Basal-like Breast Cancer Cell Lines Research Articles

Triggering a switch from basal- to luminal-like breast cancer subtype by the small-molecule diptoindonesin G via induction of GABARAPL1

Breast cancer is a heterogeneous disease that includes different molecular subtypes. The basal-like subtype has a poor prognosis and a high recurrence rate, whereas the luminal-like subtype confers a more favorable patient prognosis partially due to anti-hormone therapy responsiveness. Here, we demonstrate that diptoindonesin G (Dip G), a natural product, exhibits robust differentiation-inducing activity in basal-like breast cancer cell lines and animal models. Specifically, Dip G treatment caused a partial transcriptome shift from basal to luminal gene expression signatures and prompted sensitization of basal-like breast tumors to tamoxifen therapy. Dip G upregulated the expression of both GABARAPL1 (GABAA receptor-associated protein-like 1) and ERβ. We revealed a previously unappreciated role of GABARAPL1 as a regulator in the specification of breast cancer subtypes that is dependent on ERβ levels. Our findings shed light on new therapeutic opportunities for basal-like breast cancer via a phenotype switch and indicate that Dip G may serve as a leading compound for the therapy of basal-like breast cancer.

ANXA4 Activates JAK-STAT3 Signaling by Interacting with ANXA1 in Basal-Like Breast Cancer.

Annexin A4 (encoded by the ANXA4 gene) is a calcium ion (Ca2+)- and phospholipid-binding protein of the Annexin family. In this study, we checked the expression profile of ANXA4 in basal-like breast cancer (BLBC) and its association with survival outcomes using pan-cancer data from The Cancer Genome Atlas (TCGA) and the Genotype-Tissue Expression (GTEx) project. Then, using MDA-MB-231 and MDA-MB-468 cells, we explored the functional role of ANXA4 in regulating a cancer-related signaling pathway and identified potential partners of ANXA4. The results showed that expression of total ANXA4 and the two dominant ANXA4 protein-coding transcripts (ENST00000409920.5 and ENST00000394295.4) was consistently upregulated in tumor tissues compared with normal breast tissues. BLBC patients with high ANXA4 expression had significantly worse overall survival, progression-free survival, and disease-free survival than those with low ANXA4 expression. ANXA4 could positively modulate cyclin D1 expression and G1/S progression in the two cell lines. An in vivo tumor model showed that ANXA4 inhibition significantly slowed the growth of tumors derived from the two BLBC cell lines. ANXA4 could increase JAK1 expression and STAT3 phosphorylation (Y705). ANXA4 colocalized with ANXA1 in some MDA-MB-231 cells. A co-immunoprecipitation assay confirmed direct binding between ANXA4 and ANXA1. Knockdown of ANXA1 reduced JAK1 expression and STAT3 phosphorylation and impaired ANXA4-induced upregulation of JAK1 and p-STAT3. In conclusion, this study revealed that aberrant ANXA4 upregulation is associated with poor survival in BLBC. ANXA4 could activate JAK-STAT3 signaling by elevating the expression of JAK1 and p-STAT3, which was mediated by direct interaction with ANXA1.

FAK activates AKT-mTOR signaling to promote the growth and progression of MMTV-Wnt1-driven basal-like mammary tumors

BackgroundBreast cancer is a heterogeneous disease. Hence, stratification of patients based on the subtype of breast cancer is key to its successful treatment. Among all the breast cancer subtypes, basal-like breast cancer is the most aggressive subtype with limited treatment options. Interestingly, we found focal adhesion kinase (FAK), a cytoplasmic tyrosine kinase, is highly overexpressed and activated in basal-like breast cancer.MethodsTo understand the role of FAK in this subtype, we generated mice with conditional deletion of FAK and a knock-in mutation in its kinase domain in MMTV-Wnt1-driven basal-like mammary tumors. Tumor initiation, growth, and metastasis were characterized for these mice cohorts. Immunohistochemical and transcriptomic analysis of Wnt1-driven tumors were also performed to elucidate the mechanisms underlying FAK-dependent phenotypes. Pharmacological inhibition of FAK and mTOR in human basal-like breast cancer cell lines was also tested.ResultsWe found that in the absence of FAK or its kinase function, growth and metastasis of the tumors were significantly suppressed. Furthermore, immunohistochemical analyses of cleaved caspase 3 revealed that loss of FAK results in increased tumor cell apoptosis. To further investigate the mechanism by which FAK regulates survival of the Wnt1-driven tumor cells, we prepared an isogenic pair of mammary tumor cells with and without FAK and found that FAK ablation increased their sensitivity to ER stress-induced cell death, as well as reduced tumor cell migration and tumor sphere formation. Comparative transcriptomic profiling of the pair of tumor cells and gene set enrichment analysis suggested mTOR pathway to be downregulated upon loss of FAK. Immunoblot analyses further confirmed that absence of FAK results in reduction of AKT and downstream mTOR pathways. We also found that inhibition of FAK and mTOR pathways both induces apoptosis, indicating the importance of these pathways in regulating cell survival.ConclusionsIn summary, our studies show that in a basal-like tumor model, FAK is required for survival of the tumor cells and can serve as a potential therapeutic target.

A dual role of Irf1 in maintaining epithelial identity but also enabling EMT and metastasis formation of breast cancer cells.

An epithelial to mesenchymal transition (EMT) is an embryonic dedifferentiation program which is aberrantly activated in cancer cells to acquire cellular plasticity. This plasticity increases the ability of breast cancer cells to invade into surrounding tissue, to seed metastasis at distant sites and to resist to chemotherapy. In this study, we have observed a higher expression of interferon-related factors in basal-like and claudin-low subtypes of breast cancer in patients, known to be associated with EMT. Notably, Irf1 exerts essential functions during the EMT process, yet it is also required for the maintenance of an epithelial differentiation status of mammary gland epithelial cells: RNAi-mediated ablation of Irf1 in mammary epithelial cells results in the expression of mesenchymal factors and Smad transcriptional activity. Conversely, ablation of Irf1 during TGFβ-induced EMT prevents a mesenchymal transition and stabilizes the expression of E-cadherin. In the basal-like murine breast cancer cell line 4T1, RNAi-mediated ablation of Irf1 reduces colony formation and cell migration in vitro and shedding of circulating tumor cells and metastasis formation in vivo. This context-dependent dual role of Irf1 in the regulation of epithelial-mesenchymal plasticity provides important new insights into the functional contribution and therapeutic potential of interferon-regulated factors in breast cancer.

Diallyl Trisulfide Inhibits Leptin-induced Oncogenic Signaling in Human Breast Cancer Cells but Fails to Prevent Chemically-induced Luminal-type Cancer in Rats.

Previous studies have demonstrated inhibitory effect of garlic component diallyl trisulfide (DATS) on growth of breast cancer cells in vitro and in vivo. This study investigated the effect of DATS on oncogenic signaling regulated by leptin, which plays an important role in breast carcinogenesis. Leptin-induced phosphorylation and nuclear translocation of STAT3 was inhibited significantly in the presence of DATS in MCF-7 (a luminal-type human breast cancer cell line) and MDA-MB-231 (a basal-like human breast cancer cell line). Leptin-stimulated cell proliferation, clonogenic cell survival, and migration and/or invasion ability in MCF-7 and/or MDA-MB-231 cells were also suppressed by DATS treatment. DATS exposure resulted in inhibition of leptin-stimulated expression of protein and/or mRNA levels of Bcl-2, Bcl-xL, Cyclin D1, vascular endothelial growth factor, and matrix metalloproteinase-2. Western blotting revealed a decrease in protein levels of phosphorylated STAT3 in breast cancer xenografts from DATS-treated mice when compared to controls in vivo. However, the incidence of N-methyl-N-nitrosourea-induced luminal-type breast cancer development in rats was not affected by oral administration of 5 mg/kg or 25 mg/kg DATS. The present study reveals that oncogenic signaling induced by leptin is inhibited in the presence of DATS but higher doses of this phytochemical may be required to achieve chemopreventive activity.

Transcompp: understanding phenotypic plasticity by estimating Markov transition rates for cell state transitions.

Gradual population-level changes in tissues can be driven by stochastic plasticity, meaning rare stochastic transitions of single-cell phenotype. Quantifying the rates of these stochastic transitions requires time-intensive experiments, and analysis is generally confounded by simultaneous bidirectional transitions and asymmetric proliferation kinetics. To quantify cellular plasticity, we developed Transcompp (Transition Rate ANalysis of Single Cells to Observe and Measure Phenotypic Plasticity), a Markov modeling algorithm that uses optimization and resampling to compute best-fit rates and statistical intervals for stochastic cell-state transitions. We applied Transcompp to time-series datasets in which purified subpopulations of stem-like or non-stem cancer cells were exposed to various cell culture environments, and allowed to re-equilibrate spontaneously over time. Results revealed that commonly used cell culture reagents hydrocortisone and cholera toxin shifted the cell population equilibrium toward stem-like or non-stem states, respectively, in the basal-like breast cancer cell line MCF10CA1a. In addition, applying Transcompp to patient-derived cells showed that transition rates computed from short-term experiments could predict long-term trajectories and equilibrium convergence of the cultured cell population. Freely available for download at http://github.com/nsuhasj/Transcompp. Supplementary data are available at Bioinformatics online.

254P - Cyclin E1 is a shared biomarker of subsets of high grade serous ovarian cancer (HGSOC) and basal like breast cancer (BLBC)

BackgroundHGSOC and BLBC are aggressive cancers that share aetiology, co-occur frequently and share molecular signatures including cyclin E1 deregulation. In HGSOC, we have described two cyclin E1 high groups, with and without associated gene amplification. We aimed to determine which subsets of these disease have the greatest similarity in terms of cyclin E1 as a biomarkers of response with the ultimate aim of groping similar subsets in the future for therapeutic purposes. MethodsUsing automated tissue based assays, we assessed the expression and the amplification status of cyclin E1 in a cohort enriched for familial cancer mutations against each of the expression of cyclin E1 degradation associated proteins, i.e., FBXW7 and USP28, BRCA status and clinical outcome and in comparison with cyclin E1 high subsets of HGSOC. ResultsFor BLBC cyclin E1 overexpression but not CCNE1 amplification was found to be prognostic for overall survival. By comparison, both cyclin E1 expression and CCNE1 gene amplification are prognostic for HGSOC, which we establish with a meta-analysis of existing studies. We examined the nature of CCNE1 amplification to find that HGSOC has an enhanced degree of amplification of CCNE1 with 5.6x copies compared to 3.9x CCNE1 copies in BLBC, and this was associated with widely different expression of cyclin E1 protein. We find that BLBC cancers, with or without CCNE1 amplification, have similarities in dysregulation of regulators of cyclin E1 protein stability and those shared more features with the non-amplified cyclin E1 high HGSOC subset. Since cyclin E1 is only expressed at high levels in HGSOC and not BLBC, we examined the consequence of very high cyclin E1 expression in a BLBC cell line, MDA-MB-468. Moderate overexpression of cyclin E1 showed increased survival upon treatment with Paclitaxel, however very high expression of cyclin E1 gave less survival benefit. ConclusionsWe conclude that cyclin E1 high BLBC shared features with the non-amplified cyclin E1 high subset, high cyclin E1 expression associated with CCNE1 amplification gives rise to a unique phenotype in HGSOC, but otherwise the high expression of cyclin E1 protein may be a similar prognostic marker for BLBC and HGSOC. Legal entity responsible for the studyThe authors. FundingVentana Medical System.D. Aziz: Research grant / Funding (self), provided reagents and technical support: Ventana Medical System. P.M. Waring: Research grant / Funding (institution), Full / Part-time employment, provided reagents and technical support, worked for about 10 months for ventana during the project: Ventana Medical System. All other authors have declared no conflicts of interest.

An integrative bioinformatics analysis identified miR-375 as a candidate key regulator of malignant breast cancer.

MicroRNAs (miRNAs) are key regulators that play important biological roles in carcinogenesis and are promising biomarkers for cancer diagnosis and therapy. hsa-miR-375-3p (miR-375) has been suggested to serve as a tumor suppressor or oncogene in various tumor types; however, its specific expression and potential regulatory role in malignant breast cancer remain unclear. In this study, the results from noncoding RNA microarray analysis indicated that the miR-375 expression level is significantly decreased in malignant basal-like breast cancer compared with luminal-like breast cancer. A total of 1895 co-downregulated and 1645 co-upregulated genes were identified in miR-375 mimic-transfected basal-like breast cancer cell lines. Predicted miR-375 targets were obtained from the online databases TargetScan and DIANA-microT-CDS. Combined KEGG enrichment analysis for coregulated genes and predicted miR-375 targets provided information and revealed differences in potential dynamic signaling pathways regulated by miR-375 and also indicated specific regulatory pathways, such as RNA transport and processing, in basal-like breast cancer. Additionally, gene expression microarray analysis accompanied by UALCAN analysis was performed to screen upregulated genes in the basal-like subtype. Four potential key genes, including LDHB, CPNE8, QKI, and EIF5A2, were identified as candidate target genes of miR-375. Therefore, the present study demonstrated that miR-375 may be a potential key regulator and provide a promising direction for diagnostic and therapeutic developments for malignant breast cancer.

Abstract 2926: Nomination and characterization of TTK for radiosensitization in basal-like breast cancers

Abstract Background: Increased rates of locoregional recurrence have been observed in basal-like breast cancer despite the use of radiation therapy (RT), therefore approaches that result in radiosensitization of basal-like breast cancer are critically needed. Studies detailing the poor response of basal-like tumors to adjuvant RT underscore the biologic differences and as of yet undefined oncogenic drivers of these particular types of breast cancer. Methods: 4 independent datasets were used to correlate gene expression with local recurrence (LR) after 3 years. Kaplan-Meier analysis was used to validate the impact of TTK expression on LR. The TCGA and institutional breast cancer dataset were used to determine TTK expression in BC subtypes. TTK RNA and protein levels were measured using qPCR and western blot at baseline and correlated to intrinsic radiosensitivity. Clonogenic survival assays were used to determine the radiosensitization of cell lines after TTK inhibition (TTKi). Mice models were used to assess TTKi in combination with RT in vivo. DNA damage was quantified using γH2AX staining. HR and NHEJ efficiency assays were performed using HR/NHEJ specific reporter systems. HR competency was also assessed using RAD51 foci formation assays. Results: Ten genes were found to significantly correlate with early LR (≤3 years) across 4 distinct datasets (N=896 pts) (OR of recurrence > 2, p-value <0.000001), with TTK, a cell cycle kinase, ranked the highest. Kaplan-Meier survival analysis in 3 cohorts demonstrated that higher than median TTK expression correlates with decrease LR free survival after RT (HR 1.70-2.42, p<0.01 for all 3 cohorts). Subtype association analysis demonstrated that TTK expression was most elevated in basal-like breast cancer (p<0.0001). Clonogenic survival assays, using inducible shRNA models, show the combination of TTK knockdown and RT increases radiosensitivity in MDA-MB-231 and BT-549 basal-like breast cancer cell lines (rER 1.21-1.63). Additionally, TTKi using, Bayer 1161909 (B909), enhanced radiosensitivity in both cell lines (rER 1.10-1.39). In vivo, TTKi, using shRNA and B909 in combination with RT led to a significant increased time to tumor tripling. Using γH2AX foci formation assays, increased DNA damage was found after combination treatment of TTKi and RT compared to RT alone, indicating that DNA damage repair mechanisms may be compromised by TTKi. We found that the efficiency of the double strand DNA damage repair mechanism, homologous recombination (HR), but not non-homologous end joining (NHEJ), was reduced upon TTKi in HR/NHEJ specific reporter systems. Additionally, RAD51 foci formation was reduced by TTKi after RT compared to RT alone. Conclusion: These data support TTK inhibition as a rationale radiosensitizing strategy for clinical development in basal-like breast cancer patients and that the mechanism of radiosensitization is, at least in part, through impaired HR repair. Citation Format: Benjamin C. Chandler, Leah Moubadder, Cassie Ritter, Kari Wilder-Romans, Meleah Cameron, Meilan Liu, Shyam Nyati, Andrea Pesch, Anna Michmerhuizen, Eric Olsen, Yashar Niknafs, Arul Chinnaiyan, Corey Speers. Nomination and characterization of TTK for radiosensitization in basal-like breast cancers [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 2926.

Abstract LB-183: Anti-PD1 blunts obesity associated triple negative breast cancer progression

Abstract Few targeted therapies exist for triple negative breast cancer (TNBC), resulting in elevated recurrence and metastasis and a great unmet clinical need. Immune checkpoint therapy is promising; a recent clinical trial reported improved responses with anti-PD-L1 in advanced TNBC. Obesity increases poor outcomes in TNBC due to elevated invasion, metastasis, and mortality. Despite being an inflammatory state, obesity also can increase immune checkpoint ligands PD-1 and PD-L1 in some tumors and expression on PD-L1 on T cells and adipocytes, which drive immunosuppression. Greater PD-1 and PD-L1 may be due to hypoxia and inflammatory cytokines through increased expression and stability. Critically, immune checkpoint inhibitors appear to be more effective in obese patients, but underlying mechanisms remain unclear. Thus, we hypothesize that hypoxia and inflammation may synergistically increase immune checkpoint ligands which would alter immune checkpoint inhibitor efficacy in TNBC. To address this problem, syngeneic tumors were generated via orthotopic engraftment of E0771 basal-like breast cancer cell line into age-matched immune-competent C57BL/6J female littermates made obese on high fat diet (HFD, 60% kcal from fat) or lean on low fat diet (LFD, 10% fat). Mice were started on diets at weaning. By sacrifice at 27 weeks of age, lean and obese mice weighed 24.39g or 45.54g, respectively (p value <0.0001, N=15-16). Body composition showed no change in lean mass; obese mice had 48.64% adiposity (g fat/g body weight) compared to 15.99% in lean mice as 27 weeks (P=<0.0001). E0771 cells (0.25 x106) were injected after 24 weeks on diet and mice were sacrificed 24 days later. Mice were treated with anti-mouse anti-PD-1 or isotype control (IgG2a) (200 ug/kg i.p.) every 3rd day until sacrifice. Eleven out of 16 obese mice developed tumors (68.8% incidence). Surprisingly, only 2 tumors arose in N=16 lean mice (12.5% incidence) despite randomized same-day cell injections of lean and obese mice. Importantly, anti-PD-1 significantly reduced tumor volume in obese mice as measured by IVIS and caliper (P=0.0147, N=4-5, 22 days after tumor implantation). Ongoing studies include comprehensive evaluation of the immune microenvironment of both the tumors and normal mammary gland by flow and RNAseq. Immunohistology quantification of tumor, immune cell, and microenvironment characteristics will determine to what extent anti-PD-1 altered modifiers of tumor progression such as proliferation, apoptosis/necrosis, angiogenesis, stem cell content, epithelial to mesenchymal transition parameters. In conclusion, these results suggest that obesity-induced mammary microenvironmental effects augmented tumor incidence in immune intact C57BL/6J mice. Furthermore, immune checkpoint inhibitor successfully reduced tumor progression in obese mice. Potential inflammatory changes unique to the mammary gland or tumor microenvironment could be targeted in concert with checkpoint inhibitors in future interventions. Citation Format: Ajeeth Pingili, Emily Miller, Bin Teng, Liza Makowski. Anti-PD1 blunts obesity associated triple negative breast cancer progression [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 LB-183.

Abstract 3683: IL32 expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1

Abstract Metastatic basal-like breast cancers are believed to correspond with EpCAM-/Cd49f- cancer stem cell (CSC) enrichment. As well, basal-like breast cancers typically correspond with tumor inflammation and immunoediting phenotypes. However, the exact interplay between CSCs and the inflammatory signature of basal-like breast cancers is not well understood. To provide insight regarding the clinical overlap between breast cancer stem cells and tumor inflammation, we compared the 450K DNA methylation profile of EpCAM-/CD49f- CSCs from the isogenic MCF10A p53-/PTEN- breast cell line against the corresponding EpCAM+/CD49f+ and EpCAM-/CD49f+ subpopulations to determine whether differential DNA methylation occurred within the promoters of immune-related genes in CSCs. In addition, we also overlapped the 450K DNA methylation profile from 16 established breast cancer cell lines of varying EpCAM-/CD49f- concentrations to compare against the isolated CSCs. Based on our results, we identified 1432 differentially methylated promoter regions overall (ANOVA FDR p-value <0.001) and found IL32 to be differentially hypomethylated in the EpCAM-/CD49f- enriched cell lines. This hypomethylation of IL32 corresponded with increased expression of the beta isoform of IL32. Results from the cell lines were mirrored in The Cancer Genome Atlas (TCGA) breast cancer datasets, which revealed decreased promoter DNA methylation and increased gene expression of IL32 in basal-like patients. Further analysis of TCGA data using Gene Set Enrichment Analysis (GSEA) revealed that transcripts that tightly correlate with IL32 expression were preferentially involved in NF-kappaB mediated inflammation, with specific examples including REL, CCL5, PIK3CD, and IDO1. Furthermore, publicly available H3K27Ac and BRD4 ChIPseq data revealed that the IL32 promoter in the basal-like breast cancer cell line SUM159PT contains a high presence of H3K27 acetylation and BRD4 recruitment, with the latter event being disrupted by JQ1 treatment. These results complemented qRT-PCR results showing the IL32-beta isoform being quickly suppressed by 1uM JQ1 in SUM159PT as well as chick chorioallanotoic membrane (CAM) xenograft assays demonstrating suppressed metastasis and neovascularization of SUM159PT treated with JQ1. Collectively, these findings highlight the potential impact of IL32 promoter hypomethylation in basal-like breast cancer stem cells and how the overall epigenetic signature may predispose CSCs towards an immunomodulatory phenotype. Citation Format: Emma V. Gray, Caroline E. Dyar, Maria Ouzounova, Max S. Wicha, Hasan Korkaya, Austin Y. Shull. IL32 expression is epigenetically regulated in EpCAM-/Cd49f- basal-like breast cancers and can be suppressed by the bromodomain inhibitor JQ1 [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 3683.

Screening of immunosuppressive factors for biomarkers of breast cancer malignancy phenotypes and subtype-specific targeted therapy.

We aimed to screen and validate immunosuppressive factors in luminal- and basal-like breast cancer cell lines and tissue samples associated with malignant phenotypes. The mRNA microarray datasets, GSE40057 and GSE1561, were downloaded and remodeled, and differentially expressed genes were identified. Weighted gene co-expression network analysis (WGCNA) and gene ontology (GO) and KEGG pathway enrichment analysis were performed to explore the immune-related events related to the basal-like breast cancer. The online resources, GOBO, Kaplan–Meier Plotter and UALCAN, were employed to screen for immunosuppressive factors associated with breast cancer malignant phenotypes. Immunohistochemistry was used to evaluate VEGFA and MIF levels in breast tumors and normal breast tissues; qPCRs and western blots were used to validate the expression of clinical immuno-oncology (IO) therapeutic targets CD274 (PD-L1) and IL8 in cell lines. The results showed that various immune-related events contribute to basal-like breast cancer. First, TGFβ1 and IL8 had higher average expression levels in more malignant cell lines; second, MIF and VEGFA had higher average expression levels in more malignant breast cancer tissues, and the high expression levels were associated with poor survival rate. Third, IO targets CD274 and IL8 which were confirmed to be more suitable for the treatment of basal-like breast cancer. In view of the above, during the formation and development of breast cancer, immune-related genes are always activated, and immunosuppressive factors, IL8, TGFβ1, MIF, and VEGFA are up-regulated. Such molecules could be used as biomarkers for breast cancer prognosis. However, because individual immune-related factors can play several biological roles, the mechanistic relationship between immunosuppressive factors and breast cancer malignant phenotypes and the feasibility of their application as drug targets require further investigation.

FOXM1 Deubiquitination by USP21 Regulates Cell Cycle Progression and Paclitaxel Sensitivity in Basal-like Breast Cancer

SUMMARYThe transcription factor FOXM1 contributes to cell cycle progression and is significantly upregulated in basal-like breast cancer (BLBC). Despite its importance in normal and cancer cell cycles, we lack a complete understanding of mechanisms that regulate FOXM1. We identified USP21 in an RNAi-based screen for deubiquitinases that control FOXM1 abundance. USP21 increases the stability of FOXM1, and USP21 binds and deubiquitinates FOXM1 in vivo and in vitro, indicating a direct enzyme-substrate relationship. Depleting USP21 downregulates the FOXM1 transcriptional network and causes a signifi-cant delay in cell cycle progression. Significantly, USP21 depletion sensitized BLBC cell lines and mouse xenograft tumors to paclitaxel, an anti-mitotic, frontline therapy in BLBC treatment. USP21 is the most frequently amplified deubiquitinase in BLBC patient tumors, and its amplification co-occurs with the upregulation of FOXM1 protein. Altogether, these data suggest a role for USP21 in the proliferation and potentially treatment of FOXM1-high, USP21-high BLBC.

Abstract P2-01-11: A peptide with the conserved amino acid residue of integrin α6 inhibits metastasis through disruption of complex formation in breast cancer cells

Abstract Metastasis inhibition may improve survival of breast cancer patients. Our previous study revealed that a transcription factor SALL4 promotes cell migration for metastasis through up-regulation of integrin α6β1 in metastatic basal-like breast cancer cells. In these cells, integrin α6β1 modulates Rho GTPase activity, which enhances focal adhesion dynamics for cell migration. We therefore focused on integrin α6 as a therapeutic target for metastasis. We investigated the expression level of integrin α6 in breast cancer cell lines and a tissue microarray of breast cancer patients. In both experiments, we observed higher integrin α6 expression in basal-like breast cancer cells than in luminal ones. We performed shRNA-mediated integrin α6 knockdown in basal-like breast cancer cells and conducted Boyden chamber assays. In the results, reduced cell migration was observed in integrin α6 knocked-down cells. Reciprocally, we introduced integrin α6 overexpression in low-migratory luminal breast cancer cell lines and observed increased cell migration. These results indicate that integrin α6 promotes cell migration in breast cancer cells. To identify the functional residue of integrin α6, we analyzed its amino acid sequence by in silico analyses. First, we extracted integrin α6 specific residues in integrin α family in the human genome. Then, we compared integrin α6 sequences among vertebrates. In the result, we obtained an integrin α6-specific and vertebrate-conserved sequence, Asp-358, as a candidate for the functional residue. To inhibit integrin α6 function, we designed an 8-amino acid peptide with the sequence around Asp-358. Basal-like breast cancer cells treated with the peptide showed reduced cell migration in a dose-dependent manner. The peptide did not reduce cell migration in integrin α6 knocked-down cells, suggesting that the peptide inhibits integrin α6 function. To determine whether the peptide inhibits metastasis, we performed zebrafish metastasis assays, and observed reduced metastasis rate in the peptide-treated group. These results indicate that the peptide inhibits metastasis through reduction in cell migration. To understand the effect of the peptide, we performed chemical cross-liking assay in a basal-like breast cancer cell line. Protein samples from the cells treated with a cross-linker showed bands of integrin α6 complexes, and the intensities of these bands were reduced in the peptide-treated group. Moreover, we immunostained cells with an antibody for focal adhesion marker, phospho-paxillin, because integrins regulates focal adhesion formation for cell migration. In the results, the peptide reduced the number of focal adhesions. These observations indicate that the peptide inhibits integrin α6 function. In the future, our findings may contribute to development of a metastasis inhibition therapy. Citation Format: Itou J, Tanaka S, Senda N, Iida A, Sehara-Fujisawa A, Sato F, Toi M. A peptide with the conserved amino acid residue of integrin α6 inhibits metastasis through disruption of complex formation in breast cancer cells [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P2-01-11.

CCR2 Chemokine Receptors Enhance Growth and Cell-Cycle Progression of Breast Cancer Cells through SRC and PKC Activation.

Basal-like breast cancers are an aggressive breast cancer subtype, which often lack estrogen receptor, progesterone receptor, and Her2 expression, and are resistant to antihormonal and targeted therapy, resulting in few treatment options. Understanding the underlying mechanisms that regulate progression of basal-like breast cancers would lead to new therapeutic targets and improved treatment strategies. Breast cancer progression is characterized by inflammatory responses, regulated in part by chemokines. The CCL2/CCR2 chemokine pathway is best known for regulating breast cancer progression through macrophage-dependent mechanisms. Here, we demonstrated important biological roles for CCL2/CCR2 signaling in breast cancer cells. Using the MCF10CA1d xenograft model of basal-like breast cancer, primary tumor growth was significantly increased with cotransplantation of patient-derived fibroblasts expressing high levels of CCL2, and was inhibited with CRISP/R gene ablation of stromal CCL2. CRISP/R gene ablation of CCR2 in MCF10CA1d breast cancer cells inhibited breast tumor growth and M2 macrophage recruitment and validated through CCR2 shRNA knockdown in the 4T1 model. Reverse phase protein array analysis revealed that cell-cycle protein expression was associated with CCR2 expression in basal-like breast cancer cells. CCL2 treatment of basal-like breast cancer cell lines increased proliferation and cell-cycle progression associated with SRC and PKC activation. Through pharmacologic approaches, we demonstrated that SRC and PKC negatively regulated expression of the cell-cycle inhibitor protein p27KIP1, and are necessary for CCL2-induced breast cancer cell proliferation. IMPLICATIONS: This report sheds novel light on CCL2/CCR2 chemokine signaling as a mitogenic pathway and cell-cycle regulator in breast cancer cells.

Inhibition of super enhancer downregulates the expression of KLF5 in basal-like breast cancers.

The transcription factor KLF5 (Krüpple-like factor 5) is highly expressed in basal-like breast cancer (BLBC), which promotes cell proliferation, survival, migration and stemness, serving as a potential therapeutic target. In the current study, a super-enhancer (SE) was identified to be located downstream of the KLF5 gene in BLBC cell lines, HCC1806 and HCC1937. JQ-1, a BRD4 inhibitor, inhibits the expression and activity of KLF5 in both HCC1806 and HCC1937 cells in a time- and dose-dependent manner. Compound 870, an in-house BRD4 inhibitor, exhibited higher potency than JQ-1 to inhibit KLF5 and BLBC growth by arresting cells in G1 phase. Additionally, THZ1, a CDK7 inhibitor, also inhibits KLF5 and BLBC growth in a similar manner. Our findings suggested that KLF5 is regulated by SE, and modulation of SE could be an effective therapeutic strategy for treating BLBC.

Glyoxalase 1 gene is highly expressed in basal-like human breast cancers and contributes to survival of ALDH1-positive breast cancer stem cells.

Glyoxalase 1 (GLO1) is a ubiquitous enzyme involved in the detoxification of methylglyoxal, a cytotoxic byproduct of glycolysis that induces apoptosis. In this study, we found that GLO1 gene expression correlates with neoplasm histologic grade (χ2 test, p = 0.002) and is elevated in human basal-like breast cancer tissues. Approximately 90% of basal-like cancers were grade 3 tumors highly expressing both GLO1 and the cancer stem cell marker ALDH1A3. ALDH1high cells derived from the MDA-MB 157 and MDA-MB 468 human basal-like breast cancer cell lines showed elevated GLO1 activity. GLO1 inhibition using TLSC702 suppressed ALDH1high cell viability as well as the formation of tumor-spheres by ALDH1high cells. GLO1 knockdown using specific siRNAs also suppressed ALDH1high cell viability, and both TLSC702 and GLO1 siRNA induced apoptosis in ALDH1high cells. These results suggest GLO1 is essential for the survival of ALDH1-positive breast cancer stem cells. We therefore conclude that GLO1 is a potential therapeutic target for treatment of basal-like breast cancers.

Epigenetic activation of HORMAD1 in basal-like breast cancer: role in Rucaparib sensitivity.

Basal-like breast cancer (BLBC) is an aggressive breast cancer subtype with features similar to the basal cells surrounding the mammary ducts. Treatment of patients with BLBC has been challenging due to the lack of well-defined molecular targets. Due to the clinical and pathological similarities of BLBC with BRCA-deficient breast cancers, the effectiveness of Poly (ADP-ribose) polymerase inhibitors (PARPi) has been tested in early phase clinical trials for patients with advanced BLBC, with limited clinical responses. Recently, it was reported that HORMAD1 overexpression sensitizes BLBC to HR-targeting agents by suppressing homologous recombination. Our independent analysis suggests that HORMAD1 is aberrantly overexpressed in about 80% of BLBC, and its expression in normal tissues is restricted to testis. Our experimental data suggests that HORMAD1 overexpression correlates with focal hypomethylation in BLBC. On the other hand, investigation of the Genomics of Drug Sensitivity in Cancer dataset revealed significantly reduced sensitivity of HORMAD1-overexpressing BLBC cell lines to Rucaparib, a commonly used PARPi. To further assess the role of HORMAD1 in PARPi sensitivity, we generated three HORMAD1-overexpressing xenograft models using the HORMAD1-low BLBC cell lines HCC1954, HCC1806, and BT20; we then subjected these xenograft models to Rucaparib treatment. Ectopic expression of HORMAD1 enhances tumor formations in two of these models, and significantly reduces sensitivity to Rucaparib in the HCC1954 model. Taken together, our data suggest that epigenetic activation of HORMAD1 by hypomethylation in BLBC may endow reduced sensitivity to Rucaparib treatment in some tumor models.

Abstract 404: Amplification of SOX4 promotes PI3K/Akt signaling in human breast cancer

Abstract Basal-like or triple-negative breast cancer, if considered its own disease, would rank as the 5th leading cause of cancer deaths for women in the USA each year with ~10,000 deaths annually. Given the lack of drug-able targets expressed by TNBC tumors, few therapeutic options exist beyond currently utilized cytotoxic therapies, and the overall prognosis for these patients remains poor. The oncogenic PI3K/Akt signaling axis contributes to the dysregulation of many dominant features in breast cancer including cell proliferation, survival, metabolism, motility, and genomic instability. While multiple studies have demonstrated that basal-like or triple-negative breast tumors have uniformly high PI3K/Akt activity, genomic alterations that mediate dysregulation of this pathway in this subset of highly aggressive breast tumors remain to be determined. In this study, we present an integrated genomic analysis based on the use of a PI3K gene expression signature as a framework to analyze orthogonal genomic data in human breast tumors from TCGA (The Cancer Genome Atlas) including RNA expression, DNA copy number alterations, and protein expression. These analyses identified copy number alterations associated with PI3K activity in human breast tumors (n=1,031), including known and potentially novel drivers of PI3K signaling. Data from a genome-wide RNA interference (RNAi) screen were used to discriminate between essential and non-essential genes within identified amplicons. Integrating DNA copy number and RNAi analyses identified amplified SOX4 as a novel and essential regulator of PI3K/Akt signaling. Importantly, by analyzing protein expression data (n=733), we determined that SOX4 amplified tumors have increased activation of pAkt substrates. Moreover, depleting SOX4 levels in HCC38 and HCC1143, basal-like breast cancer cell lines with high PI3K activity and high SOX4 expression, demonstrated significant abrogation of p-Akt levels. This validated our in silico findings and confirmed the role of SOX4 in regulating PI3K pathway activity. To identify the potential mechanism(s) by which SOX4 mediates PI3K pathway activity, we performed genome-wide analyses of these cell lines following SOX4 inhibition. Transcriptomic profiling by RNA-sequencing, protein profiling by Reverse Phase Protein Array (RPPA) and Kinome profiling through multiplexed kinase inhibitor beads and mass spectrometry (MIB-MS) based approach identified TGFBR2, as a potential regulator of SOX4-mediated PI3K/Akt signaling. Further studies will elucidate the mechanism by which SOX4 modulates TGFBR2 expression and drives the oncogenic PI3K/Akt signaling pathway in basal-like breast cancer. Citation Format: Gaurav Mehta, Michael Gatza, Steve Angus, Gary Johnson. Amplification of SOX4 promotes PI3K/Akt signaling in human breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 404.

Increased α-actinin-1 destabilizes E-cadherin-based adhesions and associates with poor prognosis in basal-like breast cancer.

The controlled formation and stabilization of E-cadherin-based adhesions is vital for epithelial integrity. This requires co-operation between the E-cadherin-based adhesions and the associated actin cytoskeleton. In cancer, this co-operation often fails, predisposing cells to migration through molecular mechanisms that have only been partially characterized. Here, we demonstrate that the actin filament cross-linker α-actinin-1 is frequently increased in human breast cancer. In mammary epithelial cells, the increased α-actinin-1 levels promote cell migration and induce disorganized acini-like structures in Matrigel. This is accompanied by a major reorganization of the actin cytoskeleton and the associated E-cadherin-based adhesions. Increased expression of α-actinin-1 is particularly noted in basal-like breast cancer cell lines, and in breast cancer patients it associates with poor prognosis in basal-like subtypes. Downregulation of α-actinin-1 in E-cadherin expressing basal-like breast cancer cells demonstrate that α-actinin-1-assembled actin fibers destabilize E-cadherin-based adhesions. Taken together, these results indicate that increased α-actinin-1 expression destabilizes E-cadherin-based adhesions, which is likely to promote the migratory potential of breast cancer cells. Furthermore, our results identify α-actinin-1 as a candidate prognostic biomarker in basal-like breast cancer.