Signaling In Triple-negative Breast Cancer Research Articles

Thymoquinone inhibits cell proliferation, migration, and invasion by regulating the elongation factor 2 kinase (eEF-2K) signaling axis in triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is the most aggressive and chemoresistant subtype of breast cancer. Therefore, new molecular targets and treatments need to be developed to improve poor patient prognosis and survival. We have previously shown that eukaryotic elongation factor 2 kinase (eEF-2K) is highly expressed in TNBC cells, is associated with poor patient survival and prognosis, and promotes cell proliferation, migration, and invasion. In vivo targeting of eEF-2K significantly reduces the tumor growth of orthotopic TNBC xenograft mouse models, suggesting that eEF-2K may serve as a potential novel therapeutic target. In the current study, we identified thymoquinone (TQ), an active ingredient of Nigella sativa, as a potential safe and effective eEF-2K inhibitor in TNBC. We demonstrated for the first time that TQ inhibits the protein and mRNA expression of eEF-2K, as well as the clinically relevant downstream targets, including Src/FAK and Akt, and induces the tumor suppressor miR-603, in response to NF-kB inhibition. This effect was associated with a significant decrease in the proliferation, colony formation, migration, and invasion of TNBC cells. Furthermore, systemic in vivo injection of TQ (20 and 100mg/kg) significantly reduced the growth of MDA-MB-231 tumors and inhibited the eEF-2K expression in an orthotopic tumor model in mice. Our study provides first evidence that TQ treatment inhibits cell proliferation, migration/invasion, and tumor growth, in part through the inhibition of eEF-2K signaling in TNBC. Thus, our findings suggest that systemic TQ treatment may be used as a targeted therapeutic strategy for the inhibition of eEF-2K in TNBC tumor growth and progression.

Abstract 1900: Disruption of EGFR and integrin b1 signaling in triple-negative breast cancer through targeting SHC1

Abstract Triple-negative breast cancer (TNBC) accounts for 15-20% of breast cancer cases and is more prevalent among young women and African American women. SHC1 (Src Homology 2 Domain Containing Transforming Protein 1) is an adaptor protein that can transmit cell surface receptor signaling and activate the Ras-ERK pathway. SHC1 is found to be overexpressed in breast cancers and is important in the regulation of tumor progression and drug resistance in ER+ breast cancer. Elevated SHC1 correlates with poor prognosis in breast cancer patients. However, the biological significance of SHC1 in TNBC is not known. In this study, we performed siRNA-mediated knockdown to determine the importance of SHC1 in cell proliferation and drug response in TNBC. We found that although siSHC1 slightly inhibited the tumor growth of TNBC cells, siSHC1 + paclitaxel significantly decreased cell proliferation and increased caspase-3/7 activity in vitro. In invasion assays, siSHC1 significantly decreased the invasion of TNBC cells. To better understand the systemic regulation by SHC1, proteomic analysis was performed after siSHC1 or Scr transfection. Pathway analysis revealed the involvement of PI3K/AKT, FAK, and actin cytoskeleton pathways, by disruption of EGFR and integrin β1 signaling, in the control of TNBC progression and drug sensitivity. In tumor explant studies, siSHC1 significantly enhanced paclitaxel-induced tumor shrinkage and reduced lung metastasis. Our data suggest that SHC1 is a potential therapeutic target in TNBC. Citation Format: Ya-Fang Chang, Elizabeth Cordell, Yuhao Wang, Geoffrey Greene. Disruption of EGFR and integrin b1 signaling in triple-negative breast cancer through targeting SHC1 [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 1900.

Abstract 5470: IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative breast cancer

Abstract Metastatic breast cancer carries a poor prognosis despite the success of newly targeted therapies and treatment options remain especially limited for the subtype of triple negative breast cancer (TNBC). Several signaling pathways, including NF-κB, are altered in TNBC. Given that ΙΚΚε behaves as an oncogene in breast cancer we hypothesized that IKKε regulates classical or alternative NF-κB signaling to control diverse oncogenic functions in TNBC. Vector expression and RNA interference were used to investigate the functional role of IKKε in triple-negative breast cancer cells. Viability, protein expression, NF-κB binding activity, invasion, anoikis, and spheroid formation were examined in cells expressing high or low levels of IKKε in conjunction with alternative NF-κB p52 RNA interference or MEK inhibition. Our data show that p52 protein levels and binding activity are inversely proportional to ΙΚΚε. Moreover, knockdown of IKKε leads to increased expression of p52 and the p52-regulated gene CXCL1. This interaction was confirmed by CHiP-PCR experiments showing increased binding of p52 in the CXCL1 promoter of cells with IKKε knockdown relative to control. We further found that IKKε and MEK were required for growth in anchorage supportive conditions whereas p52 was required in anchorage resistant conditions. Western blots confirm diminished IKKε expression and enhanced p52 processing in anchorage resistant conditions relative to anchorage supportive conditions. In this model, IKKε and MEK cooperate to support growth of cancer cells in the attached, anchored environment, whereas p52 is required for growth in unattached 3-D environment. These studies reveal novel information about the role of IKKε in TNBC and highlight the adaptability of NF-κB signaling in maintaining cancer cell survival under different growth conditions. Current studies are underway to clarify the mechanism of IKKε regulation of p52. A better understanding of the diversity of NF-κB signaling may ultimately improve the development of novel therapeutic regimens for TNBC. Citation Format: Carrie D. House, Valentina Grajales, Michelle Ozaki, Elizabeth Jordan, Helmae Wubneh, Danielle Kimble, Marianne Kim, Christina M. Annunziata. IKK-epsilon supports anchorage independent growth via alternative NF-kB signaling in triple-negative 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 5470.

Preferential Inhibition of Wnt/β-Catenin Signaling by Novel Benzimidazole Compounds in Triple-Negative Breast Cancer.

Wnt/β-catenin signaling is upregulated in triple-negative breast cancer (TNBC) compared to other breast cancer subtypes and normal tissues. Current Wnt/β-catenin inhibitors, such as niclosamide, target the pathway nonspecifically and exhibit poor pharmacokinetics/pharmacodynamics in vivo. Niclosamide targets other pathways, including mTOR, STAT3 and Notch. Novel benzimidazoles have been developed to inhibit Wnt/β-catenin signaling with greater specificity. The compounds SRI33576 and SRI35889 were discovered to produce more cytotoxicity in TNBC cell lines than in noncancerous cells. The agents also downregulated Wnt/β-catenin signaling mediators LRP6, cyclin D1, survivin and nuclear active β-catenin. In addition, SRI33576 did not affect mTOR, STAT3 and Notch signaling in TNBC and noncancerous cells. SRI35889 inhibited mTOR signaling less in noncancerous than in cancerous cells, while not affecting STAT3 and Notch pathways. Compounds SRI32529, SRI35357 and SRI35361 were not selectively cytotoxic against TNBC cell lines compared to MCF10A cells. While SRI32529 inhibited Wnt/β-catenin signaling, the compound also mitigated mTOR, STAT3 and Notch signaling. SRI33576 and SRI35889 were identified as cytotoxic and selective inhibitors of Wnt/β-catenin signaling with therapeutic potential to treat TNBC in vivo.

Characterization and Targeting Cancer Stem‐like Cells in Triple Negative Breast Cancer

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive diseases. TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore, CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of canonical‐Notch signaling in TNBC, however the role of non‐canonical Notch signaling in TNBC remains unknown. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal (M) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is dependent on recruitment to the cIAP‐2 promoter of NF‐κB subunits, IKKα and Notch1. Short term exposure of MDA‐MB‐231 cells (M, PTEN wild‐type), but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by AKT inhibitors, IKK inhibitors, and dual mTORC1/2 inhibitors but not an mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Importantly, we find co‐localization of Notch1 with Mitochondria in MDA‐MB‐231 cells by confocal microscopy and Western blot of isolated mitochondrial fractions. We demonstrate that recombinant Jagged1 increases the metabolism of TNBC cells. Knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, and oxidative metabolism compared to non‐stem cells. AKT inhibition or IKK inhibition decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by gamma secretase inhibitor (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or IKK inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low sorted secondary mammospheres formation. Notably, we find similar results in TNBC patient derived xenograft (PDX) models. However, Notch inhibition may have adverse effects on tumor immunity, due to the role of Notch in CD4 and CD8 T‐cell activation. Thus, we explored an immunocompetent tumor mouse model to study the effects of these pharmacological interventions in that setting. We developed an immunocompetent TNBC mouse model by injecting TNBC mouse cells (C0321) into mammary fat pads of isogenic FVB female mice. Combination treatments with GSI (PF‐03084014) and AKT inhibitor (MK‐2206) significantly delayed tumor growth as well as decreased CSCs number in that model. These data suggest that combination treatments affecting the intersection of Notch and AKT pathways have potential therapeutic applications in targeting CSCs in TNBC cases with high Notch1 expression. The possible immunological effects of such combination treatments require additional investigation.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Nitro-fatty acid inhibition of triple-negative breast cancer cell viability, migration, invasion, and tumor growth

Triple-negative breast cancer (TNBC) comprises ∼20% of all breast cancers and is the most aggressive mammary cancer subtype. Devoid of the estrogen and progesterone receptors, along with the receptor tyrosine kinase ERB2 (HER2), that define most mammary cancers, there are no targeted therapies for patients with TNBC. This, combined with a high metastatic rate and a lower 5-year survival rate than for other breast cancer phenotypes, means there is significant unmet need for new therapeutic strategies. Herein, the anti-neoplastic effects of the electrophilic fatty acid nitroalkene derivative, 10-nitro-octadec-9-enoic acid (nitro-oleic acid, NO2-OA), were investigated in multiple preclinical models of TNBC. NO2-OA reduced TNBC cell growth and viability in vitro, attenuated TNFα-induced TNBC cell migration and invasion, and inhibited the tumor growth of MDA-MB-231 TNBC cell xenografts in the mammary fat pads of female nude mice. The up-regulation of these aggressive tumor cell growth, migration, and invasion phenotypes is mediated in part by the constitutive activation of pro-inflammatory nuclear factor κB (NF-κB) signaling in TNBC. NO2-OA inhibited TNFα-induced NF-κB transcriptional activity in human TNBC cells and suppressed downstream NF-κB target gene expression, including the metastasis-related proteins intercellular adhesion molecule-1 and urokinase-type plasminogen activator. The mechanisms accounting for NF-κB signaling inhibition by NO2-OA in TNBC cells were multifaceted, as NO2-OA (a) inhibited the inhibitor of NF-κB subunit kinase β phosphorylation and downstream inhibitor of NF-κB degradation, (b) alkylated the NF-κB RelA protein to prevent DNA binding, and (c) promoted RelA polyubiquitination and proteasomal degradation. Comparisons with non-tumorigenic human breast epithelial MCF-10A and MCF7 cells revealed that NO2-OA more selectively inhibited TNBC function. This was attributed to more facile mechanisms for maintaining redox homeostasis in normal breast epithelium, including a more favorable thiol/disulfide balance, greater extents of multidrug resistance protein-1 (MRP1) expression, and greater MRP1-mediated efflux of NO2-OA-glutathione conjugates. These observations reveal that electrophilic fatty acid nitroalkenes react with more alkylation-sensitive targets in TNBC cells to inhibit growth and viability.

New Insights into the Role of Exercise in Inhibiting mTOR Signaling in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) does not express estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 and is characterized by its aggressive nature, lack of targets for targeted therapies, and early peak of recurrence. Due to these specific characteristics, chemotherapy does not usually yield substantial improvements and new target therapies and alternative strategies are needed. The beneficial responses of TNBC survivors to regular exercise, including a reduction in the rate of tumor growth, are becoming increasingly apparent. Physiological adaptations to exercise occur in skeletal muscle but have an impact on the entire body through systemic control of energy homeostasis and metabolism, which in turn influence the TNBC tumor microenvironment. Gaining insights into the causal mechanisms of the therapeutic cancer control properties of regular exercise is important to improve the prescription and implementation of exercise and training in TNBC survivors. Here, we provide new evidence of the effects of exercise on TNBC prevention, control, and outcomes, based on the inhibition of the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (PKB also known as Akt)/mammalian target of rapamycin (mTOR) (PI3K-Akt-mTOR) signaling. These findings have wide-ranging clinical implications for cancer treatment, including recurrence and case management.

Targeting the androgen receptor in triple-negative breast cancer: current perspectives.

Triple-negative breast cancer (TNBC) is an aggressive subtype associated with frequent recurrence and metastasis. Unlike hormone receptor-positive subtypes, treatment of TNBC is currently limited by the lack of clinically available targeted therapies. Androgen signaling is necessary for normal breast development, and its dysregulation has been implicated in breast tumorigenesis. In recent years, gene expression studies have identified a subset of TNBC that is enriched for androgen receptor (AR) signaling. Interference with androgen signaling in TNBC is promising, and AR-inhibiting drugs have shown antitumorigenic activity in preclinical and proof of concept clinical studies. Recent advances in our understanding of androgenic signaling in TNBC, along with the identification of interacting pathways, are allowing development of the next generation of clinical trials with AR inhibitors. As novel AR-targeting agents are developed and evaluated in clinical trials, it is equally important to establish a robust set of biomarkers for identification of TNBC tumors that are most likely to respond to AR inhibition.

PAD1 promotes epithelial-mesenchymal transition and metastasis in triple-negative breast cancer cells by regulating MEK1-ERK1/2-MMP2 signaling

Peptidylargininedeiminase 1 (PAD1) catalyzes protein for citrullination, and this activity has been linked to the epidermal cornification. However, a role for PAD1 in tumorigenesis, including breast cancers has not been previously explored. Here we first showed that PAD1 is overexpressed in human triple negative breast cancer (TNBC). In cultured cells and xenograft mouse models, PAD1 depletion or inhibition reduced cell proliferation, suppressed epithelial-mesenchymal transition, and prevented metastasis of MDA-MB-231 cells. These changes were correlated with a dramatic decrease in MMP2/9 expression. Furthermore, ERK1/2 and P38 MAPK signaling pathways are activated upon PAD1 silencing. Treatment with MEK1/2 inhibitor in PAD1 knockdown cells significantly recovered MMP2 expression, while inhibiting P38 activation only slightly elevated MMP9 levels. We then showed that PAD1 interacts with and citrullinates MEK1 thereby disrupting MEK1-catalyzed ERK1/2 phosphorylation, thus leading to the MMP2 overexpression. Collectively, our data indicate that PAD1 appears to promote tumorigenesis by regulating MEK1-ERK1/2-MMP2 signaling in TNBC. These results also raise the possibility that PAD1 may function as an important new biomarker for TNBC tumors and suggest that PAD1-specific inhibitors could potentially be utilized to treat metastatic breast cancer.

Expression of the MHC class II in triple-negative breast cancer is associated with tumor-infiltrating lymphocytes and interferon signaling.

Tumor-infiltrating lymphocytes (TILs) have been known for their strong prognostic and predictive significance in triple-negative breast cancer (TNBC). Several mechanisms for TIL influx in TNBC have been elucidated. Major histocompatibility complex class II (MHC-II) is an essential component of the adaptive immune system and is generally restricted to the surface of antigen-presenting cells. However, it has been reported that interferon-gamma signaling may induce MHC-II in almost all cell types, including those derived from cancer. We aimed to examine the relationship between MHC-II expression in tumor cells and the amount of TILs in 681 patients with TNBC. Further, the prognostic significance of MHC-II and the association of MHC-II with a couple of molecules involved in the interferon signaling pathway were investigated using immunohistochemical staining. Higher MHC-II expression in tumor cells was associated with the absence of lymphovascular invasion (p = 0.042); larger amounts of TILs (p < 0.001); frequent formations of tertiary lymphoid structures (p < 0.001); higher expression of myxovirus resistance gene A, one of the main mediators of the interferon signaling pathway (p < 0.001); and higher expression of double-stranded RNA-activated protein kinase, which can be induced by interferons (p = 0.008). Moreover, tumors that showed high MHC class I expression and any positivity for MHC-II had larger amounts of CD4- and CD8-positive T lymphocytes (p < 0.001). Positive MHC-II expression in tumor cells was associated with better disease-free survival in patients who had lymph node metastasis (p = 0.009). In conclusion, MHC-II expression in tumor cells was closely associated with an increase in TIL number and interferon signaling in TNBC. Further studies are warranted to improve our understanding regarding TIL influx, as well as patients’ responses to immunotherapy.

Abstract 1507: Fatty acid oxidation mediated autophagy regulation in triple negative breast cancer

Abstract Compared to hormone regulated/responsive (ER+) breast cancer (BC), triple negative BC (TNBC) patients have a worse overall survival, a significantly shorter disease-free survival, and a shorter post-recurrence survival. Using transmitochondrial cybrid (cybrid) (compares different mitochondria under a common defined nuclear background)-based discovery and validation in cell lines, patient derived xenografts (PDXs) and BC clinical tissues/data, we recently published that fatty acid oxidation (FAO) is a major energy pathway in TNBC. We also showed that FAO is a major regulator of Src activation by autophosphorylation at Src Y419 in metastatic TNBC. Our data suggest that inhibition of FAO induce cell growth inhibition. Short-term treatment of FAO inhibitors induces both cell cycle arrest and apoptosis. We observed cell cycle arrest in G1-S phase in FAO inhibitor treated TNBC cells. Apoptotic markers like cleaved Caspase-3 and cleaved PARP induced in FAO inhibited cells. Short-term treatment of FAO inhibitor also showed induction of AKT pathway. Further analysis on the role of FAO in TNBC suggests that FAO is a critical player in autophagy signaling in TNBC. Long-term treatment with FAO inhibitors or knockdown of FAO rate-limiting proteins carnitine palmitoyltransferase I (CPT1) induce autophagic cell death with beclin-1 and LC3-II induction in TNBC. However, no such phenomenon observed in estrogen receptor positive (ER+) breast cancer cell lines. Treatment with autophagy inhibitor 3-methyladenine (3-MA) abolished the FAO-induced cell growth inhibition and reduced beclin-1 and LC3-II induction. Ongoing studies using knock-down and over expression approaches focus on the role of specific pathways that are responsible for mitochondrial energy reprogramming regulated autophagy signaling in metastatic TNBC. Citation Format: Kwang Hwa Jung, Jun Hyoung Park, Tirupataiah Sirupangi, Sajna Vithayathil, Lee-Jun Wong, Benny A. Kaipparettu. Fatty acid oxidation mediated autophagy regulation in 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 1507. doi:10.1158/1538-7445.AM2017-1507

Abstract 4302: Disulfiram possesses antitumor activity and inhibits cancer stem-like properties via inhibition of STAT3 activation in triple-negative breast cancer cells

Abstract Disulfiram (DSF), a clinically used treatment for alcohol dependence, is considered as a potential anti-cancer agent due to its cytotoxicity in many cancer cell types on copper (Cu)-dependent processes. However, the molecular mechanisms underlying DSF-induced antitumor activity in triple-negative breast cancer (TNBC) are still poorly understood. Here, we hypothesized that DSF induces antitumor effects by targeting cancer stem cells (CSCs) and STAT3 signaling in TNBC. We then examined the effects of DSF in combination with Cu on the cell viability and apoptosis, cell proliferation and survival, as well as CSC-like properties in MDA-MB-231 and 4T1 TNBC cell lines. DSF/Cu effectively induced cytotoxicity and apoptotic cell death, concomitant with caspase-3 activation and PARP cleavage. DSF/Cu also significantly suppressed cell migration and mammosphere formation, which are accompanied by a remarkable decrease in the CD44+/CD24- (or CD49f+/CD24+ in mouse 4T1 cells) stem-like population and ALDH1 activity. These effects were functionally associated with the inhibition of STAT3 phosphorylation (Tyr705). Furthermore, DSF/Cu treatment resulted in the downregulation of cyclin D1 and survivin expression, which are downstream targets of STAT3 signaling. Interestingly, phosphorylated STAT3 and STAT3 downstream signaling were highly upregulated in the mammospheres, and these upregulated molecules were significantly decreased by DSF/Cu treatment. In MDA-MB-231-derived xenograft model, DSF administration elicited a significant reduction in tumor growth with a noticeable downregulation of CD44, ALDH1 and phosphorylated STAT3 levels. Taken together, our results suggest that therapeutic treatment to DSF can effectively manifest antitumor activity by reducing CSC-like properties, and that STAT3 could be a key molecule of aggressive TNBC that is targeted by DSF. Note: This abstract was not presented at the meeting. Citation Format: Yoon-Jae Kim, Nahyun Lee, Eunhye Oh, Daeil Sung, Tae-Min Cho, Ji Young Kim, Jae Hong Seo. Disulfiram possesses antitumor activity and inhibits cancer stem-like properties via inhibition of STAT3 activation in triple-negative breast cancer cells [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 4302. doi:10.1158/1538-7445.AM2017-4302

Abstract 1334: Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in triple negative breast cancer

Abstract Recent studies correlate Hedgehog (Hh) signaling with reduced survival rates in triple negative breast cancer (TNBC) patients. Activation of hedgehog signaling in the adjacent mesenchyme has been shown to promote tumor growth and it is a poor prognosis factor for TNBC. We developed a novel tumor-mesenchymal in vitro model of hedgehog signaling in TNBC to evaluate the role of mesenchymal cell sub-populations in the proliferative potential and stem cell markers of breast cancer cells using a custom designed multiwell array. As a source of mesenchymal cell sub-populations we evaluated myofibroblasts, mesenchymal stem cells derived from bone marrow or adipose tissue and tumor cells that undergo epithelial-mesenchymal transition (EMT). Tumor cells were culture with 1 or 2 mesenchymal cell sub-populations in adjacent compartments +/- SHH ligand for 72-96hrs. Active Hh signaling was confirmed by up-regulation of main Hh target genes (e.g. Gli1, Patch1). Paracrine Hh signaling only in bone marrow-derived mesenchymal stem cell and EMT significantly increased proliferation (10-15%) of TNBC and normal breast cancer cell lines. Addition of adipose-derived mesenchymal stem cells or pharmacological Hh inhibitors (Cyclopamine and Gant61) partially abolished tumor growth indicating that other non-canonical signals are involve in paracrine Hh-driven tumors. Cancer stem cell markers were selectively modulated in co-cultures. Our results suggest that paracrine Hh signaling-driven by bone marrow and EMT mesenchymal are potential therapeutic targets to treating TNBC. Citation Format: Karla P. Ramos, Maribella Domenech. Mesenchymal cell sub-populations selectively modulate paracrine hedgehog signaling in 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 1334. doi:10.1158/1538-7445.AM2017-1334

Non‐cannonical notch signaling pathways regulate breast cancer stem‐like cells function in triple negative breast cancer

Triple negative breast cancer (TNBC) is a heterogeneous group of clinically aggressive form of breast cancers, characterized by lack of expression of estrogen receptor α (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2).TNBC patients have high risk of recurrence and metastasis, and current treatment options remain limited. Cancer stem‐like cells (CSCs) have been linked to cancer initiation, progression and chemotherapy resistance. Therefore CSC‐targeted therapies are keenly sought. There is strong evidence for the involvement of Notch signaling in TNBC. Notch1 is highly expressed in Basal‐like 1 (BL1) and especially Mesenchymal‐Stem‐Like (MSL) TNBCs. Expression of Notch1 and its ligand Jagged1 correlate with poor prognosis. Moreover, strong evidence supports key roles of different Notch paralogs in breast CSCs. Here, we demonstrate that Notch1 promotes cell survival in MDA‐MB‐231 cells, representative of MSL TNBC, in part by activating NF‐κB. Notch activation by Jagged1‐expressing stromal cells enhances transcription of the anti‐apoptotic gene cIAP‐2 (BIRC3), a known NF‐κB target. This event is rely on Notch dependent recruitment of NF‐κB subunits, IKKα to the cIAP‐2 promoter. Short term exposure of MDA‐MB‐231 cells [MSL, phosphatase and tensin homolog (PTEN) wild‐type], but not MDA‐MB‐468 cells (BL1, PTEN‐null) to recombinant Jagged1 leads to AKT phosphorylation. This is suppressed by gamma secretase inhibitor (GSI), dual mTORC1/2 inhibitors, AKT inhibitors and IKKα inhibitors but not mTORC1‐selective inhibitor. These observations support a model where canonical and non‐canonical mechanisms downstream of Notch1 trigger AKT phosphorylation and NF‐κB activation in PTEN wild type TNBC cells. Rapid AKT phosphorylation downstream of Notch1 requires mTORC2, PI3K and IKKα, and contributes to NF‐κB activation. This suggests a bidirectional crosstalk between the IKKα and AKT arms of this Jagged1‐activated Notch pathway. We demonstrate that recombinant Jagged1 increases the cellular metabolism of TNBC cells and knockdown of Notch1 or IKKα by siRNA decreases mitochondrial respiration and glycolysis. We have found that CSCs derived from MDA‐MB‐231 cells have increased Notch1, p‐AKT, p‐IKKα, and oxidative metabolism. AKT inhibition or IKKα inhibition alone or in combination with GSI (PF‐03084014) decreases both mitochondrial respiration and glycolysis of TNBC derived CSCs. Pharmacological inhibition of Notch cleavage by GSI (PF‐03084014) in combination with AKT inhibitor (MK‐2206) or NF‐κB inhibitor (Bay11‐7082) blocks CD90hi or CD44+CD24low (CSCs marker) sorted secondary mammospheres formation. These data suggest that blocking of non‐canonical Notch‐ NF‐κB ‐AKT pathways have potential therapeutic importance in targeting CSCs of TNBC. Our data indicate that therapeutic regimens targeting this pathway deserve further investigation in TNBCs.Support or Funding InformationFunding: NIH 3P01CA166009‐04S1

AR Signaling in Breast Cancer.

Androgen receptor (AR, a member of the steroid hormone receptor family) status has become increasingly important as both a prognostic marker and potential therapeutic target in breast cancer. AR is expressed in up to 90% of estrogen receptor (ER) positive breast cancer, and to a lesser degree, human epidermal growth factor 2 (HER2) amplified tumors. In the former, AR signaling has been correlated with a better prognosis given its inhibitory activity in estrogen dependent disease, though conversely has also been shown to increase resistance to anti-estrogen therapies such as tamoxifen. AR blockade can mitigate this resistance, and thus serves as a potential target in ER-positive breast cancer. In HER2 amplified breast cancer, studies are somewhat conflicting, though most show either no effect or are associated with poorer survival. Much of the available data on AR signaling is in triple-negative breast cancer (TNBC), which is an aggressive disease with inferior outcomes comparative to other breast cancer subtypes. At present, there are no approved targeted therapies in TNBC, making study of the AR signaling pathway compelling. Gene expression profiling studies have also identified a luminal androgen receptor (LAR) subtype that is dependent on AR signaling in TNBC. Regardless, there seems to be an association between AR expression and improved outcomes in TNBC. Despite lower pathologic complete response (pCR) rates with neoadjuvant therapy, patients with AR-expressing TNBC have been shown to have a better prognosis than those that are AR-negative. Clinical studies targeting AR have shown somewhat promising results. In this paper we review the literature on the biology of AR in breast cancer and its prognostic and predictive roles. We also present our thoughts on therapeutic strategies.

The Androgen Receptor Promotes Cellular Proliferation by Suppression of G-Protein Coupled Estrogen Receptor Signaling in Triple-Negative Breast Cancer

Background/Aims: The targeted therapy for triple-negative breast cancer (TNBC) is still challenging due to poor understanding on its molecular etiology. The androgen receptor (AR) has recently emerged as a prognostic and treatment-predictive marker in breast cancer. However, the role of AR in TNBC remained elusive. Methods: Immunohistochemistry (IHC) was used to detect AR and G-protein coupled estrogen receptor (GPER) expression in tissue microarrays of 165 TNBC patients. Microarray analysis of mRNAs was performed to identify downstream regulators of AR. TNBC cells were cultured with dihydrotestosterone (DHT) alone or in combination with AR knockdown performed with AR shRNA. Cell viability and colony formation were assessed. Western blotting and qRT-PCR were used to examine protein and mRNA expression, respectively. The potential mechanism of AR-mediated GPER suppression was identified by Chromatin immunoprecipitation (ChIP) assay. AR and GPER expressions were also assessed in nude mouse xenografts by IHC. Results: IHC staining showed that the expression of AR was positively associated with tumor size, lymph node metastasis and high-grade tumor in TNBC patients. AR activation triggered by DHT suppressed GPER expression, to promote cell growth of TNBC. G-1, a GPER agonist, inhibited DHT-stimulated proliferation. Further experiments illustrated that AR suppressed GPER activation via binding directly to the promoter of GPER. Moreover, a negative correlation between AR and GPER was observed in MDA-MB-231 tumor cell xenografts and TNBC patient samples. Conclusions: The suppression of GPER via AR may be involved in the positive actions towards the TNBC progression, making it a promising therapeutic target for TNBC treatment.

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.

Aurora A kinase activates YAP signaling in triple-negative breast cancer.

The Yes-associated protein (YAP) is an effector that transduces the output of the Hippo pathway to transcriptional modulation. Considering the role of YAP in cancers, this protein has emerged as a key node in malignancy development. In this study, we determined that Aurora A kinase acts as a positive regulator for YAP-mediated transcriptional machinery. Specifically, YAP associates with Aurora A predominantly in the nucleus. Activation of Aurora A can impinge on YAP activity through direct phosphorylation. Moreover, aberrant expression of YAP and Aurora A signaling is highly correlated with triple-negative breast cancer (TNBC). We herein provide evidence to establish the functional relevance of this newly discovered regulatory axis in TNBC.

The hedgehog pathway in triple-negative breast cancer.

Treatment of triple‐negative breast cancer (TNBC) remains challenging due to the underlying heterogeneity of this disease coupled with the lack of predictive biomarkers and effective targeted therapies. Intratumoral heterogeneity, particularly enrichment for breast cancer stem cell‐like subpopulations, has emerged as a leading hypothesis for systemic therapy resistance and clinically aggressive course of poor prognosis TNBC. A growing body of literature supports the role of the stem cell renewal Hedgehog (Hh) pathway in breast cancer. Emerging preclinical data also implicate Hh signaling in TNBC pathogenesis. Herein, we review the evidence for a pathophysiologic role of Hh signaling in TNBC and explore mechanisms of crosstalk between the Hh pathway and other key signaling networks as well as their potential implications for Hh‐targeted interventions in TNBC.

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.