Claudin-low Triple-negative Breast Cancer Research Articles

HER3 targeting augments the efficacy of panobinostat in claudin-low triple-negative breast cancer cells

Patients with triple-negative breast cancer (TNBC) have a poor prognosis and high relapse rate due to limited therapeutic options. This study was conducted to determine the mechanisms of action of panobinostat, a pan-inhibitor of histone deacetylase (HDAC) and FDA-approved medication for multiple myeloma, in TNBC and to provide a rationale for effective drug combinations against this aggressive disease. RNA sequencing analyses of the claudin-low (CL) TNBC (MDA-MB-231) cells untreated or treated with panobinostat were performed to identify the differentially expressed genes. Adaptive alterations in gene expression were analyzed and validated in additional CL TNBC cells. Tumor xenograft models were used to test the in vivo antitumor activity of panobinostat alone or its combinations with gefitinib, an EGFR-tyrosine kinase inhibitor (TKI). Panobinostat potently inhibited proliferation and induced apoptosis in all TNBC cells tested. However, in CL TNBC cells, this HDAC inhibitor markedly enhanced expression of HER3, which interacted with EGFR to activate both receptors and Akt signaling pathways. Combinations of panobinostat and gefitinib synergistically suppressed CL TNBC cell proliferation and promoted apoptosis in vitro and in vivo. Upregulation of HER3 compromises the efficacy of panobinostat in CL TNBC. Inactivation of HER3 combined with panobinostat represents a practical approach to combat CL TNBC.

ADAM12 abrogation alters immune cell infiltration and improves response to checkpoint blockade therapy in the T11 murine model of triple-negative breast cancer

ABSTRACT Immunosuppressive tumor microenvironment (TME) impedes anti-tumor immune responses and contributes to immunotherapy resistance in triple-negative breast cancer (TNBC). ADAM12, a member of cell surface metalloproteases, is selectively upregulated in mesenchymal/claudin-low TNBCs, where its expression is largely restricted to tumor cells. The role of cancer cell-expressed ADAM12 in modulating the immune TME is not known. We show that Adam12 knockout in the T11 mouse syngeneic transplantation model of claudin-low TNBC leads to decreased numbers of tumor-infiltrating neutrophils (TINs)/polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) and increased numbers of tumor-infiltrating B cells and T cells. ADAM12 loss in cancer cells increases chemotaxis of B cells in vitro and this effect is eliminated by inhibition of CXCR4, a receptor for CXCL12, or anti-CXCL12 blocking antibody. Importantly, ADAM12 loss in T11 cancer cells sensitizes tumors to anti-PD1/anti-CTLA4 combination therapy, although the initial responsiveness is followed by acquired therapy resistance. Depletion of B cells in mice eliminates the improved response to immune checkpoint blockade of Adam12 knockout T11 tumors. Analysis of gene expression data for claudin-low TNBCs from the METABRIC patient cohort shows significant inverse correlations between ADAM12 and gene expression signatures of several anti-tumor immune cell populations, as well as a significant positive correlation between ADAM12 and gene expression signature of TINs/PMN-MDSCs. Collectively, these results implicate ADAM12 in immunosuppression within the TME in TNBC.

MALT1 Is a Targetable Driver of Epithelial-to-Mesenchymal Transition in Claudin-Low, Triple-Negative Breast Cancer.

This study nominates a GPCR/MALT1 signaling axis as a pathway that can be pharmaceutically targeted to abrogate EMT and metastatic progression in TNBC, an aggressive form of breast cancer that currently lacks targeted therapies.

Targeting PVT1 Exon 9 Re-Expresses Claudin 4 Protein and Inhibits Migration by Claudin-Low Triple Negative Breast Cancer Cells.

Simple SummaryTriple negative breast cancer accounts for 10–15% of all breast cancers. Specific molecular characteristics have led to the identification of six subtypes of triple negative breast cancer, with one in particular being claudin-low. PVT1, a non-protein coding gene, has been demonstrated to play an oncogenic role in various cancers. Specifically, PVT1 exon 9 has been shown to have oncogenic capability. In this study, we aimed to assess the role of PVT1 exon 9 in triple negative breast cancer cells. We have observed that siRNA targeting of PVT1 exon 9 in claudin-low triple negative breast cancer cells resulted in re-expression of claudin 4 protein, and inhibition of migration. These findings indicate that PVT1 exon 9 regulates claudin 4 expression and migration in triple negative breast cancer cells.PVT1 is a long non-coding RNA transcribed from a gene located at the 8q24 chromosomal region that has been implicated in multiple cancers including breast cancer (BC). Amplification of the 8q24 chromosomal region is a common event in BC and is associated with poor clinical outcomes. Claudin–low (CL) triple negative breast cancer (TNBC) is a subtype of BC with a particularly dismal outcome. We assessed PVT1 exon 9 expression in the T47D estrogen receptor positive BC cell line, and in the MDA MB 468 and MDA MB 231 TNBC cell lines, followed by the assessment of the expression of claudins 1, 3, 4 and 7, in MDA MB 468 and MDA MB 231 (TNBC) cells. We found that MDA MB 231 TNBC cells significantly express less claudin 1, 3, 4, and 7 than MDA MB 468 TNBC cells. PVT1 exon 9 is significantly upregulated in MDA MB 231 CL TNBC cells, and significantly downregulated in MDA MB 468 claudin high (CH) TNBC cells, in comparison to T47D estrogen receptor positive BC cells. We then analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells. Notably, siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. Taken together, our data indicate that PVT1 exon 9 regulates claudin 4 expression and migration in CL TNBC cells, and may have clinical implications in CL TNBC.

Abstract PO-126: PVT1 exon 9 transcript regulates claudin 4 expression and migration in triple negative breast cancer

Abstract Breast cancer (BC) is a heterogeneous disease that is classically driven by the estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (EGFR2/HER2) signaling pathways. Triple negative breast cancer (TNBC), is a lethal subtype of invasive BC tumors that are ER-, PR- and HER2-. A subtype of TNBC is claudin low (CL). Dysregulation of claudin proteins disrupts tight junctions, consequently inducing the epithelial-to-mesenchymal transition (EMT) in cancers. This leads to enhanced motility and metastasis. Patients with CL TNBC have worse prognosis than patients with other BC subtypes. PVT1 is a long noncoding RNA (lncRNA) transcribed from the 8q24 genomic locus that has been implicated in multiple cancers including BC. Amplification of the 8q24 gene locus is a common event in many malignant diseases and is associated with poor clinical outcomes. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC was previously unknown. We assessed PVT1 expression in BC, and we observed that PVT1 exons 4A, 4B, and 9 are significantly upregulated in MDA MB 231 cells (claudin low) and significantly downregulated in MDA MB 468 cells (claudin high), in comparison to T47D (ER+). We have confirmed that claudin expression, specifically claudins 1, 3, 4 and 7, are significantly higher in MDA MB 468 cells and significantly lower in MDA MB 231 cells. Knockdown of PVT1 exon 9 expression in the MDA MB 231 cell line, led to a significant reduction in migration when compared to cells transfected with a control scramble siRNA, indicating that PVT1 exon 9 regulates migration in CL TNBC. Interestingly, we observed that claudin 4 expression, and not claudins 1, 3 and 7, was increased in cells where PVT1 exon 9 was knocked down when compared to the control cells. This indicates that PVT1 exon 9 regulates claudin 4 protein stability in CL TNBC. We also assessed the expression of EMT markers (vimentin, fibronectin, and E-cadherin) in MDA MB 231 cells. We observed no changes in EMT markers when PVT1 exon 9 is knocked down; however, our data suggests that EMT markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC. Citation Format: Fayola Levine, Olorunseun Ogunwobi. PVT1 exon 9 transcript regulates claudin 4 expression and migration in triple negative breast cancer [abstract]. In: Proceedings of the AACR Virtual Conference: Thirteenth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2020 Oct 2-4. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(12 Suppl):Abstract nr PO-126.

Abstract 1401: PVT1 exon 9 transcript regulates claudin 4 expression and migration in triple negative breast cancer

Abstract Breast cancer (BC) is a heterogeneous disease that is classically driven by the estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (EGFR2/HER2) signaling pathways. Triple negative breast cancer (TNBC), is a lethal subtype of invasive BC tumors that are ER-, PR- and HER2-. A subtype of TNBC is claudin low (CL). Dysregulation of claudin proteins disrupts tight junctions, consequently inducing the epithelial-to-mesenchymal transition (EMT) in cancers. This leads to enhanced motility and metastasis. Patients with CL TNBC have worse prognosis than patients with other BC subtypes. PVT1 is a long noncoding RNA (lncRNA) transcribed from the 8q24 genomic locus that has been implicated in multiple cancers including BC. Amplification of the 8q24 gene locus is a common event in many malignant diseases and is associated with poor clinical outcomes. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC was previously unknown. We assessed PVT1 expression in BC, and we observed that PVT1 exons 4A, 4B, and 9 are significantly upregulated in MDA MB 231 cells (claudin low) and significantly downregulated in MDA MB 468 cells (claudin high), in comparison to T47D (ER+). We have confirmed that claudin expression, specifically claudins 1, 3, 4 and 7, are significantly higher in MDA MB 468 cells and significantly lower in MDA MB 231 cells. Knockdown of PVT1 exon 9 expression in the MDA MB 231 cell line, led to a significant reduction in migration when compared to cells transfected with a control scramble siRNA, indicating that PVT1 exon 9 regulates migration in CL TNBC. Interestingly, we observed that claudin 4 expression, and not claudins 1, 3 and 7, was increased in cells where PVT1 exon 9 was knocked down when compared to the control cells. This indicates that PVT1 exon 9 regulates claudin 4 protein stability in CL TNBC. We also assessed the expression of EMT markers (vimentin, fibronection, and caveolin) in MDA MB 231 cells. We observed no changes in EMT markers when PVT1 exon 9 is knocked down; however, our data suggests that EMT markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC. Citation Format: Fayola A. Levine, Olorunseun O. Ogunwobi. PVT1 exon 9 transcript regulates claudin 4 expression and migration in triple negative breast cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1401.

Abstract B071: PVT1 regulation of claudin expression in triple-negative breast cancer

Abstract Breast cancer (BC) is a heterogeneous disease that is classically driven by the estrogen receptor (ER), progesterone receptor (PR) and human epithelial growth factor receptor 2 (EGFR2/HER2) signaling pathways. Triple negative breast cancer (TNBC), is a lethal subtype of invasive BC tumors that are ER-, PR- and HER2-. A subtype of TNBC is claudin low (CL). Dysregulation of claudin proteins disrupts tight junctions, consequently inducing the epithelial-to-mesenchymal transition (EMT) in cancers. This leads to enhanced motility and metastasis. Patients with CL TNBC have worse prognosis than patients with other BC subtypes. African American (AA) women with TNBC account for almost 20% of all BC cases and premenopausal African American (AA) women are more likely to die from the disease. PVT1 is a long noncoding RNA (lncRNA) transcribed from the 8q24 genomic locus that has been implicated in multiple cancers including BC. Amplification of the 8q24 gene locus is a common event in many malignant diseases and is associated with poor survival rate among patients. Although previous research demonstrates a critical functional role which PVT1 plays in BC, the underlying molecular mechanisms of PVT1 in CL TNBC was previously unknown. We assessed PVT1 expression in BC, and we observed that PVT1 exons 4A, 4B, and 9 are significantly upregulated in MDA MB 231 cells (claudin low) and significantly downregulated in MDA MB 468 cells (claudin high), in comparison to T47D (ER+). We have confirmed that claudin expression, specifically claudins 1, 3, 4 and 7, are higher in MDA MB 468 and lower in MDA MB 231. When PVT1 exon 9 expression is silenced in the MDA MB 231 CL TNBC cell line, we observed a significant reduction in migration when compared to cells transfected with a control scramble siRNA, suggesting that PVT1 exon 9 may be regulating migration in CL TNBC. Further investigation could lead to an understanding of the molecular mechanisms by which PVT1 exon 9 regulates claudin expression and consequently clinical outcome in TNBC. Citation Format: Fayola Levine, Olorunseun Ogunwobi. PVT1 regulation of claudin expression in triple-negative breast cancer [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr B071.

Functional Link between miR-200a and ELK3 Regulates the Metastatic Nature of Breast Cancer.

Triple-negative breast cancer (TNBC) refers to breast cancer that does not have receptors for estrogen, progesterone, and HER2 protein. TNBC accounts for 10–20% of all cases of breast cancers and is characterized by its metastatic aggressiveness, poor prognosis, and limited treatment options. Here, we show that the metastatic nature of TNBC is critically regulated by a functional link between miR-200a and the transcription factor ELK3. We found that the expression levels of miR-200a and the ELK3 mRNA were negatively correlated in the luminal and TNBC subtypes of breast cancer cells. In vitro experiments revealed that miR-200a directly targets the 3’ untranslated region (UTR) of the ELK3 mRNA to destabilize the transcripts. Furthermore, ectopic expression of miR-200a impaired the migration and invasion of TNBC cells by reducing the expression level of the ELK3 mRNA. In in vivo studies, transfection of MDA-MB 231 cells (a claudin-low TNBC cell type) with exogenous miR-200a reduced their extravasation into the lung during 48 h after tail vein injection, and co-transfection of the cells with an expression plasmid harboring ELK3 that lacked an intact 3’UTR recovered their extravasation ability. Overall, our findings provide evidences that miR-200a and ELK3 is functionally linked to regulate invasive characteristics of breast cancers.

MYC regulates fatty acid metabolism through a multigenic program in claudin-low triple negative breast cancer

BackgroundRecent studies have suggested that fatty acid oxidation (FAO) is a key metabolic pathway for the growth of triple negative breast cancers (TNBCs), particularly those that have high expression of MYC. However, the underlying mechanism by which MYC promotes FAO remains poorly understood.MethodsWe used a combination of metabolomics, transcriptomics, bioinformatics, and microscopy to elucidate a potential mechanism by which MYC regulates FAO in TNBC.ResultsWe propose that MYC induces a multigenic program that involves changes in intracellular calcium signalling and fatty acid metabolism. We determined key roles for fatty acid transporters (CD36), lipases (LPL), and kinases (PDGFRB, CAMKK2, and AMPK) that each contribute to promoting FAO in human mammary epithelial cells that express oncogenic levels of MYC. Bioinformatic analysis further showed that this multigenic program is highly expressed and predicts poor survival in the claudin-low molecular subtype of TNBC, but not other subtypes of TNBCs, suggesting that efforts to target FAO in the clinic may best serve claudin-low TNBC patients.ConclusionWe identified critical pieces of the FAO machinery that have the potential to be targeted for improved treatment of patients with TNBC, especially the claudin-low molecular subtype.

Abstract 2147: Galectin-1: A marker for claudin-low breast cancer

Abstract Metastasis and mortality among patients with triple negative breast cancer (TNBC) of the claudin-low subtype remain high because these tumors are clinically aggressive, chemoresistant, and lack targeted therapies. We have previously used proteomic approaches to analyze orthotopic and metastatic tumors from the syngeneic murine T11 tumor model, which displays gene expression profiles that mirror human claudin-low TNBC. These studies identified Galectin-1 (Gal-1), an N-acetyllactosamine-binding protein, as an overexpressed protein in cultured T11 cells, primary tumors, and lung metastases compared to normal lung and mammary fat pad tissue from healthy control mice. These results were confirmed by Western immunoblot and immunohistochemistry and were further supported by analysis of murine tumor microarray data sets, which revealed Gal-1 overexpression in the claudin-low subtype compared to other breast cancer subtypes. Here we extend our murine studies to investigate Gal-1 expression in breast cancer patients, with specific inclusion of claudin-low tumors. To compare differential Gal-1 expression across breast cancer subtypes, we initially analyzed published human microarray data sets from normal breast and primary breast tumors (total n=423, of which 51 are the claudin-low subtype): the highest Gal-1 expression was observed in claudin-low breast cancer. In order to validate these findings, we are examining breast cancer specimens from a unique cohort of ethnically diverse patients from rural Eastern North Carolina with a median follow-up of over 8 years. RNASeq data from a subset of this cohort (n=121) was used to assign subtypes (using PAM50 and the claudin-low classifier). Further studies are in progress to compare Gal-1 expression between subtypes and correlate with clinicopathologic factors and outcome. Immunohistochemical Gal-1 staining of primary tumor tissue blocks is underway to confirm expression in each subtype at the protein level. Immunohistochemistry will also define Gal-1 expression in the cell types comprising the heterogeneous tumor microenvironment (tumor, stromal, immune cells) and its subcellular localization (nuclear, cytoplasmic, membrane-associated). Through this ongoing work evaluating Gal-1 across breast cancer subtypes, we will help corroborate initial findings from the T11 claudin-low mouse model and yield further insights into the role of Gal-1 in breast cancer progression. Citation Format: Kassondra Balestrieri, Moses McDaniel, Christiana Shoopman, Nasreen Vohra, Kathryn Verbanac. Galectin-1: A marker for claudin-low breast cancer [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 2147.

Abstract A25: TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer

Abstract Lacking marked expression of human epidermal growth factor receptor 2 (HER2), estrogen receptor (ER), and progesterone receptor (PR), triple-negative breast cancer (TNBC) is a breast cancer subtype in desperate need of targeted therapy options. Tumor endothelial marker 8 (TEM8), initially identified as a tumor endothelium marker in colon cancer, has been shown to be upregulated in TNBC. To confirm this, we stained primary TNBC tissues for TEM8; in all cases TEM8 was expressed with no expression in normal breast tissue. TEM8 is expressed by TNBC cell lines as indicated by flow cytometry and Western blot. We thus engineered chimeric antigen receptor (CAR) T cells to specifically target TEM8 in TNBC. TEM8 CAR T cells distinctly recognized TEM8, secreted immunostimulatory cytokines, and killed TEM8-positive TNBC cells in vitro. In vivo, the adoptive transfer of TEM8 CAR T cells induced regression against orthotopic patient-derived xenograft (PDX) models, including the aggressive claudin-low TNBC PDX, WHIM12. Systemic administration of TEM8 CAR T cells also induced regression against a lung metastasis TNBC model. In all models, treatment with TEM8 CAR T cells resulted in a survival advantage in mice compared to controls. Hence, TEM8 may serve as an attractive targeted immunotherapy of TNBC. Citation Format: Tiara Byrd, Kristen Fousek, Antonella Pignata, Christopher Szot, Heba Samaha, Lacey Dobrolecki, Htoo Zarni Oo, Poul Sorensen, Matthew Ellis, Michael Lewis, Meenakshi Hegde, Bradley Fletcher, Brad St. Croix, Nabil Ahmed. TEM8 specific CAR T cells induce regression of patient-derived xenograft and metastatic models of triple-negative breast cancer [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr A25.

Abstract 2703: Proteomic identification of Galectin-1 in murine primary and metastatic triple-negative breast tumors

Abstract The purpose of this study was to identify proteins associated with Triple Negative Breast Cancer (TNBC) metastases using a proteomics approach, in order to gain insight into pathways and mechanisms that could help identify biomarkers or targets for intervention. For our investigations, we used a murine claudin-low tumor model that mirrors human claudin-low TNBC molecular profiles, T11. Cultured T11 cells were lysed, trypsin-digested, and analyzed by liquid chromatography mass spectrometry (LC/MS). A total of 104 proteins were identified with >95% confidence using Protein Pilot software. Among these proteins was Galectin-1 (Gal-1); seven distinct Gal-1 peptides were identified with protein coverage of 58%. Galectins are a family of ß-galactoside-binding proteins that share homology in the carbohydrate binding domain. Gal-1 was selected for further investigation due to its reported roles in cancer progression and immune evasion. Gal-1 expression was next examined in naïve murine lung tissue and in lung metastases from mice orthotopically implanted with T11. Lung tissue was homogenized, trypsin-digested and analyzed by LC/MS. Although Gal-1 was not identified in naïve lungs, three Gal-1 peptides (28% protein coverage) were identified with >95% confidence in lungs with macrometastases. LC/MS analysis did not identify any other galectin family members in primary tumor or lung metastases. Matrix-assisted laser desorption ionization (MALDI) imaging of trypsin-digested tissue sections revealed two distinct Gal-1 peptides localized to tumor regions within metastatic lung tissue. Immunohistochemistry (IHC) analysis showed higher Gal-1 expression in orthotopic T11 primary tumors compared to naïve mammary fat pad, and also higher Gal-1 expression in T11 lung metastases compared to naïve lung. Within tumors, Gal-1 was detected in extracellular matrix, in the cytoplasm of tumor cells and associated with tumor cell membranes. These findings are supported by our analysis of both murine and human genomic data sets, which showed Gal-1 overexpression in claudin-low TNBC compared to other breast cancer subtypes and normal breast tissue. There are several reports that exogenous Gal-1 induces an M2-like phenotype in myeloid cells, consistent with our prior findings that primary tumors, pre-metastatic and metastatic lungs exhibit significant M2-like myeloid cell infiltrates. Future investigations in our lab will focus on determining the role of tumor-produced Gal-1 in the regulation of myeloid cell function to promote cancer progression. Citation Format: Kassondra Balestrieri, Kim Kew, Mohamed Ramez, Keith Pittman, Nasreen Vohra, Kathryn Verbanac. Proteomic identification of Galectin-1 in murine primary and metastatic triple-negative breast tumors [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 2703.

Panobinostat suppresses the mesenchymal phenotype in a novel claudin-low triple negative patient-derived breast cancer model.

Claudin-low triple negative breast cancer (CL-TNBC) is a clinically aggressive molecular TNBC subtype characterized by a propensity to metastasize, recur and acquire chemoresistance. CL-TNBC has a diverse intra- and extracellular composition and microenvironment, and currently there are no clinically approved targeted therapies. Histone deacetylase inhibitors (HDACi) have been investigated as therapeutic agents targeting invasive TNBC phenotypes. However, further studies are required to evaluate HDAC inhibition in CL-TNBC. Here, we utilize a novel CL- TNBC patient-derived xenograft model to study the various and diverse therapeutic potential targets within CL-TNBC tumors. To evaluate effects of the pan-HDACi panobinostat on metastasis and the mesenchymal phenotype of CL-TNBC, we utilize immunohistochemistry staining and qRT-PCR in in vitro, ex vivo and in vivo studies. Further, we evaluate pan-HDAC inhibition on stem-like subpopulations using 3D mammosphere culture techniques and quantification. Finally, we show that pan- HDACi suppresses collagen expression in CL-TNBC. In this study, we provide evidence that pan-HDAC inhibition has effects on various components of the CL-TNBC subtype, and we demonstrate the potential of our novel CL-TNBC PDX model in therapeutic discovery research.

A novel patient-derived xenograft model for claudin-low triple-negative breast cancer.

Triple-negative breast cancer (TNBC) subtypes are clinically aggressive andcannot be treated with targeted therapeutics commonly used in other breast cancer subtypes. The claudin-low (CL) molecular subtype of TNBC has high rates of metastases, chemoresistance and recurrence. There exists an urgent need to identify novel therapeutic targets in TNBC; however, existing models utilized in target discovery research are limited. Patient-derived xenograft (PDX) models have emerged as superior models for target discovery experiments because they recapitulate features of patient tumors that are limited by cell-line derivedxenograft methods. We utilize immunohistochemistry, qRT-PCR and Western Blot to visualize tumor architecture, cellular composition, genomic and protein expressions of a new CL-TNBC PDX model (TU-BcX-2O0). We utilize tissue decellularization techniques to examine extracellular matrix composition of TU-BcX-2O0. Our laboratory successfully established a TNBC PDX tumor, TU-BCX-2O0, which represents a CL-TNBC subtype and maintains this phenotype throughout subsequent passaging. We dissected TU-BCx-2O0 to examine aspects of this complex tumor that can be targeted by developing therapeutics, including the whole andintact breast tumor, specific cell populations within the tumor, andthe extracellular matrix. Here, we characterize a claudin-low TNBC patient-derived xenograft model that can be utilized for therapeutic research studies.

Neutralization of IL-8 decreases tumor PMN-MDSCs and reduces mesenchymalization of claudin-low triple-negative breast cancer.

The complex signaling networks of the tumor microenvironment that facilitate tumor growth and progression toward metastatic disease are becoming a focus of potential therapeutic options. The chemokine IL-8 is overexpressed in multiple cancer types, including triple-negative breast cancer (TNBC), where it promotes the acquisition of mesenchymal features, stemness, resistance to therapies, and the recruitment of immune-suppressive cells to the tumor site. The present study explores the utility of a clinical-stage monoclonal antibody that neutralizes IL-8 (HuMax-IL8) as a potential therapeutic option for TNBC. HuMax-IL8 was shown to revert mesenchymalization in claudin-low TNBC models both in vitro and in vivo as well as to significantly decrease the recruitment of polymorphonuclear myeloid-derived suppressor cells (PMN-MDSCs) at the tumor site, an effect substantiated when used in combination with docetaxel. In addition, HuMax-IL8 enhanced the susceptibility of claudin-low breast cancer cells to immune-mediated lysis with NK and antigen-specific T cells in vitro. These results demonstrate the multifaceted way in which neutralizing this single chemokine reverts mesenchymalization, decreases recruitment of MDSCs at the tumor site, assists in immune-mediated killing, and forms the rationale for using HuMax-IL8 in combination with chemotherapy or immune-based therapies for the treatment of TNBC.

H-Ferritin Enriches the Curcumin Uptake and Improves the Therapeutic Efficacy in Triple Negative Breast Cancer Cells.

Triple negative breast cancer (TNBC) is a highly aggressive, invasive, and metastatic tumor. Although it is reported to be sensitive to cytotoxic chemotherapeutics, frequent relapse and chemoresistance often result in treatment failure. In this study, we developed a biomimetic nanodrug consisting of a self-assembling variant (HFn) of human apoferritin loaded with curcumin. HFn nanocage improved the solubility, chemical stability, and bioavailability of curcumin, allowing us to reliably carry out several experiments in the attempt to establish the potential of this molecule as a therapeutic agent and elucidate the mechanism of action in TNBC. HFn biopolymer was designed to bind selectively to the TfR1 receptor overexpressed in TNBC cells. HFn-curcumin (CFn) proved to be more effective in viability assays compared to the drug alone using MDA-MB-468 and MDA-MB-231 cell lines, representative of basal and claudin-low TNBC subtypes, respectively. Cellular uptake of CFn was demonstrated by flow cytometry and label-free confocal Raman imaging. CFn could act as a chemosensitizer enhancing the cytotoxic effect of doxorubicin by interfering with the activity of multidrug resistance transporters. In addition, CFn exhibited different cell cycle effects on these two TNBC cell lines, blocking MDA-MB-231 in G0/G1 phase, whereas MDA-MB-468 accumulated in G2/M phase. CFn was able to inhibit the Akt phosphorylation, suggesting that the effect on the proliferation and cell cycle involved the alteration of PI3K/Akt pathway.

Abstract 3890: Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance

Abstract Cytotoxic chemotherapy is the standard of care for patients with triple negative breast cancer (TNBC). Most patients with advanced TNBC progress after chemotherapy and die from metastatic disease. MCL1 is an anti-apoptotic Bcl-2 family member known to sequester and inactivate pro-apoptotic Bcl-2 family proteins and, thus, contribute to chemotherapy resistance. We previously reported that ~45% of residual TNBCs that remain in the breast after neoadjuvant chemotherapy harbor MCL1 amplification, suggesting a causal role for MCL1 in drug resistance. A recent report (Goodwin et al. 2015) suggested that siRNA-mediated ablation of MCL1 does not induce apoptosis in claudin-low TNBC cells with a cancer stem cell (CSC) gene expression signature. CSCs comprise a rare population of cells with tumor-initiating properties and refractoriness to chemotherapy. In this study, we showed that MCL1 expression is elevated in claudin-low TNBC SUM159PT and MDA436 CSCs as measured by ALDH+ by flow cytometry and ability to form mammospheres. RNA interference of MCL1 in SUM159PT cells reduced CSCs and attenuated tumor formation in vivo. Mitochondrial oxidative phosphorylation (mtOXPHOS) plays a crucial role in maintenance of CSCs. MCL1 has been shown to localize in the mitochondrial matrix and contribute to mitochondrial respiration. Thus, we hypothesized that MCL1 contributes to enrichment of TNBC CSCs and chemotherapy resistance via mitochondrial regulation. Stable transfection and overexpression of MCL1 in MDA468 cells increased oxygen consumption ratio, mitochondrial membrane potential, and production of reactive oxygen species (ROS), all features of activated mtOXPHOS. Conversely, RNAi-mediated ablation of MCL1 in SUM159PT and MDA436 cells repressed these markers of activated mtOXPHOS. A mutant of MCL1 lacking its mitochondrial target sequences (MTS) was unable to localize in mitochondria and, when transfected into MDA468 cells, reduced the CD44high/CD24low fraction and mammosphere formation. We next tested VU0659158, a BH3 mimetic in development at Vanderbilt that disrupts MCL1 interactions with BH3 domain-containing proteins, such as BID, BIM, NOXA and PUMA. Treatment of SUM159PT cells with VU0659158 increased caspase activity but did not attenuate mammosphere formation. Analysis of mRNA expression in TCGA revealed that genes induced by mtOXPHOS involved in the hypoxia pathway are significantly up-regulated in MCL1 amplified breast cancers. Finally, pharmacological inhibition of HIF-1α, a key regulator of hypoxia, with digoxin decreased CSCs and attenuated tumor formation in vivo. These data suggest that 1) MCL1 confers resistance to chemotherapy by expanding CSCs via mtOXPHOS independent of its BH3 domain-mediated, anti-apoptotic function, and 2) targeting mitochondrial respiration and the hypoxia pathway may delay or reverse chemotherapy resistance in MCL1 amplified TNBC. Citation Format: Kyung-min Lee, Jennifer Giltnane, Justin Balko, Luis Schwarz, Angel Guerrero, Katie Hutchinson, Mellissa Hicks, Violeta Sanchez, Melinda Sanders, Taekyu Lee, Edward Olejniczak, Stephen Fesik, Carlos Arteaga. Mitochondrial MCL1 maintains triple negative breast cancer stem cells and contributes to chemotherapy resistance [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 3890. doi:10.1158/1538-7445.AM2017-3890

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

Abstract 5051: Comparison of microbeam versus conventional broadbeam radiation therapy on tumor delivery enhancement of PEGylated liposomal doxorubicin in a triple negative breast cancer mouse model

Abstract Background: Recent meta-analyses have reported that the tumor delivery of nanoparticles (NPs) is lower than expected and inefficient. Thus, there is a strong need to develop new methods to enhance the tumor delivery of NPs without increasing toxicity. Microbeam radiation therapy (MRT) is an experimental therapy utilizing an array of parallel microplaner X-ray beams to deliver periodically oscillating high and low dose regions in the treatment volume. MRT has been shown to eradicate tumors and causes significantly less toxicity compared to equivalent radiation doses delivered by conventional broadbeam radiation therapy (BRT). Our prior studies reported that MRT significantly altered tumor microvasculature and enhanced the tumor delivery of PEGylated liposomal doxorubicin (PLD) compared to PLD alone in mice (Chang et al. AACR 2015). In this study we compared MRT and the clinically widely used BRT in their ability to enhance the tumor delivery of PLD in a preclinical murine tumor model. Methods: Plasma and tumor PK studies of PLD were performed in a genetically engineered mouse model of claudin-low triple-negative breast cancer (T11) in 4 treatment regimens: 1) PLD alone, 2) BRT 28 Gy + PLD, 3) MRT 28 Gy + PLD, 4) MRT 100 Gy + PLD. Mice received a single radiation treatment 24 h prior to administration of PLD at 6 mg/kg IVP x 1 via a tail vein. Following administration of PLD, mice were harvested at 5 min and 24 h. Encapsulated and released doxorubicin concentrations (conc) in plasma and sum total (encapsulated + released) doxorubicin conc in tumor were measured by HPLC with fluorescence. Results: The mean ± SD conc of sum total doxorubicin in tumor at 24 h after administration of PLD alone, BRT 28 Gy + PLD, MRT 28 Gy + PLD, and MRT 100 Gy + PLD were 2,575 ± 459, 8,601 ± 1,552*, 7,579 ± 4,428, and 12,911 ± 3,445* ng/mL, respectively (*P<0.05 vs PLD alone). In addition, the ratio of tumor sum total conc to plasma encapsulated conc at 24 h for PLD alone, BRT 28 Gy + PLD, MRT 28 Gy + PLD, and MRT 100 Gy + PLD were 0.21 ± 0.04, 0.51 ± 0.17*, 0.39 ± 0.22, and 1.45 ± 0.70*, respectively (*P<0.05 vs PLD alone). The exposures of sum total doxorubicin in tumor at 5 min were similar in all groups. The plasma PK of PLD was also similar in all groups. Conclusions: MRT and BRT administered 24 h prior to PLD increased the tumor exposure of sum total doxorubicin compared to PLD alone with 100Gy MRT having the greatest increase in tumor delivery of PLD. In addition, MRT exhibits significantly lower toxicity to normal tissues in comparison to BRT allowing for enhanced PLD tumor delivery with low toxicity. Studies are ongoing to evaluate the mechanism(s) for the enhanced tumor uptake of PLD induced by MRT. Future studies include investigating dose dependence of MRT-induced tumor delivery enhancement and effects on other NP agents and tumor models. Citation Format: Sha X. Chang, Judith N. Rivera, Leah B. Herity, Lauren S. Price, Andrew J. Madden, Jose R. Roques, Charlene Santos, David Darr, William C. Zamboni. Comparison of microbeam versus conventional broadbeam radiation therapy on tumor delivery enhancement of PEGylated liposomal doxorubicin in a triple negative breast cancer mouse model [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 5051. doi:10.1158/1538-7445.AM2017-5051

Genome-wide effects of MELK-inhibitor in triple-negative breast cancer cells indicate context-dependent response with p53 as a key determinant

Triple-negative breast cancer (TNBC) is an aggressive, highly recurrent breast cancer subtype, affecting approximately one-fifth of all breast cancer patients. Subpopulations of treatment-resistant cancer stem cells within the tumors are considered to contribute to disease recurrence. A potential druggable target for such cells is the maternal embryonic leucine-zipper kinase (MELK). MELK expression is upregulated in mammary stem cells and in undifferentiated cancers, where it correlates with poor prognosis and potentially mediates treatment resistance. Several MELK inhibitors have been developed, of which one, OTSSP167, is currently in clinical trials. In order to better understand how MELK and its inhibition influence TNBC, we verified its anti-proliferative and apoptotic effects in claudin-low TNBC cell lines MDA-MB-231 and SUM-159 using MTS assays and/or trypan blue viability assays together with analysis of PARP cleavage. Then, using microarrays, we explored which genes were affected by OTSSP167. We demonstrate that different sets of genes are regulated in MDA-MB-231 and SUM-159, but in both cell lines genes involved in cell cycle, mitosis and protein metabolism and folding were regulated. We identified p53 (TP53) as a potential upstream regulator of the regulated genes. Using western blot we found that OTSSP167 downregulates mutant p53 in all tested TNBC cell lines (MDA-MB-231, SUM-159, and BT-549), but upregulates wild-type p53 in the luminal A subtype MCF-7 cell line. We propose that OTSSP167 might have context-dependent or off-target effects, but that one consistent mechanism of action could involve the destabilization of mutant p53.