Basal-like Triple-negative Breast Cancer Research Articles

Impact of inducible nitric oxide synthase (iNOS) expression on triple negative breast cancer outcome and activation of EGFR and ERK signaling pathways.

Inflammation is implicated in triple negative breast cancer (TNBC) progression. TNBC carries a worse prognosis than other breast cancer subtypes, and with the clinical and molecular heterogeneity of TNBC, there is a lack of effective therapeutic targets available. Identification of molecular targets for TNBC subtypes is crucial towards personalized patient stratification. Inducible nitric oxide synthase (iNOS) has been shown to induce p53 mutation accumulation, basal-like gene signature enrichment and transactivation of the epidermal growth factor receptor (EGFR) via s-nitrosylation. Herein we report that iNOS is associated with disease recurrence, distant metastasis and decreased breast cancer specific survival in 209 cases of TNBC. Employing TNBC cell lines representing normal basal breast, and basal-like 1 and basal-like 2 tumors, we demonstrate that nitric oxide (NO) induces EGFR-dependent ERK phosphorylation in basal-like TNBC cell lines. Moreover NO mediated cell migration and cell invasion was found to be dependent on EGFR and ERK activation particularly in basal-like 2 TBNC cells. This occurred in conjunction with NF-κB activation and increased secretion of pro-inflammatory cytokines IL-8, IL-1β and TNF-α. This provides substantial evidence for EGFR as a therapeutic target to be taken into consideration in the treatment of a specific subset of basal-like TNBC overexpressing iNOS.

Abstract 2876: Loss of the stem cell and basal lineage regulator LBH delays onset of basal-like triple negative breast cancer

Abstract There is increasing evidence that basal-like triple negative breast cancer (TNBC) originates from luminal mammary epithelial cells. Specific gene signatures have already been experimentally proven to serve as genetic lineage switches that, when overexpressed, transform pre-malignant luminal cells to oncogenic cells with basal, stem-like characteristics. Conversely, depletion of these genes in oncogenic cells with basal, stem-like characteristics leads to luminal differentiation. These studies suggest that during the earliest steps of neoplastic transformation, specific gene sets can alter cell fate decisions and differentiation status in mammary epithelial cells, which ultimately contributes to the heterogeneity of breast tumors. Our lab has identified a novel Wnt/β-Catenin target gene, Limb bud and heart (LBH) that is majorly overexpressed in TNBC. LBH is a regulator of the basal mammary stem cell lineage and repressor of luminal differentiation via induction of ΔNp63 and repression of estrogen receptor alpha. LBH is required for the self-renewal and maintenance of adult basal mammary stem cells, which tend to be enriched in TNBC. Knockout studies in mice have shown that genetic ablation of LBH does not impair embryogenesis or normal adult organ function, making it a possible therapeutic target. Using crosses between MMTV-Wnt-1 transgenic mice and K14Cre LBHloxP knockout mice, we are studying the effect of LBH ablation in the basal cells of the mammary epithelium downstream of ectopic Wnt expression in the mammary gland. We postulated that LBH may be an effector of Wnt-driven TNBC, therefore its inhibition would lead to decreased Wnt-induced mammary gland hyperplasia and tumor formation. In our model, LBH ablation in the basal mammary epithelium of female Wnt transgenic mice reduces mammary gland hyperplasia and delays tumor onset. MMTV-Wnt-1 driven, LBH-null tumors also exhibit histopathological differences indicative of a luminal to basal conversion. There is an urgent need to elucidate mechanisms underlying TNBC development and progression and find reliable avenues for treatment. Our data indicates a role for Lbh as a novel effector for Wnt-driven TNBC and further studies may prove antagonism of Lbh to be a novel method to control TNBC progression. Citation Format: Kilan C. Ashad-Bishop, Karoline Briegel. Loss of the stem cell and basal lineage regulator LBH delays onset of basal-like triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2876. doi:10.1158/1538-7445.AM2017-2876

Abstract 4786: Integrated eoxme and transcriptome sequencing identifies a novel candidate for breast cancer

Abstract Many somatic mutations, structural alterations, and gene expression changes are causally implicated in oncogenesis and tumor progression, and as a result, affect clinical outcome. Although majority of breast cancer patients have benefits from therapeutics targeting tumor biology still many patients suffer from disease recurrence and metastasis. To find novel therapeutic target in breast cancer, here we examine the both whole exome and whole transcriptome of fresh-frozen primary breast cancer tissues from 120 patients whose clinical, pathological, and survival data are available. We identified 11,684 putative somatic mutations in 7,373 genes. We hypothesized that therapeutic target has to have recurrent mutation and gene expression at least more than average expression. With a cut-off of 3 or more mutations in each gene, 1,116 genes were selected. After the filtering of Volume>0.3, 696 genes were selected. Finally, 55 genes were selected which are druggable or potentially druggable using drug database. Interestingly, 18 of the 55 were genes involved in metabolism. Among them we further focused PSMD2 and interrogated their oncogenic roles, a subunit of the 19S regulatory complex of proteasomes, as a constituent of a signature is a candidate driven factor in breast cancer. PSMD2 is required for proliferation both in vitro and in vivo, as shRNA-mediated PSMD2 silencing attenuated tumor growth. Further, PMSD2 is sufficient to induce stem cell characteristics, as forced PSMD2 expression facilitated mammosphere formation and increased the CD44+ breast CSC-like population. Mechanistically, PSMD2 inhibition were associated with changes in epithelial-mesenchymal transition (EMT) phenotype. Knockdown of PSMD2 induced dramatic morphological changes, the typical spindle-like appearance cells were replaced with the round shape, like epithelial morphology. Moreover, treatment of TGF-β1, the main inducer of EMT, lead to increased expression of PSMD2. PSMD2 silencing up-regulated E-cadherin and down-regulated expression of vimentin, N-cadherin and snail, mesenchymal markers. Clinically, PSMD2 expression was elevated in basal-like TNBC (triple negative breast cancer), correlated with poor prognosis, insilico analysis on a dataset comprising over 2413 microarrays of the web application bc-GenExMiner. Altogether, our findings suggest that PSMD2 may be a good molecular target candidate and PSMD2 inhibitor may kills breast cancer stem cells and ameliorates poor prognosis of breast cancer. Citation Format: Kyung-Min Lee, Dong-Young Noh, Wonshik Han. Integrated eoxme and transcriptome sequencing identifies a novel candidate for breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4786. doi:10.1158/1538-7445.AM2017-4786

Abstract 3064: Retinoic acid: An effective therapy for basal-like breast cancer

Abstract We have identified a novel strategy to identify breast cancer patients who will benefit from an existing anti-cancer agent, retinoic acid (RA). While RA has not yet achieved success in the treatment of breast cancers, we hypothesized that it can be an effective therapy for a subset of triple-negative breast cancer (TNBC) patients. TNBC is among the most aggressive breast cancers, and lacks targeted therapies. TNBCs can be further subtyped into basal-like and claudin-low, which differ in gene expression and drug sensitivities. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. To test this hypothesis, we performed tumor growth assays on TNBC cell lines and patient-derived xenografts (PDXs). We found that RA treatment decreased the tumor growth of four basal-like TNBC cell lines (MDA-MB-468, HCC70, SUM149, HCC1937). In contrast, RA increased the tumor growth of two claudin-low TNBC cell lines (MDA-MB-231, MDA-MB-436). Gene expression and methylation analysis of these affected cell lines revealed subtype-specific expression of RA-inducible genes due to silencing by DNA methylation, e.g. of the RA-inducible tumor-suppressor gene RARRES1. RARRES1 is silenced by methylation in claudin-low cell lines, but is hypomethylated and expressed in basal-like cells. Use of the subtype-specific expression and methylation profiles allowed us to accurately predict the response of 4 PDXs to RA treatment. Continued classification of TNBCs into these two subtypes will enable clinical use of RA, in part due to the subtype-specific hypomethylation of RA-inducible tumor suppressor genes including RARRES1. We have identified additional subtype-specific biomarkers which can predict the response of patient tumors to RA treatment, thus identifying a novel targeted therapy strategy for TNBCs. Citation Format: Krysta Mila Coyle, Cheryl A. Dean, Dejan Vidovic, Ian C. Weaver, Carman A. Giacomantonio, Paola Marcato. Retinoic acid: An effective therapy for basal-like breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3064. doi:10.1158/1538-7445.AM2017-3064

Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

Cancer cells reprogram their metabolism to increase the synthesis of macromolecules for rapid proliferation. Compared to fatty acids, much less is known about the synthesis of phospholipids, which is essential for membrane biogenesis in cancer cells. We found that LPIN1, which encodes lipin-1, a phosphatidic acid phosphatase (PAP) controlling the rate-limiting step in the phospholipid synthesis pathway, is highly up-regulated in basal-like triple-negative breast cancer (TNBC). Moreover, high LPIN1 expression correlates with the poor prognosis of these patients. Knockdown of LPIN1 increases apoptosis in basal-like TNBC cell lines, whereas it has minimal or less effect on normal human mammary gland epithelial cells (HMECs) and estrogen receptor-positive breast cancer cell lines. Fatty acid incorporation and lipidomics analyses showed that LPIN1 knockdown blocks phospholipid synthesis and changes membrane lipid compositions that ultimately induce the activation of 1 of the 3 branches of unfolded protein responses, the inositol-requiring enzyme-1α pathway. We also show for the first time, to our knowledge, that lipin-1 knockdown significantly inhibits tumor growth in vivo using an orthotopic xenograft breast mouse model. Our results suggest that lipin-1 is a potential target for cancer therapy.-He, J., Zhang, F., Tay, L. W. R., Boroda, S., Nian, W., Levental, K. R., Levental, I., Harris, T. E., Chang, J. T., Du, G. Lipin-1 regulation of phospholipid synthesis maintains endoplasmic reticulum homeostasis and is critical for triple-negative breast cancer cell survival.

ΔNp63 activates EGFR signaling to induce loss of adhesion in triple-negative basal-like breast cancer cells.

The basal-A subtype of triple-negative breast cancer is characterized by high levels of ΔNp63. Various functions have been proposed for p63 in breast cancer initiation and growth, and p63 mediates chemotherapeutic response in a subset of triple-negative breast cancers. We investigated the signaling pathways that are controlled by ΔNp63 in basal-A triple-negative breast cancer. Human basal-A triple-negative breast cancer cell lines with ΔNp63α induction or inhibition were studied, along with primary human triple-negative breast cancer tissues. Proteomic, phospho-kinase array, mRNA measurements, and immunohistochemistry were employed. Global phosphoproteomics identified increased EGFR phosphorylation in MDA-MB-468 cells expressing ΔNp63α. ΔNp63α expression increased EGFR mRNA, total EGFR protein, and phospho-EGFR(Y1086), whereas silencing endogenous ΔNp63 in HCC1806 cells reduced both total and phospho-EGFR levels and inhibited the ability of EGF to activate EGFR. EGFR pathway gene expression analysis indicated that ΔNp63 alters EGFR-regulated genes involved in cell adhesion, migration, and angiogenesis. Addition of EGF or neutralizing EGFR antibodies demonstrated that EGFR activation is responsible for ΔNp63-mediated loss of cellular adhesion. Finally, immunohistochemical staining showed that p63-positive triple-negative breast cancers were more likely to express high levels of EGFR than p63-negative cancers, corroborated by in silico analysis of gene expression profiling data. These data identify EGFR as a major target for ΔNp63 regulation that influences cancer cell adhesion in basal-like triple-negative breast cancer.

Abstract ES9-3: ES9-3 (Neo)adjuvant systemic therapy of TNBC: How much is enough?

Abstract The use of neoadjuvant chemotherapy in ER-negative cancers has become quite popular in patients with stage II and III breast cancers, since it gives us important information on long-term prognosis, based on the amount of residual disease burden at the time of definitive surgical resection. Two relatively large phase II randomized neoadjuvant clinical trials have explored the addition of carboplatin to an anthracycline/taxane based chemotherapy regimen for patients with stage II or III triple-negative breast cancers (TNBC): GeparSixto, in Europe, and CALGB40603, in the US. Despite significant differences in chemotherapy backbone, and frequency and duration of carbo administration, both trials showed that the addition of carboplatin significantly increased the rate of pCR from ∼ 40% to 54%. Both trials also reported a significant amount of added toxicity for the carboplatin arms, with a significant amount of patient not being able to complete their planned treatment. However, whereas the GeparSixto trial reported an improvement in 3-year disease-free survival (DFS), the CALGB40603 did not. Since neither of these studies was powered to address disease-free or overall survival benefit from the addition of carboplatin to neoadjuvant systemic chemotherapy, lack of precision from small sample sizes are likely responsible for the difference seen in DFS between these two trials. Additionally, there's not yet a confirmed correlation between increases in frequency of pCR and event-free survival (EFS). A recently pooled analysis by Cortazar et al. could not validate pCR as a surrogate endpoint for improved EFS and overall survival (OS): as some patients who achieve a pCR go on to relapse and many with residual disease never experience a recurrence, small differences in pCR may never translate to improvements in EFS. Long term effects of the added toxicity of carboplatin are also not known. At present, upon completion of neoadjuvant therapy, the standard of care for patients with TNBC (who have no clinical evidence of metastatic disease after surgical excision of the cancer regardless of burden of residual disease) is observation. But recently, preliminary data from a Japanese phase III trial (CREATE-X) that randomized patients with HER2-negative breast cancer with lack of pCR post-neoadjuvant chemotherapy to observation or capecitabine x 8 cycles showed that the addition of capecitabine provided a statistically significantly higher 5-year DFS and OS in the TNBC group. EA1131, an ongoing adjuvant randomized phase III clinical trial seeks to address if patients with basal-like TNBC, with high risk of recurrence due to residual disease post completion of neoadjuvant chemotherapy, have a higher DFS benefit from a platinum agent than from capecitabine. The results of this trial will ultimately help avoid exposing upfront unselected patients, many of whom will never relapse, to toxic therapy in the absence of long-term known benefit. In summary, the addition of a platinum agent routinely in the neoadjuvant setting in TNBC is still an individualized decision and not yet standard of care for all patients. The decision to use capecitabine in patients with residual disease post-neoadjuvant chemo may still be slightly premature, confirmatory results of EA1131 are eagerly awaited. Citation Format: Mayer IA. ES9-3 (Neo)adjuvant systemic therapy of TNBC: How much is enough? [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr ES9-3.

Abstract P6-07-26: ST8SIA1 is over-expressed in triple negative breast cancer and associated with p53 mutations

Abstract Cancer stem cells constitute a small fraction of cells present in primary tumors, are highly tumorigenic, drug resistant and cause metastases. We discovered Ganglioside GD2 as a breast cancer stem cell (BCSCs) marker in triple negative breast cancer (TNBC). GD2 biosynthesis is tightly regulated by the enzyme ST8SIA1 (GD3 synthase). However, expression of ST8SIA and its association with different breast cancer sub-types is not known. Here we hypothesize that ST8SIA1 is up-regulated in TNBC or basal-type breast tumors and associated with stemness in primary breast tumors. Frist, we examined ST8SIA1 protein expression in breast cancer cell lines (n=12) and found up-regulation of ST8SIA1 in basal compared to luminal-type breast cancer cells. To investigate ST8SIA1's expression in primary tumors, we analyzed RNAseq data from the cancer genome atlas (TCGA) data base which has expression data from 1105 primary and metastatic breast tumors as well as adjacent normal tissues. We found that ST8SIA1 expression varies widely among different breast tumors and that 'basal' type tumors express highest levels of ST8SIA1 compared to all other types of breast cancers including luminal-A or luminal-B or HER2 enriched (p<0.01) tumors. In addition, triple negative breast cancer (TNBC, n=115) express 4.63fold higher ST8SIA1 levels compared to hormone receptors positive tumors including ER+ or PR+ or HER2+ tumors (n=852, p<0.001). Survival analysis by log-rank test indicates that patients with ST8SIA1high tumors survive shorter (median survival 2.6 years) compared to patients with ST8SIA1low tumors (median survival 4.3 years). These data suggests that ST8SIA1 expression is associated with basal-like TNBC tumor types and high ST8SIA1 levels in tumors may shorten survival in TNBC patients. To investigate the association between ST8SIA1expression and the most commonly mutated genes in breast cancer, we chose the top 20 most frequently mutated genes in TCGA dataset and examined their correlation with ST8SIA1 mRNA expression. We found that, among the top 20 mutations, p53 has a very strong positive correlation with ST8SIA1 expression (p<0.00001). The expression of ST8SIA1 is >2 fold higher in p53 mutated tumors compared to p53 wild type tumors. The other positively correlated mutation was a nuclear envelop protein called Spectrin Repeat Containing, Nuclear Envelope 1 (SYNE1; p<0.05). Mutations in GATA3 are the most negatively correlated (p<0.001) with ST8SIA1 expression. Interestingly, GATA3 plays a role in epithelial cell differentiation in the mammary gland, supporting the notion that ST8SIA1 is a stem cell-associated gene. In addition, correlation of ST8SIA1 mRNA expression with other genes revealed that FOXA1, the protein which is co-expressed with GATA3 and serves as negative predictor of basal type of breast cancer, is down regulated in ST8SIA1high tumors. In conclusion, ST8SIA1 is associated with basal-type TNBC tumors and has a strong positive correlation with p53 mutations and negative correlation with GATA3 mutations. Knowing that TP53 mutations have a major role in tumorigenesis and drug resistance, these data suggest ST8SIA1 as a potential therapeutic target in TNBC patients with p53 mutations. Citation Format: Yan Y, Nguyen K, Do K-A, Ueno N, Andreeff M, Battula VL. ST8SIA1 is over-expressed in triple negative breast cancer and associated with p53 mutations [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-07-26.

Correlation between epithelial-mesenchymal transition related markers and poor clinical outcome of triple-negative breast cancer

Objective To investigate the expression of epithelial-mesenchymal transition (EMT) related molecular markers (vimentin, smooth muscle actin, osteonectin, N-cadherin, and E-cadherin loss) and EMT inducer zinc-finger E-box binding homeobox 1 (ZEB1) in triple-negative breast cancer (TNBC) samples and inquire the correlation between their expression and clinicopathological features of the tumors. Methods We analyzed immunohistochemical expression of EMT-related markers mentioned above in 173 TNBC samples and correlation between their expression with clinicopathological features of the tumors, including clinical outcome. Results The expression levels of vimentin were more frequent in basal-like TNBC (87/154, 56.5%) than in non-basal-like TNBC (3/17, 17.6%). The ZEB1 expression was correlated only with the expression of smooth muscle actin (P=0.002). The survival analyses revealed that vimentin (P=0.515), smooth muscle actin (P=0.739), osteonectin (P=0.094), E-cadherin (P=0.925) and N-cadherin (P=0.509) were not associated with patients’ clinical outcomes. However, the ZEB1 expression was found to be an independent prognostic factor for poor disease-free survival. Conclusion These findings indicate that the expression of EMT-related markers in TNBC can be a signature of a certain subgroup of TNBC. Key words: Triple-negative breast cancer; Epithelial-mesenchymal transition; Vimentin; Smooth muscle actin; Osteonectin; N-cadherin; E-cadherin

Strong Correlation of Indoleamine 2,3-Dioxygenase 1 Expression with Basal-Like Phenotype and Increased Lymphocytic Infiltration in Triple-Negative Breast Cancer.

Indoleamine 2,3-dioxygenase 1 (IDO1) is an immunosuppressive enzyme involved in tumor immune escape. Blockade of the IDO1 pathway is an emerging modality of cancer immunotherapy. Triple-negative breast cancer (TNBC) lacks established therapeutic targets and may be a good candidate for this novel immunotherapeutic agent. The purpose of this study was to evaluate the clinicopathologic characteristics of the IDO1-expressing TNBC subset. A tissue microarray was constructed from 200 patients with TNBC. Immunohistochemistry (IHC) for IDO1 and TNBC molecular subtype-surrogate markers (AR, GCDFP-15, claudin-3, E-cadherin, CK5/6, and EGFR) was performed using this tissue microarray. Real-time polymerase chain reaction was performed to confirm the IDO1 mRNA expression level in 16 fresh-frozen TNBC samples. Two hundred TNBCs were classified into four subtypes based on surrogate IHC results: 22 luminal androgen receptor type (11.0%), 23 claudin-low type (11.4%), 103 basal-like type (51.5%), and 52 mixed type (26.0%). IDO1 positivity (defined as expression of >10% tumor cells) was observed in 37% of all TNBCs. IDO1 IHC expression was well correlated with mRNA expression. IDO1 positivity was significantly associated with smaller tumor size, dense stromal lymphocytic infiltration, and basal-like phenotype; however, it did not affect the patients' prognosis. IDO1 expression in basal-like TNBCs is considered an immune inhibitory signal that counterbalances active immunity and may reflect the high mutational load of these tumors. Our results suggest which patients with TNBC would be more efficaciously treated with IDO1 blockade.

A novel Ubc9 -dependent pathway regulates SIRT1- ER-α Axis and BRCA1-associated TNBC lung metastasis.

Triple negative breast cancer (TNBC) is a heterogeneous disease and has a higher rate of recurrence and distant metastasis. African-American (AA) women have a higher frequency of BRCA1 mutations and TNBC compared to other populations. Basal-like tumors have a higher rate of brain, lung and distant nodal metastasis more than other TNBC subtypes, contributing to higher mortality rate. Our previous work suggested Ubc9, a SUMO E2-conjugating enzyme to induce proliferation and migration of BRCA1-incompetent TNBC cells and TNBC cell lines established from the pleural effusion metastasis of a woman with TNBC. To understand the downstream signaling axis involved in distant metastasis we have used clinically relevant BRCA1 mutant and lung metastatic TNBC cell lines and our results show deregulated expression of caveolin-1, VEGF and SIRT1 in these cells compared to normal mammary epithelial cells by immunofluorescence analysis. We observed SIRT1 to be induced by wild type BRCA1a and BRCA1a I26A mutant unlike the disease associated Ubc9 binding mutants in TNBC cells. Knock down of Ubc9 induced SIRT1 expression in TNBC and ER-α expression in breast cancer cells. This is the first report demonstrating a role for Ubc9 in repressing both SIRT1 and ER-α expression in BRCA1 associated TNBC cells. It also suggests that the BARD-dependent E3 Ubiquitin ligase and HR (homologous recombination) activity of BRCA1 may not be required for inducing SIRT1 expression. Our results suggest for the first time that in BRCA1 mutant TNBC Ubc9-mediated induction of VEGF, inhibition of caveolin-1, SIRT1 and ER-α expression as a novel molecular mechanism underlying TNBC EMT (epithelial mesenchymal transition) leading to lung metastasis with pleural effusion. Drugs that target Ubc9 to both induce SIRT1 and ER-α or using SIRT1 agonists in combination with chemotherapy can be used as a promising targeted therapeutic approach for treating basal-like metastatic BRCA1-linked TNBC thus reducing the mortality in patients with TNBC.

Knockdown of CAVEOLIN-1 Sensitizes Human Basal-Like Triple-Negative Breast Cancer Cells to Radiation

Background/Aims: Triple-negative breast cancer (TNBC) is a high-risk breast cancer phenotype without specific targeted therapy options and is significantly associated with increased local recurrence in patients treated with radiotherapy. CAVEOLIN-1 (CAV-1)-mediated epidermal growth factor receptor (EGFR) nuclear translocation following irradiation promotes DNA repair and thus induces radiation resistance. In this study, we aimed to determine whether knockdown of CAV-1 enhances the radiosensitivity of basal-like TNBC cell lines and to explore the possible mechanisms. Methods: Western blotting was used to compare protein expression in a panel of breast cancer cell lines. Nuclear accumulation of EGFR as well as DNA repair and damage at multiple time points following irradiation with or without CAV-1 siRNA pretreatment were investigated using western blotting and confocal microscopy. The radiosensitizing effect of CAV-1 siRNA was evaluated using a clonogenic assay. Flowcytometry was performed to analyse cell apoptosis and cell cycle alteration. Results: We found that CAV-1 is over-expressed in basal-like TNBC cell lines and barely expressed in HER-2-positive cells; additionally, we observed that HER-2-positive cell lines are more sensitive to irradiation than basal-like TNBC cells. Our findings revealed that radiation-induced EGFR nuclear translocation was impaired by knockdown of CAV-1. In parallel, radiation-induced elevation of DNA repair proteins was also hampered by pretreatment with CAV-1 siRNA before irradiation. Silencing of CAV-1 also promoted DNA damage 24 h after irradiation. Colony formation assays verified that cells could be radiosensitized after knockdown of CAV-1. Furthermore, G₂/M cell cycle arrest and apoptosis enhancement may also contribute to the radiosensitizing effect of CAV-1 siRNA. Conclusion: Our results support the hypothesis that CAV-1 knockdown by siRNA causes increased radiosensitivity in basal-like TNBC cells. The mechanisms associated with this effect are reduced DNA repair through delayed CAV-1-associated EGFR nuclear accumulation and induction of G₂/M arrest and apoptosis through the combined effects of CAV-1 siRNA and radiation.

C-Jun N-terminal kinase promotes stem cell phenotype in triple-negative breast cancer through upregulation of Notch1 via activation of c-Jun.

c-Jun N-terminal kinase (JNK) plays a vital role in malignant transformation of different cancers, and JNK is highly activated in basal-like triple-negative breast cancer (TNBC). However, the roles of JNK in regulating cancer stem-like cell (CSC) phenotype and tumorigenesis in TNBC are not well defined. JNK is known to mediate many cellular events via activating c-Jun. Here, we found that JNK regulated c-Jun activation in TNBC cells and that JNK activation correlated with c-Jun activation in TNBC tumors. Furthermore, the expression level of c-Jun was significantly higher in TNBC tumors than in non-TNBC tumors, and high c-Jun mRNA level was associated with shorter disease-free survival of patients with TNBC. Thus, we hypothesized that the JNK/c-Jun signaling pathway contributes to TNBC tumorigenesis. We found that knockdown of JNK1 or JNK2 or treatment with JNK-IN-8, an adenosine triphosphate-competitive irreversible pan-JNK inhibitor, significantly reduced cell proliferation, the ALDH1+ and CD44+/CD24- CSC subpopulations, and mammosphere formation, indicating that JNK promotes CSC self-renewal and maintenance in TNBC. We further demonstrated that both JNK1 and JNK2 regulated Notch1 transcription via activation of c-Jun and that the JNK/c-Jun signaling pathway promoted CSC phenotype through Notch1 signaling in TNBC. In a TNBC xenograft mouse model, JNK-IN-8 significantly suppressed tumor growth in a dose-dependent manner by inhibiting acquisition of the CSC phenotype. Taken together, our data demonstrate that JNK regulates TNBC tumorigenesis by promoting CSC phenotype through Notch1 signaling via activation of c-Jun and indicate that JNK/c-Jun/Notch1 signaling is a potential therapeutic target for TNBC.

Abstract 4149: Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1

Abstract BACKGROUND: We previously reported that direct co-culture of triple negative breast cancer cell line MDA-MB-231 and immune cells results in reduction of EGFR expression on cell surface of MDA-MB-231 (AACR 2015 #2349). However, a role of reduction of EGFR on MDA-MB-231 via co-culture with immune cells still remains unclear. Therefore, we evaluated a role of reduction of EGFR on MDA-MB-231 at the immunological point of view by testing expression of immune related genes including PD-L1. METHODS: In order to verify the importance of direct immune cell contact to breast cancer cell, MDA-MB-231 cells were co-cultured directly or indirectly with THP-1 cells (human monocytic cell line) at 1:50 cellular ratios. In order to study indirect co-culture assay, we used cell culture insert to avoid direct cancer cell-immune cell contact. We analyzed gene expression by quantitative real time PCR and membrane protein expression by flow cytometry of EGFR, also other HER family on MDA-MB-231 which is directly or indirectly co-cultured with THP-1. We also evaluated the expression of immune related genes including PD-L1. RESULTS: Both mRNA and protein level of EGFR on MDA-MB-231 cells directly co-cultured with THP-1 were significantly decreased as compared to those with indirectly co-cultured MDA-MB-231 cells. There are no significant differences in EGFR expression between indirectly co-cultured MDA-MB-231 cells and control MDA-MB-231 cells. Importantly, PD-L1 expression on MDA-MB-231 cells directly co-cultured with THP-1 was significantly decreased as compared to that with indirectly co-cultured MDA-MB-231 cells. CONCLUSION: It has been reported that PD-L1 expression in cancers is regulated by phosphatidylinositol 3-kinase (PI3K) and Akt signaling. Thus, our findings may give a novel insight on regulation of PD-L1 expression on cancer cells in tumor microenvironment that tumor infiltrated immune cell directly contact with cancer cells and EGFR down-regulation leads to reduction of PD-L1 expression on cancer cells. Further investigation is needed to elucidate the mechanism of reduction of EGFR by direct immune cell contact to cancer cells and its interaction with modulation of PD-L1 expression. This will provide novel aspects for immune therapy of breast cancer patients. Citation Format: Ayane Yamaguchi, Eiji Suzuki, Kosuke Kawaguchi, Mariko Nishie, Moe Tsuda, Masakazu Toi. Direct immune cell contact to basal-like triple negative breast cancer cells evokes downregulation of EGFR and PD-L1. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4149.

Abstract 4596: Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer

Abstract Although the basal-like 1 (BL1) and BL2 subtypes of triple negative breast cancer (TNBC) share similar genetic and biological characteristics, patients with BL2 type tumors have markedly poorer prognosis. In addition, despite the overexpression of epidermal growth factor receptor (EGFR) characteristically observed in basal-like TNBC, EGFR inhibitors have performed poorly in clinical trials. In order to understand the mechanism of resistance to EGFR inhibition in these tumors, our laboratory utilized cell line models of BL2 (SUM-149 and SUM-229) and BL1 (MDA-MB-468; MM468) TNBC, all of which overexpress EGFR, are PTEN null and exhibit elevated AKT phosphorylation (p-AKT). Compared to MCF10A and MM-468 cells, AKT phosphorylation in the SUM-149 and SUM-229 cell lines was resistant to treatment with the EGFR inhibitor gefitinib, as well as to the PI3K inhibitor BKM120 (BKM) and the mTOR inhibitor KU-0063794 (KU). Even treatment with the allosteric AKT inhibitor MK2206 (MK) only partially reduced p-AKT. RT-PCR and immunoblot analysis of AKT isoform expression showed that in SUM-149 and SUM-229 cells, AKT3 is expressed at higher levels than AKT1 or AKT2 at both the protein and message level. Because AKT3 has a higher IC50 for MK than AKT1 or AKT2, increasing doses of MK were used in an effort to abrogate AKT phosphorylation. The results showed that MK treatment at higher concentrations (5 μM) decreased p-AKT. In addition, shRNA-mediated knock-down of AKT3 sensitized the cells to inhibition of AKT by BKM, KU and MK. Interestingly, MK treatment alone had little effect on colony forming efficiency, even at higher doses, but when added in combination with gefitinib, MK dramatically reduced colony formation. We then investigated the effect of PTEN re-expression on signaling in these cell lines. PTEN re-expression alone had little effect on p-AKT in SUM-149 and SUM-229 cells, but resulted in a dramatic reduction in p-AKT in MM-468 cells. Although PTEN expression alone had little effect on p-AKT in SUM-149 and 229 cells, these cells exhibited decreased p-AKT following treatment with gefitinib and were sensitive to inhibition of AKT by MK. In addition, treatment of SUM-149/PTEN cells with gefitinib markedly reduced colony-forming efficiency. Our laboratory has previously demonstrated that in SUM-149 cells, a set of genes that are essential for survival, including PLK1, are uncoupled from regulation by EGFR. Thus, we examined the effect of AKT inhibition on expression of PLK1 and found that inhibition of AKT or re-expression of PTEN was able to recouple EGFR-mediated regulation of PLK1 expression. Our results demonstrate that PI-3 kinase and mTORC2 drive phosphorylation of AKT1 but not AKT3 in SUM-149 cells, which explains their relative resistance to these targeted drugs. Citation Format: Christiana S. Kappler, Ericka L. Smith, Stephen Ethier. Regulation of epidermal growth factor receptor signaling in triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4596.

Abstract 3928: Caveolin-1: Beyond a marker for basal-like breast cancers

Abstract Introduction: Caveolin-1 (Cav1) is associated with basal-like triple-negative (ER-/PR-/Her2-) breast cancers (TNBC). Its biological contribution to this subtype has not been fully explored and controversy persists regarding the molecular role of Cav1 in carcinogenesis. Experimental Procedures: Thirty-four TNBC (17 Cav1+/17 Cav1-) patients molecularly-profiled with a commercial assay (Caris Life Sciences, AZ) were evaluated retrospectively. Cav1 status was determined by immunohistochemistry (caveolin-1 polyclonal; ≥2+ ≥50%). The majority of specimens (28/34) used for profiling were from primary breast sites and contained ≥50% neoplastic cells. The transcriptomes were profiled using Illumina's HumanHT-12 microarray (v4). Data were normalized using mean normalization procedure. Differential expression analysis was performed using R's Limma package. Pathway analysis was carried out using R's signaling pathway impact analysis (SPIA) package with 69 cancer, immunity, and cell signaling related KEGG pathways. Results: Using a cutoff of two-fold and adjusted p-value of 0.05, we identified 954 genes differentially expressed between Cav1+/- TNBC patients. Included in these were 31 genes which were found to be up-regulated by over five- fold and 3 genes down-regulated by over five fold in Cav1+ TNBC. Genes of notable interest for their role in cell signaling, cell adhesion, tumor invasion and metastasis, included an up-regulation of TGFBR2, SPARC, integrins (ITGA11, ITGB5, ITGBL1), cell adhesion proteins (LAMB3, COL5A3) and molecules which facilitate tumor invasion (LAMB3, MMP1, MMP2, MMP9). In addition, genes found to be down-regulated in Cav1+ patients and notable for their roles in promoting epithelial-mesenchymal-transition (EMT) included Claudin 3(CLD3) and CA125/MUC16 (Mucin 16). We also detected an approximately two-fold down-regulation of CDKN2A in Cav1+ patients. Using SPIA pathway analysis, 12 pathways were found to be differentially activated in Cav1+ vs. Cav1- TNBC. The most differentially activated pathways were the focal adhesion pathway (p = 4.51E-18), PI3k-Akt signaling pathway (p = 2.01E-6) and TGF-β and MAPK signaling pathways (p = 0.005, 0.014, respectively). Conclusions: Differential gene expression patterns and pathway analyses provide evidence for distinct profiles for gene expression between Cav1+/- TNBC. Cav1+ TNBC patients exhibit up-regulation of genes important for cell signaling, extracellular matrix remodeling and tumor invasion, and down-regulation of genes that may facilitate EMT and loss of cell cycle control. The focal adhesion pathway, as well as TGF-β, PI3K and MAPK signaling pathways, were identified as differentially activated among Cav1+/- TNBC. Taken together, these data support the role of Cav1+ in identifying a subtype of TNBC that may have a greater risk for invasion and metastasis. The correlation of this subtype with prognosis and drug response should be investigated in future studies. Citation Format: Rebecca A. Feldman, Zoran Gatalica, Semir Vranic, Ryan Bender, Sandeep Reddy, Anatole Ghazalpour. Caveolin-1: Beyond a marker for basal-like breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3928.

Abstract 3661: ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers

Abstract The retinoic acid (RA) signalling pathway plays an important role in breast cancer progression and has either a pro-tumorigenic or tumor-suppressive role depending upon the effector function of RA-inducible genes that are expressed or epigenetically silenced. To study this paradigm in breast cancer, we focused on a controversial RA-inducible gene, the retinoic acid receptor responder 1 (RARRES1) protein that is often hypermethylated in cancer and has been reported to have tumor-suppressive function in prostate and nasopharyngeal carcinomas. However, in a study focused on a rare subtype of breast cancer, inflammatory breast cancer, RARRES1 is pro-tumorigenic. This functional discrepancy requires further investigation to determine its role in breast cancer in general. First, analysis of patient data sets revealed that RARRES1 is predominantly expressed in triple-negative breast cancers (TNBCs). Knockdown of RARRES1 in claudin-low MDA-MB-231 and basal-like MDA-MB-468 and HCC1937 significantly increased tumor growth and cell proliferation, suggesting RARRES1 has a tumor suppressive function in (TNBC), regardless of position on the differentiation hierarchy. Expression analyses of 24 breast cancer cell lines (including 18 TNBC and 2 normal-like cell lines) revealed that RARRES1 is predominantly expressed in basal-like TNBC cells We found that RARRES1 expression is dependent on, and strongly correlates with, the cancer stem cell marker, and RA-producing, ALDH1A3 in fixed breast cancer patient samples. Immunohistochemistry of the same patient tumor samples revealed RARRES1 expression is localized to the endoplasmic reticulum. Importantly, however, the presence of ALDH1A3 or RA is not sufficient to induce RARRES1 expression. RARRES1 is hypermethylated in claudin-low breast cancer cell lines, and release of this silencing is required for full induction of RARRES1 expression. We have identified sites of regulation by methylation in RARRES1 using Illumina 450K methylation arrays and 5-methylcytosine ChIP. We conclude that RARRES1 is an ALDH1A3/RA-inducible tumor suppressor in TNBC with methylation and expression profiles distinct to the differentiation hierarchy observed in breast cancer. Citation Format: Krysta M. Coyle, Patrick Murphy, Dejan Vidovic, Cheryl A. Dean, Margaret L. Thomas, Derek Clements, Mohammad Sultan, Ahmad Vaghar-Kashani, Carman Giacomantonio, Lucy Helyer, Ian Weaver, Shashi Gujar, Patrick WK Lee, Paola Marcato. ALDH1A3-inducible RARRES1 is a tumor suppressor in triple-negative breast cancer and is methylated in claudin-low breast cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3661.

Abstract 4587: Dynactin dysregulation-induced increase in EGFR on basal-like breast cancer cells

Abstract Aggressive triple-negative basal-like breast cancer (BLBC) cells are often characterized with overabundance of the epidermal growth factor receptor (EGFR). Major objective of our research is to explore the possibility that dysregulation of the function of the dynactin complex as a mechanism of over-abundance of EGFR on BLBC cell surface. Our notion is that molecular defects in the dynactin-mediated retrograde EGFR recycling pathway in the BLBC cells causes the accumulation of this receptor on their cell surface making them hyper-responsive to EGF signaling. Our comparative transcriptome analysis between BLBC cells with higher levels of EGFR and the non-BLBC cells with lower levels of EGFR revealed that the expression of dynactin complex subunits 1, 2, 3, 4 and 6 (DCTN1, 2, 3, 4, 6), are significantly reduced in the EGFR-high aggressive BLBC cells. In addition, EGFR-high BLBC cells also have lower levels of two other dynactin-associated proteins, CLIP1 and LRRK1. Interestingly, elevation of surface EGFR levels in BLBC cells are associated with higher levels of the long non-coding RNA PVT1. Six miRNAs are generated from the precursor of PVT1 RNA during cis-splicing. They are miR-1204, miR-1205, miR-1206, miR-1207-3p, miR-1207-5p and miR-1208. We found that relative levels of these miRNAs are significantly higher in EGFR-high BLBC cells and tissues in comparison to the non-aggressive EGFR-low BLBC cells. Bioinformatics analysis followed by experimental validation revealed that miR-1207-5p inhibits DCTN1, DCTN3 and ACTR1A; DCTN2 is inhibited by miR-1205 and miR-1208. MiR-1208 also inhibits DCTN6 and CLIP1 whereas LRRK1 and CAPZA1 are inhibited by miR-1207-3p and miR-1206, respectively. Moreover beta-spectrin (SPTB), a protein associated with dynactin complex, is also inhibited by miR-1205 and miR-1207-3p. Treatment of the cells with the antagomirs of the miRNAs increased the levels of their respective target proteins with concomitant decrease in the surface level of EGFR. Our data suggest that BLBC cells maintain a robust level of EGFR on the cell surface by repressing the retrograde transport of EGFR through the repression of the proteins in the dynactin complex and thus sustain their higher growth rates. This research is supported in part by DOD-CDMRP IDEA Expansion Grant# BC103645 and NIH/NCI grant 1R21CA181920-01 to GC. Citation Format: Gautam Chaudhuri, Smita Misra. Dynactin dysregulation-induced increase in EGFR on basal-like breast cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4587.

Maximiscin Induces DNA Damage, Activates DNA Damage Response Pathways, and Has Selective Cytotoxic Activity against a Subtype of Triple-Negative Breast Cancer.

Triple-negative breast cancers are highly aggressive, and patients with these types of tumors have poor long-term survival. These breast cancers do not express estrogen or progesterone receptors and do not have gene amplification of human epidermal growth factor receptor 2; therefore, they do not respond to available targeted therapies. The lack of targeted therapies for triple-negative breast cancers stems from their heterogeneous nature and lack of a clear definition of driver defects. Studies have recently identified triple-negative breast cancer molecular subtypes based on gene expression profiling and representative cell lines, allowing for the identification of subtype-specific drug leads and molecular targets. We previously reported the identification of a new fungal metabolite named maximiscin (1) identified through a crowdsourcing program. New results show that 1 has selective cytotoxic efficacy against basal-like 1 MDA-MB-468 cells compared to cell lines modeling other triple-negative breast cancer molecular subtypes. This compound also exhibited antitumor efficacy in a xenograft mouse model. The mechanisms of action of 1 in MDA-MB-468 cells were investigated to identify potential molecular targets and affected pathways. Compound 1 caused accumulation of cells in the G1 phase of the cell cycle, suggesting induction of DNA damage. Indeed, treatment with 1 caused DNA double-strand breaks with concomitant activation of the DNA damage response pathways, indicated by phosphorylation of p53, Chk1, and Chk2. Collectively, these results suggest basal-like triple-negative breast cancer may be inherently sensitive to DNA-damaging agents relative to other triple-negative breast cancer subtypes. These results also demonstrate the potential of our citizen crowdsourcing program to identify new lead molecules for treating the subtypes of triple-negative breast cancer.

Breast cancer subtype dictates DNA methylation and ALDH1A3-mediated expression of tumor suppressor RARRES1.

Breast cancer subtyping, based on the expression of hormone receptors and other genes, can determine patient prognosis and potential options for targeted therapy. Among breast cancer subtypes, tumors of basal-like and claudin-low subtypes are typically associated with worse patient outcomes, are primarily classified as triple-negative breast cancers (TNBC), and cannot be treated with existing hormone-receptor-targeted therapies. Understanding the molecular basis of these subtypes will lead to the development of more effective treatment options for TNBC. In this study, we focus on retinoic acid receptor responder 1 (RARRES1) as a paradigm to determine if breast cancer subtype dictates protein function and gene expression regulation. Patient tumor dataset analysis and gene expression studies of a 26 cell-line panel, representing the five breast cancer subtypes, demonstrate that RARRES1 expression is greatest in basal-like TNBCs. Cell proliferation and tumor growth assays reveal that RARRES1 is a tumor suppressor in TNBC. Furthermore, gene expression studies, Illumina HumanMethylation450 arrays, and chromatin immunoprecipitation demonstrate that expression of RARRES1 is retained in basal-like breast cancers due to hypomethylation of the promoter. Additionally, expression of the cancer stem cell marker, aldehyde dehydrogenase 1A3, which provides the required ligand (retinoic acid) for RARRES1 transcription, is also specific to the basal-like subtype. We functionally demonstrate that the combination of promoter methylation and retinoic acid signaling dictates expression of tumor suppressor RARRES1 in a subtype-specific manner. These findings provide a precedent for a therapeutically-inducible tumor suppressor and suggest novel avenues of therapeutic intervention for patients with basal-like breast cancer.