/MKP-1 axis represents a potential therapeutic target in breast cancer.

Adipocytes Promote Breast Cancer Cell Survival and Migration through Autophagy Activation.

Simple SummaryBreast tumours are in direct contact with the adipose tissue of the mammary gland. Although the interactions between breast cancer cells and adipocytes that secrete tumour-promoting factors are well known, the molecular mechanisms remain under investigation. The aim of our study was to understand whether and how adipocytes regulate a cell-recycling pathway in breast cancer cells—autophagy. We show that adipocytes promote autophagy in breast cancer cells through the acidification of lysosomes, leading to cancer cell survival in nutrient-deprived conditions and to cancer cell migration. In this study, we have identified a new mechanism, which can link adipose tissue with breast cancer progression.White adipose tissue interacts closely with breast cancers through the secretion of soluble factors such as cytokines, growth factors or fatty acids. However, the molecular mechanisms of these interactions and their roles in cancer progression remain poorly understood. In this study, we investigated the role of fatty acids in the cooperation between adipocytes and breast cancer cells using a co-culture model. We report that adipocytes increase autophagy in breast cancer cells through the acidification of lysosomes, leading to cancer cell survival in nutrient-deprived conditions and to cancer cell migration. Mechanistically, the disturbance of membrane phospholipid composition with a decrease in arachidonic acid content is responsible for autophagy activation in breast cancer cells induced by adipocytes. Therefore, autophagy might be a central cellular mechanism of white adipose tissue interactions with cancer cells and thus participate in cancer progression.

High BMI1 Expression with Low CD8+ and CD4+ T Cell Activity Could Promote Breast Cancer Cell Survival: A Machine Learning Approach.

BMI1 is known to play a key role in the regulation of stem cell self-renewal in both endogenous and cancer stem cells. High BMI1 expression has been associated with poor prognosis in a variety of human tumors. The aim of this study was to reveal the correlations of BMI1 with survival rates, genetic alterations, and immune activities, and to validate the results using machine learning. We investigated the survival rates according to BMI1 expression in 389 and 789 breast cancer patients from Kangbuk Samsung Medical Center (KBSMC) and The Cancer Genome Atlas, respectively. We performed gene set enrichment analysis (GSEA) with pathway-based network analysis, investigated the immune response, and performed in vitro drug screening assays. The survival prediction model was evaluated through a gradient boosting machine (GBM) approach incorporating BMI1. High BMI1 expression was correlated with poor survival in patients with breast cancer. In GSEA and in in silico flow cytometry, high BMI1 expression was associated with factors indicating a weak immune response, such as decreased CD8+ T cell and CD4+ T cell counts. In pathway-based network analysis, BMI1 was directly linked to transcriptional regulation and indirectly linked to inflammatory response pathways, etc. The GBM model incorporating BMI1 showed improved prognostic performance compared with the model without BMI1. We identified telomerase inhibitor IX, a drug with potent activity against breast cancer cell lines with high BMI1 expression. We suggest that high BMI1 expression could be a therapeutic target in breast cancer. These results could contribute to the design of future experimental research and drug development programs for breast cancer.

BRD4 modulates vulnerability of triple-negative breast cancer to targeting of integrin-dependent signaling pathways

PurposeStemming from a myriad of genetic and epigenetic alterations, triple-negative breast cancer (TNBC) is tied to poor clinical outcomes and aspires for individualized therapies. Here we investigated the therapeutic potential of co-inhibiting integrin-dependent signaling pathway and BRD4, a transcriptional and epigenetic mediator, for TNBC.MethodsTwo independent patient cohorts were subjected to bioinformatic and IHC examination for clinical association of candidate cancer drivers. The efficacy and biological bases for co-targeting these drivers were interrogated using cancer cell lines, a protein kinase array, chemical inhibitors, RNAi/CRISPR/Cas9 approaches, and a 4 T1-Balb/c xenograft model.ResultsWe found that amplification of the chromosome 8q24 region occurred in nearly 20% of TNBC tumors, and that it coincided with co-upregulation or amplification of c-Myc and FAK, a key effector of integrin-dependent signaling. This co-upregulation at the mRNA or protein level correlated with a poor patient survival (p < 0.0109 or p < 0.0402, respectively). Furthermore, we found that 14 TNBC cell lines exhibited high vulnerabilities to the combination of JQ1 and VS-6063, potent pharmacological antagonists of the BRD4/c-Myc and integrin/FAK-dependent pathways, respectively. We also observed a cooperative inhibitory effect of JQ1 and VS-6063 on tumor growth and infiltration of Ly6G+ myeloid-derived suppressor cells in vivo. Finally, we found that JQ1 and VS-6063 cooperatively induced apoptotic cell death by altering XIAP, Bcl2/Bcl-xl and Bim levels, impairing c-Src/p130Cas-, PI3K/Akt- and RelA-associated signaling, and were linked to EMT-inducing transcription factor Snail- and Slug-dependent regulation.ConclusionBased on our results, we conclude that the BRD4/c-Myc- and integrin/FAK-dependent pathways act in concert to promote breast cancer cell survival and poor clinical outcomes. As such, they represent promising targets for a synthetic lethal-type of therapy against TNBC.

Regulation of Autophagy by Protein Kinase C-ε in Breast Cancer Cells.

Protein kinase C-ε (PKCε), an anti-apoptotic protein, plays critical roles in breast cancer development and progression. Although autophagy is an important survival mechanism, it is not known if PKCε regulates autophagy in breast cancer cells. We have shown that silencing of PKCε by siRNA inhibited basal and starvation-induced autophagy in T47D breast cancer cells as determined by the decrease in LC3-II, increase in p62, and decrease in autophagy puncta both in the presence and absence of bafilomycin A1. The mechanistic target of rapamycin (mTOR) associates with Raptor or Rictor to form complex-1 (mTORC1) or complex-2 (mTORC2), respectively. Knockdown of PKCε attenuated an increase in autophagy caused by the depletion of Raptor and Rictor. Overexpression of PKCε in MCF-7 cells caused activation of mTORC1 and an increase in LC3-I, LC3-II, and p62. The mTORC1 inhibitor rapamycin abolished the increase in LC3-I and p62. Knockdown of mTOR and Rictor or starvation enhanced autophagy in PKCε overexpressing cells. While overexpression of PKCε in MCF-7 cells inhibited apoptosis, it induced autophagy in response to tumor necrosis factor-α. However, inhibition of autophagy by Atg5 knockdown restored apoptosis in PKCε-overexpressing cells. Thus, PKCε promotes breast cancer cell survival not only by inhibiting apoptosis but also by inducing autophagy.

Combined Anastrozole and Antiplatelet Therapy Treatment Differentially Promotes Breast Cancer Cell Survival.

Thromboembolic disorders are the second leading cause of death in breast cancer. Antiplatelet therapy combined with cancer therapy is a potential treatment strategy against cancer-associated thromboembolic disorders; however, the efficacy of such dual treatment has not been established. This study reports novel findings on the response of hormone-dependent breast cancer cell lines (MCF7/T47D) following 24 h treatment with Anastrozole, combined with Aspirin and Clopidogrel cocktail; and Atopaxar. Neutral red and lactate dehydrogenase assays were conducted to assess viability and cytotoxicity respectively. Flow cytometric Annexin-V/PI assay was used to assess the mode of cell death. Morphological alterations were studied using scanning electron microscopy. Statistical analysis was conducted using Statistica V13. Definitive outcomes were established with flow cytometric detection of phosphatidylserine exposure and propidium iodide staining, complemented with ultrastructural analysis. Results showed that a few cells were undergoing death mainly through secondary necrosis. Morphological features suggesting induced cell motility (pseudopodia/ruffled membranes) were observed in both cell lines; notably, T47D cells presented pronounced features than MCF7 cells. Overall, these findings suggest that such combined treatment may differentially promote cell survival, inducing a more aggressive breast cancer phenotype.

LncRNA CYTOR promotes tamoxifen resistance inbreast cancer cells via sponging miR‑125a‑5p.

Development of resistance to endocrine therapy, such as tamoxifen, remains a tricky clinical problem during the treatment of breast cancer. Accumulating evidence suggested that dysregulation of long noncoding (lnc_RNAs contributes to the development of tamoxifen resistance. In the current study, via screening, cytoskeleton regulator RNA (CYTOR) was identified as the most significantly elevated lncRNA in the established tamoxifen resistant MCF7 cell lines (MCF7/TAM1 and MCF7/TAM2) compared with the parental MCF7 cells (MCF7-P). The CCK-8 assay indicated that silencing of CYTOR increased the sensitivity of MCF7/TAM1 and MCF7/TAM2 to tamoxifen treatment. Using bioinformatic analysis, it was predicted that microRNA (miR)-125a-5p might bind to CYTOR and the expression of miR-125a-5p was negatively correlated with CYTOR in the tumor tissues of breast cancer. In addition, RT-qPCR and dual luciferase assays validated that CYTOR directly repressed miR-125a-5p expression in breast cancer cells. Through regulation of miR-125a-5p, CYTOR elevated serum response factor (SRF) expression and activated Hippo and mitogen associated protein kinase signaling pathways to promote breast cancer cell survival upon tamoxifen treatment. In the collected tumor tissues of breast cancer in the present study, high expression of CYTOR was detected in tissues from patients with no response to tamoxifen compared with those from patients who were not treated with tamoxifen. A positive correlation between CYTOR and SRF mRNA expression was observed in tissues collected from patients with breast cancer. In conclusion, the results of the present study demonstrated a pivotal role of CYTOR in mediating tamoxifen resistance in breast cancer.

Uev1A promotes breast cancer cell survival and chemoresistance through the AKT-FOXO1-BIM pathway

BackgroundUbiquitin-conjugating enzyme variant UEV1A is required for Ubc13-catalyzed K63-linked poly-ubiquitination that regulates several signaling pathways including NF-κB, MAPK and PI3K/AKT. Previous reports implicate UEV1A as a potential proto-oncogene and have shown that UEV1A promotes breast cancer metastasis through constitutive NF-кB activation. Ubc13-Uev1A along with TARF6 can also ubiquitinate AKT but its downstream events are unclear.MethodsIn this study, we experimentally manipulated UEV1 expression in two typical breast cancer cell lines MDA-MB-231 and MCF7 under serum starvation conditions and monitored AKT activation and its downstream protein levels, as well as cellular sensitivity to chemotherapeutic agents.ResultsWe found that overexpression of UEV1A is sufficient to activate the AKT signaling pathway that in turn inhibits FOXO1 and BIM expression to promote cell survival under serum starvation conditions and enhances cellular resistance to chemotherapy. Consistently, experimental depletion of Uev1 in breast cancer cells inhibits AKT signaling and promotes FOXO1 and BIM expression to reduce cell survival under serum starvation stress and enhance chemosensitivity.ConclusionsUev1A promotes cell survival under serum starvation stress through the AKT-FOXO1-BIM axis in breast cancer cells, which unveals a potential therapeutic target in the treatment of breast cancers.

CEMIP upregulates BiP to promote breast cancer cell survival in hypoxia.

Cell migration-inducing protein (CEMIP) and binding immunoglobulin protein (BiP) are upregulated in human cancers, where they drive cancer progression and metastasis. It has been shown that CEMIP resides in the endoplasmic reticulum (ER) where it interacts with BiP to induce cell migration, but the relationship between the two proteins was previously unknown. Here we show that CEMIP mediates activation of the BiP promoter and upregulates BiP transcript and protein levels in breast cancer cell lines. Moreover, CEMIP overexpression confers protective adaptations to cancer cells under hypoxic conditions, by decreasing apoptosis, activating autophagy, and increasing glucose uptake, to facilitate tumor growth. We demonstrate that BiP signals downstream of CEMIP, modulating cellular resistance to hypoxia. Reducing BiP in CEMIP-expressing cells sensitized cells to hypoxia treatment, decreased glucose uptake, and resulted in tumor regression in vivo. Our study provides insights into the link between CEMIP and BiP expression and the pro-survival role they play in hypoxia. Better understanding of the mechanisms behind cancer cell adaptations to harsh tumor environments could lead to development of improved cancer treatments.

Abstract 1011: Prolactin-induced EDD E3 ubiquitin ligase regulates pro/anti-apoptotic gene expression, and stimulates TORC1 signaling and drug resistance in breast cancer cells

Abstract EDD E3 ubiquitin ligase or UBR5, first identified as a progestin-inducible gene in human T47D breast cancer cells, mediates DNA damage signal transduction and is a key regulator of various cellular processes in cancer. We identified EDD as a prolactin (PRL)-inducible gene and as a novel partner of the TORC1-associated α4 phosphoprotein-PP2Ac phosphatase complex that regulates the initiation of translation and cell cycle progression. EDD targets PP2Ac for proteasomal degradation. Our aim was to further study the role of EDD in breast cancer cell survival, PRL-stimulated TORC1 signaling and drug resistance. We showed that EDD gene expression varied in breast cancer cell lines of different subtypes, such as luminal-A (MCF7, T47D), HER2-enriched (SkBR3), triple-negative claudin-low (MDA-MB-231) and basal-like (MDA-MB-436, -468). In high EDD-expressing MCF7 and T47D cells, EDD knockdown with siRNA (siEDD) significantly decreased cell viability, increased apoptosis, and arrested cells in the G2-phase of the cell cycle. Specifically, MCF-7 cells in late-stage apoptosis increased significantly (P=0.043, n=5) from a range of 2 - 8% in control cells to 20 - 35 % in siEDD-transfected cells. Although T47D cells were less affected by EDD loss, cells in early-stage apoptosis increased from 4 - 5% in control cells to 13 - 20 % in siEDD-transfected cells. SiEDD-induced apoptosis in MCF7 cells correlated with a significant increase in pro-apoptotic Bim and Bak mRNAs/proteins, leading to increased cleavage of caspase-7 at 6 - 48 h and consequently, cleavage of caspase substrate PARP-1 at 24 - 48 h, as compared to control cells. EDD loss in MCF7 cells decreased PRL-induced phosphorylation of eukaryotic initiation factor 4E-binding protein (4EBP1), a mediator of TORC1 signaling. Specifically, doublet α- and β-bands of 4EBP1 were detected in control cell lysates. A hyper-phosphorylated γ-band appeared in lysates of cells treated with PRL for 1 h but this γ-band disappeared in cells with EDD loss, implicating PRL-induced EDD enhancement of TORC1 signaling. Lastly, in low EDD-expressing MDA-MB-436 cells, EDD gain following pCMV-Tag2.EDD transfection increased cell resistance to chemotherapeutic drugs cisplatin and doxorubicin, and TORC1/TORC2 inhibitors rapamycin and INK128, as compared to controls. In contrast, EDD loss in MCF7 cells increased cell sensitivity to these drugs and to selective estrogen receptor modulator tamoxifen. In summary, PRL-induced EDD promotes breast cancer cell survival, enhances 4EBP1 phosphorylation, and increases drug resistance in vitro. These findings implicate EDD as a potential therapeutic target and support PRL receptor blockade as an additional therapy for breast tumours that express this receptor, including the aggressive HER2+ and triple-negative subtypes. Citation Format: Tyler MacDonald, Lynn N. Thomas, Emily Daze, Penelope J. Barnes, Catherine K. Too. Prolactin-induced EDD E3 ubiquitin ligase regulates pro/anti-apoptotic gene expression, and stimulates TORC1 signaling and drug resistance in breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1011.

The type 2 diabetes-associated Th17 cytokines IL-21 and IL-22 promote breast cancer cell survival.

Abstract Obesity-induced inflammation has taken center stage as a driving force in metabolic disease and type 2 diabetes (T2D). As the epidemic of obesity deepens, it has become imperative to dissect mechanisms by which obesity-induced inflammation contributes to increased cancer morbidity. Recent studies have shown that increased breast cancer mortality rates are associated with obesity-related T2D. Our goal is to understand the immunological mechanisms in T2D that contribute to increased breast cancer risk. We previously showed that PBMCs treated with T cell-targeted stimuli produce a pro-inflammatory Th17 cytokine profile that differentiates T2D from obese non-T2D subjects. We therefore tested the hypothesis that Th17 cytokines support breast cancer cells. Proliferation assays showed that IL-21 and IL-22, but not IL-17A/F, or the Th1 cytokine IL-6, maintained the proliferation of MCF7 cells, an estrogen dependent breast cancer cell line, in the absence of estrogen. Building on this result, we tested the effect of IL-21/22 on MCF7 survival. Immunoblots and mRNA arrays demonstrated that IL-21 but not IL-22 upregulated steady-state expression of BCL-2 family survival mRNA/protein. Both IL-21 and IL-22 survival was blocked by JQ1, an inhibitor of the Bromodomain and ExtraTerminal (BET) family of chromatin regulating proteins. The inferred cross-talk between BET- and Th17 cytokine-triggered pathways implicate the Th17 cytokine signature from PBMCs of patients with T2D in the higher recurrence of post-menopausal breast cancer after tamoxifen treatment. The public health impact of these data is that monitoring Th17 cytokine production by PBMCs may inform risk assessment for relapse or recurrence in breast cancer patients with T2D.

Cyclooxygenase-2 in tumor-associated macrophages promotes breast cancer cell survival by triggering a positive-feedback loop between macrophages and cancer cells.

Tumor-associated macrophages (TAMs) play an important role in cancer cell survival, however, the mechanism of which remains elusive. In this study, we found that COX-2 was abundantly expressed in breast TAMs, which was correlated to poor prognosis in breast cancer patients. Ectopic over-expression of COX-2 in TAMs enhanced breast cancer cell survival both in vitro and in vivo. COX-2 in TAMs was determined to be essential for the induction and maintenance of M2-phenotype macrophage polarity. COX-2(+) TAMs promoted breast cancer cell proliferation and survival by increasing Bcl-2 and P-gp and decreasing Bax in cancer cells. Furthermore, COX-2 in TAMs induced the expression of COX-2 in breast cancer cells, which in turn promoted M2 macrophage polarization. Inhibiting PI3K/Akt pathway in cancer cells suppressed COX-2(+) TAMs-induced cancer cell survival. These findings suggest that COX-2, functions as a key cancer promoting factor by triggering a positive-feedback loop between macrophages and cancer cells, which could be exploited for breast cancer prevention and therapy.

YAP enhances autophagic flux to promote breast cancer cell survival in response to nutrient deprivation.

The Yes-associated protein (YAP), a transcriptional coactivator inactivated by the Hippo tumor suppressor pathway, functions as an oncoprotein in a variety of cancers. However, its contribution to breast cancer remains controversial. This study investigated the role of YAP in breast cancer cells under nutrient deprivation (ND). Here, we show that YAP knockdown sensitized MCF7 breast cancer cells to nutrient deprivation-induced apoptosis. Furthermore, in response to ND, YAP increased the autolysosome degradation, thereby enhancing the cellular autophagic flux in breast cancer cells. Of note, autophagy is crucial for YAP to protect MCF7 cells from apoptosis under ND conditions. In addition, the TEA domain (TEAD) family of growth-promoting transcription factors was indispensable for YAP-mediated regulation of autophagy. Collectively, our data reveal a role for YAP in promoting breast cancer cell survival upon ND stress and uncover an unappreciated function of YAP/TEAD in the regulation of autophagy.

Identification of the PRMT1v1 and PRMT1v2 specific interactomes by quantitative mass spectrometry in breast cancer cells.

Arginine methylation is catalyzed by a family of enzymes called protein arginine methyltransferases (PRMTs). The PRMT1 gene generates at least seven distinct alternatively spliced isoforms (PRMT v1-v7), which together contribute a significant portion of the cellular arginine methylome. The distinct biochemical and biological functions of these PRMT1 isoforms have not been well characterized. Previously we have shown that while both PRMT1v1 and PRMT1v2 are overexpressed in breast cancer cells, PRMT1v2 specifically promotes breast cancer cell survival and invasion. These isoforms also have distinct subcellular localizations, PRMT1v1 is mainly nuclear and PRMT1v2 cytosolic. To gain further knowledge into their isoform-specific roles within cells we used a SILAC-based quantitative affinity purification/MS approach to identify their individual protein interactomes in breast cancer cells. This analysis has uncovered distinct interactomes for PRMT1v1 and PRMT1v2. Consistent with their distinct subcellular localizations, PRMT1v1 enriched a mainly nuclear protein interactome, while PRMT1v2 enriched predominantly cytoplasmic interactors from whole-cell extracts. Furthermore, these interactomes revealed that PRMT1v1 has a role in regulating gene expression, while PRMT1v2 functions in cytoskeletal dynamics. These results highlight the unique functions of these isoforms and the distinct roles they may play within cells, with potential implications for breast cancer and other diseases.

Fine-Scale Mapping of the 5q11.2 Breast Cancer Locus Reveals at Least Three Independent Risk Variants Regulating MAP3K1

Genome-wide association studies (GWASs) have revealed SNP rs889312 on 5q11.2 to be associated with breast cancer risk in women of European ancestry. In an attempt to identify the biologically relevant variants, we analyzed 909 genetic variants across 5q11.2 in 103,991 breast cancer individuals and control individuals from 52 studies in the Breast Cancer Association Consortium. Multiple logistic regression analyses identified three independent risk signals: the strongest associations were with 15 correlated variants (iCHAV1), where the minor allele of the best candidate, rs62355902, associated with significantly increased risks of both estrogen-receptor-positive (ER(+): odds ratio [OR] = 1.24, 95% confidence interval [CI] = 1.21-1.27, ptrend = 5.7× 10(-44)) and estrogen-receptor-negative (ER(-): OR = 1.10, 95% CI = 1.05-1.15, ptrend = 3.0× 10(-4)) tumors. After adjustment for rs62355902, we found evidence of association of a further 173 variants (iCHAV2) containing three subsets with a range of effects (the strongest was rs113317823 [pcond = 1.61× 10(-5)]) and five variants composing iCHAV3 (lead rs11949391; ER(+): OR = 0.90, 95% CI = 0.87-0.93, pcond = 1.4× 10(-4)). Twenty-six percent of the prioritized candidate variants coincided with four putative regulatory elements that interact with the MAP3K1 promoter through chromatin looping and affect MAP3K1 promoter activity. Functional analysis indicated that the cancer risk alleles of four candidates (rs74345699 and rs62355900 [iCHAV1], rs16886397 [iCHAV2a], and rs17432750 [iCHAV3]) increased MAP3K1 transcriptional activity. Chromatin immunoprecipitation analysis revealed diminished GATA3 binding to the minor (cancer-protective) allele of rs17432750, indicating a mechanism for its action. We propose that the cancer risk alleles act to increase MAP3K1 expression invivo and might promote breast cancer cell survival.

The Evaluation of WBP2NL-Related Genes Expression in Breast Cancer.

Breast cancer is the most frequent cause of mortality in women all around the world; therefore, study on molecular aspects of breast cancer is necessary for finding new biomarkers. Recent studies have shown that WW Binding Protein 2 (WBP2) is an important protein for the oncogenic property of cancer. We have previously evaluated the WW Binding Protein 2N-Terminal Like (WBP2NL) gene expression in cancerous cell line and breast tumor tissues, and reported changes in expression, which could increase tumorigenic cell growth. However, the molecular mechanisms of WBP2NL and its clinical relevance have not been investigated. In this study, the expression of WBP2NL-related genes in the invasive breast carcinoma and normal breast tissues was evaluated for the first time. Analysis of WBP2NL-related genes expression was performed with reverse transcription-PCR and real time-PCR detection method. The target genes studied were as follow: WW domain containing E3 ubiquitin protein ligase 1(WWP1), membrane associated guanylatekinase containing WW and PDZ domain-1 (MAGI1), neural precursor cell expressed developmentally down-regulated 4 (NEDD4), formin binding protein-4 (FNBP4), BCL2-associated athanogene-3 (BAG3), WW domain-containing oxidoreductase (WWOX), yes-associated protein-1 (YAP1), WW domain containing transcription regulator (WWTR1), member RAS oncogene family (RAB2A), and small G protein signaling modulator 3 (SGSM3). The expression of WWP1, BAG3, and WWTR1 was significantly increased in breast cancer. In contrast, the expression of WWOX, YAP1, RAB2A, and SGSM3 was significantly decreased. The MAGI1 and NEDD4 expression was increased, while the expression of FNBP4 was unchanged. These findings lead us to suggest that WBP2NL might play roles as an anti-apoptotic factor or co-activator to promote breast cancer cell survival and proliferation.

Abstract 3789: The alternatively spliced PRMT1 isoform PRMT1v2 promotes breast cancer cell survival and invasiveness.

Abstract Breast cancer is the leading cause of cancer-related deaths in women. Despite intensive treatment to overcome this disease, recurrence is common and often incurable due to the highly aggressive nature of the recurrent tumour. Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types, however their precise contribution to the cell transformation process and cancer progression is not well understood. Previously, we reported that the PRMT1 gene generates several alternatively spliced isoforms and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and sub-cellular localization. Here we have focused on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform and has increased relative expression in breast cancer cell lines and tumours. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in breast cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in MDA-MB-231, an aggressive breast cancer cell line, resulted in significantly decreased cell invasion. Alternatively, PRMT1v2 overexpression in a non-aggressive breast cancer cell line, MCF7, was sufficient to enhance their invasiveness. This novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. It also requires both proper subcellular localization and methylase activity. Consistent with an invasive phenotype, PRMT1v2 overexpression caused altered cell morphology and reduced cell-cell adhesion. Finally, we observed a significant reduction in β-catenin protein levels, a critical cell-cell adhesion protein, and show that this reduction is pivotal to PRMT1v2-enhanced cell invasion. Our study underscores the importance of characterizing the distinct functional differences between PRMT1 isoforms. Overall, we demonstrate a specific role for PRMT1v2 in promoting breast cancer cell survival and invasion, and propose that it may represent a promising therapeutic target. *RMB is a Postdoctoral Fellow of the Canadian Institute of Health Research Citation Format: Robert Mitchell Baldwin, Alan Morettin, Genevieve Paris, Isabelle Goulet, Jocelyn Cote. The alternatively spliced PRMT1 isoform PRMT1v2 promotes breast cancer cell survival and invasiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2013-3789