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Corticosteroids act on the glucocorticoid receptor (GR; NR3C1) to resolve inflammation and are routinely prescribed to breast cancer patients undergoing chemotherapy treatment to alleviate side effects. Triple negative breast cancers (TNBCs) account for 15-20% of diagnoses and lack expression of estrogen and progesterone receptors as well as amplified HER2, but often express high GR levels. GR is a mediator of TNBC progression to advanced metastatic disease, however the mechanisms underpinning this transition to more aggressive behavior remain elusive. We previously showed that tissue/cellular stress (hypoxia, chemotherapies) as well as factors in the tumor microenvironment (TGFβ, HGF) activate p38 MAPK, which phosphorylates GR on Ser134. In the absence of ligand, p-Ser134-GR further upregulates genes important for responses to cellular stress, including key components of the p38 MAPK pathway. Herein, we show that p-Ser134-GR is required for TNBC metastatic colonization to the lungs of female mice. To understand the mechanisms of p-Ser134-GR action in the presence of GR agonists, we examined glucocorticoid-driven transcriptomes in CRISPR knock-in models of TNBC cells expressing wild-type or phospho-mutant (S134A) GR. We identified dexamethasone- and p-Ser134-GR-dependent regulation of specific gene sets controlling TNBC migration (NEDD9, CSF1, RUNX3) and metabolic adaptation (PDK4, PGK1, PFKFB4). TNBC cells harboring S134A-GR displayed metabolic reprogramming that was phenocopied by PDK4 knockdown. PDK4 knockdown or chemical inhibition also blocked cancer cell migration. Our results reveal a convergence of GR agonists (i.e., host stress) with cellular stress signaling whereby pSer134-GR critically regulates TNBC metabolism, an exploitable target for the treatment of this deadly disease.

Western blot of MDA-MB-231 parental or HIF DKO cells demonstrating loss of regulation of PELP1 expression in response to taxol treatment in cells with HIF double-knockout.

Figure S4 shows that FACS analysis profiles of MDA-MB-231 and MDA-MB-468 TNBC cells following treatment with vehicle (Control), colchicine or paclitaxel, showing VERU-111 induces G2/M checkpoint arrest and apoptosis in TNBC cells. (A) Representative cell cycle distribution images after VERU-111 (10, 20, 50 and 100 nM), colchicine (100 nM) and paclitaxel (100 nM) treatment. Cells were treated for 24 h and stained with anti-phospho-histone H3 (Ser10) and propidium iodide (PI). (B) Representative FACS analysis profiles of apoptotic TNBC cells after 24 h treatment with VERU-111, colchicine or paclitaxel as detected by Annexin-V-FITC/PI staining. (C) Representative FACS analysis profiles of apoptotic TNBC cells after treating with 100 nM VERU-111 for 24, 48 or 72 h, cells were stained with Annexin-V-FITC and PI.

Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of patients with breast cancer; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of triple-negative breast cancer (TNBC) remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e., via knock-out or add-back) had little effect on primary tumor volume, but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in-frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor–stimulated cell motility relative to SH3-del, KM, or wild-type PTK6 controls. Reverse-phase protein array revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6+/AhR+ primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model.Implications:The SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC via RhoA and AhR, identified herein as novel therapeutic targets in PTK6+ breast tumors.

Supplementary Data from Colchicine-Binding Site Agent CH-2-77 as a Potent Tubulin Inhibitor Suppressing Triple-Negative Breast Cancer

Western blot and qPCR data of MDA-MB-231 cells treated for 0-5 days with 5FU, showing increased Brk, HIF-1a, and HIF-2a in response to 5FU.

Table S1 shows anti-proliferating effects of VERU-111 against various human cancer cell lines (mean {plus minus} SEM, n = 4 biological replicate experiments).

Figure legends describing supplementary data

Table S1. Oligonucleotide sequences used in this study. Figure S1. Amino acid sequence used to generate PTK6 domain mutant models. Figure S2. Characterization of PTK6 genetically modified MDA-MB-231 TNBC cell lines. Figure S3. PTK6 silencing reduces tumor-sphere formation. Figure S4: Dose-dependent effects of CT04 or CH223191 on growth inhibition in MDA-MB-231 and cHCI-10 TNBC cells. Figure S5: GR and AHR expression alone are not predictive of patient outcome.

Western blot and qPCR data of C57bl/6 mice treated with vehicle or dexamethasone for 6 hours, demonstrating increased Sik expression upon dex treatment.

TMA data derived from luminal, or adjacent 'normal' tissue, stained for total GR or phospho-GR expression.

Example of immunohistochemical staining of total GR and phospho-GR.

Confirmation of HIF knockout in MDA-MB-231 cells.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC.Significance:These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.

Supplementary Section: 1) Seven supplementary figures and legends: A. Supplementary figure S1: TCGA analysis of WNT10B network. B. Supplementary figure S2: Human IHC analysis of WNT10B, HMGA2 and EZH2. C. Supplementary figure S3: Mouse IHC analysis of HMGA2 and EZH2. D. Supplementary figure S4: ChIP, qPCR and IB analysis on WNT10B/�CATENIN direct target genes. E. Supplementary figure S5: Characterization of CRISPR HMGA2KO and EZH2KO cells both in vitro and in vivo. F. Supplementary figure S6: Validation of WNT10B network on TNBC PDX tumors. G. Supplementary figure S7: Isobologram curves of ICG-001 and Doxorubicin activity. 2) Supplementary Material & Methods 3) Supplementary Table 1 (ST1) qPCR primers 4) Supplementary Table 2 (ST2) ChIP primers 5) 6) Supplementary Table 3 (ST3) drug combinatorial index

DUSP1 and GILZ regulation and GR recruitment in MDA-MB-231 shControl and HIF1/2-shRNA cells.

Figure legends for supplementary figures 1 and 2.

Dex regulation of Brk mRNA and protein in TNBC cell lines and explants.

Clinicopathological characteristics of TNBC explant tumors.

Cancer cells use stress response pathways to sustain their pathogenic behavior. In breast cancer, stress response–associated phenotypes are mediated by the breast tumor kinase, Brk (PTK6), via the hypoxia-inducible factors HIF-1α and HIF-2α. Given that glucocorticoid receptor (GR) is highly expressed in triple-negative breast cancer (TNBC), we investigated cross-talk between stress hormone–driven GR signaling and HIF-regulated physiologic stress. Primary TNBC tumor explants or cell lines treated with the GR ligand dexamethasone exhibited robust induction of Brk mRNA and protein that was HIF1/2–dependent. HIF and GR coassembled on the BRK promoter in response to either hypoxia or dexamethasone, indicating that Brk is a direct GR/HIF target. Notably, HIF-2α, not HIF-1α, expression was induced by GR signaling, and the important steroid receptor coactivator PELP1 was also found to be induced in a HIF-dependent manner. Mechanistic investigations showed how PELP1 interacted with GR to activate Brk expression and demonstrated that physiologic cell stress, including hypoxia, promoted phosphorylation of GR serine 134, initiating a feed-forward signaling loop that contributed significantly to Brk upregulation. Collectively, our findings linked cellular stress (HIF) and stress hormone (cortisol) signaling in TNBC, identifying the phospho-GR/HIF/PELP1 complex as a potential therapeutic target to limit Brk-driven progression and metastasis in TNBC patients. Cancer Res; 76(6); 1653–63. ©2016 AACR.

Simple Summary Targeting dysregulated cellular metabolism is a promising avenue to treat metastatic disease. The aim of our study was to identify genes downstream of the hypoxia-inducible factor (HIF)-1 transcription factor that are amenable to therapeutic intervention to treat metastatic breast cancer (MBC). We identified creatine kinase, brain isoform (CKB) as an HIF-dependent gene that strongly promotes invasion and metastasis in estrogen-receptor (ER)-negative breast cancer models. Deletion of Ckb also repressed glycolysis and mitochondrial respiration, leading to a reduction in intracellular ATP. Either the deletion of Ckb or inhibition of creatine kinase (CK) activity using the creatine analog cyclocreatine (cCr) repressed cell invasion, the formation of invadopodia and lung metastasis. In addition, when paired with paclitaxel or doxorubicin, cCr enhanced growth inhibition in an additive or synergistic manner. cCr may be an effective anti-metastatic agent in ER-negative, HIF-1α-positive breast cancers, targeting both cellular metabolism and motility, particularly when paired with conventional cytotoxic agents. Abstract The oxygen-responsive hypoxia inducible factor (HIF)-1 promotes several steps of the metastatic cascade. A hypoxic gene signature is enriched in triple-negative breast cancers (TNBCs) and is correlated with poor patient survival. Inhibiting the HIF transcription factors with small molecules is challenging; therefore, we sought to identify genes downstream of HIF-1 that could be targeted to block invasion and metastasis. Creatine kinase brain isoform (CKB) was identified as a highly differentially expressed gene in a screen of HIF-1 wild type and knockout mammary tumor cells derived from a transgenic model of metastatic breast cancer. CKB is a cytosolic enzyme that reversibly catalyzes the phosphorylation of creatine, generating phosphocreatine (PCr) in the forward reaction, and regenerating ATP in the reverse reaction. Creatine kinase activity is inhibited by the creatine analog cyclocreatine (cCr). Loss- and gain-of-function genetic approaches were used in combination with cCr therapy to define the contribution of CKB expression or creatine kinase activity to cell proliferation, migration, invasion, and metastasis in ER-negative breast cancers. CKB was necessary for cell invasion in vitro and strongly promoted tumor growth and lung metastasis in vivo. Similarly, cyclocreatine therapy repressed cell migration, cell invasion, the formation of invadopodia and lung metastasis. Moreover, in common TNBC cell line models, the addition of cCr to conventional cytotoxic chemotherapy agents was either additive or synergistic to repress tumor cell growth.

HER2+ breast cancer accounts for 15% of all breast cancer cases. Current frontline therapy for HER2+ metastatic breast cancer relies on targeted antibodies, trastuzumab and pertuzumab, combined with microtubule inhibitors in the taxane class (paclitaxel or docetaxel). It is well known that the clinical efficacy of taxanes is limited by the development of chemoresistance and hematological and neurotoxicities. The colchicine-binding site inhibitors (CBSIs) are a class of promising alternative agents to taxane therapy. Sabizabulin (formerly known as VERU-111) is a potent CBSI that overcomes P-gp-mediated taxane resistance, is orally bioavailable, and inhibits tumor growth and distant metastasis in triple negative breast cancer (TNBC). Herein, we demonstrate the efficacy of sabizabulin in HER2+ breast cancer. In vitro, sabizabulin inhibits the proliferation of HER2+ breast cancer cell lines with low nanomolar IC50 values, inhibits clonogenicity, and induces apoptosis in a concentration-dependent manner. In vivo, sabizabulin inhibits breast tumor growth in the BT474 (ER+/PR+/HER2+) xenograft model and a HER2+ (ER-/PR-) metastatic patient-derived xenograft (PDX) model, HCI-12. We demonstrate that sabizabulin is a promising alternative agent to target tubulin in HER2+ breast cancer with similar anti-metastatic efficacy to paclitaxel, but with the advantage of oral bioavailability and lower toxicity than taxanes.

Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of patients with breast cancer; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of triple-negative breast cancer (TNBC) remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e., via knock-out or add-back) had little effect on primary tumor volume, but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in-frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor–stimulated cell motility relative to SH3-del, KM, or wild-type PTK6 controls. Reverse-phase protein array revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6+/AhR+ primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model. Implications: The SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC via RhoA and AhR, identified herein as novel therapeutic targets in PTK6+ breast tumors.

Triple negative breast cancer (TNBC) patients have higher recurrence rates and a worse prognosis relative to patients diagnosed with other breast cancer subtypes. Protein tyrosine kinase 6 (PTK6; also called Brk), a soluble tyrosine kinase, is overexpressed in 86% of breast cancer patients, however its precise function in the context of TNBC is poorly defined. PTK6 expression is elevated in TNBC models in response to both cellular and endocrine stress, coordinated transcriptionally by the Hypoxia-Inducible Factors (HIFs) and glucocorticoid receptor (GR). We showed previously that PTK6 expression, but not its intrinsic kinase activity, is required for breast cancer cell motility. To further delineate the mechanisms of PTK6 signaling, we created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domains. MDA-MB-231 cells expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were less responsive to growth factor-stimulated cell motility relative to wild type or kinase dead (KM) controls. To identify signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants, we used a reverse phase protein array (RPPA), which revealed that the SH2 domain of PTK6 mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Moreover, in TNBC cells, including a taxane-refractory TNBC model, addition of AhR or Rho inhibitors to paclitaxel (Taxol) enhanced cytotoxicity. Together, these studies reveal that the SH2-domain of PTK6 is an effector of advanced cancer phenotypes in GR+ TNBC cells and identify RhoA and AhR as novel therapeutic targets in PTK6+ tumors.

Triple-negative breast cancers (TNBCs), which lack specific targeted therapy options, evolve into highly chemo-resistant tumors that metastasize to multiple organs simultaneously. We have previously shown that TNBCs maintain an activated WNT10B-driven network that drives metastasis. Pharmacologic inhibition by ICG-001 decreases &beta, catenin-mediated proliferation of multiple TNBC cell lines and TNBC patient-derived xenograft (PDX)-derived cell lines. In vitro, ICG-001 was effective in combination with the conventional cytotoxic chemotherapeutics, cisplatin and doxorubicin, to decrease the proliferation of MDA-MB-231 cells. In contrast, in TNBC PDX-derived cells doxorubicin plus ICG-001 was synergistic, while pairing with cisplatin was not as effective. Mechanistically, cytotoxicity induced by doxorubicin, but not cisplatin, with ICG-001 was associated with increased cleavage of PARP-1 in the PDX cells only. In vivo, MDA-MB-231 and TNBC PDX orthotopic primary tumors initiated de novo simultaneous multi-organ metastases, including bone metastases. WNT monotherapy blocked multi-organ metastases as measured by luciferase imaging and histology. The loss of expression of the WNT10B/&beta, catenin direct targets HMGA2, EZH2, AXIN2, MYC, PCNA, CCND1, transcriptionally active &beta, catenin, SNAIL and vimentin both in vitro and in vivo in the primary tumors mechanistically explains loss of multi-organ metastases. WNT monotherapy induced VEGFA expression in both tumor model systems, whereas increased CD31 was observed only in the MDA-MB-231 tumors. Moreover, WNT-inhibition sensitized the anticancer response of the TNBC PDX model to doxorubicin, preventing simultaneous metastases to the liver and ovaries, as well as to bone. Our data demonstrate that WNT-inhibition sensitizes TNBC to anthracyclines and treats multi-organ metastases of TNBC.

Triple-negative breast cancer (TNBC) accounts for approximately 15% of breast cancer cases in the United States. TNBC has poorer overall prognosis relative to other molecular subtypes due to rapid onset of drug resistance to conventional chemotherapies and increased risk of visceral metastases. Taxanes like paclitaxel are standard chemotherapies that stabilize microtubules, but their clinical efficacy is often limited by drug resistance and neurotoxicities. We evaluated the preclinical efficacy of a novel, potent, and orally bioavailable tubulin inhibitor, VERU-111, in TNBC models. VERU-111 showed potent cytotoxicity against TNBC cell lines, inducing apoptosis and cell-cycle arrest in a concentration-dependent manner. VERU-111 also efficiently inhibited colony formation, cell migration, and invasion. Orally administered VERU-111 inhibited MDA-MB-231 xenograft growth in a dose-dependent manner, with similar efficacies to paclitaxel, but without acute toxicity. VERU-111 significantly reduced metastases originating from the mammary fat pad into lung, liver, and kidney metastasis in an experimental metastasis model. Moreover, VERU-111, but not paclitaxel, suppressed growth of luciferase-labeled, taxane-resistant, patient-derived metastatic TNBC tumors. In this model, VERU-111 repressed growth of preestablished axillary lymph node metastases and lung, bone, and liver metastases at study endpoint, whereas paclitaxel enhanced liver metastases relative to vehicle controls. Collectively, these studies strongly suggest that VERU-111 is not only a potent inhibitor of aggressive TNBC phenotypes, but it is also efficacious in a taxane-resistant model of metastatic TNBC. Thus, VERU-111 is a promising new generation of tubulin inhibitor for the treatment of TNBC and may be effective in patients who progress on taxanes. Results presented in this study demonstrate the efficacy of VERU-111 in vivo and provide strong rationale for future development of VERU-111 as an effective treatment for metastatic breast cancer.

Triple-negative breast cancer (TNBC) commonly develops resistance to chemotherapy, yet markers predictive of chemoresistance in this disease are lacking. Here, we define WNT10B-dependent biomarkers for β-CATENIN/HMGA2/EZH2 signaling predictive of reduced relapse-free survival. Concordant expression of HMGA2 and EZH2 proteins is observed in MMTV-Wnt10bLacZ transgenic mice during metastasis, and Hmga2 haploinsufficiency decreased EZH2 protein expression, repressing lung metastasis. A novel autoregulatory loop interdependent on HMGA2 and EZH2 expression is essential for β-CATENIN/TCF-4/LEF-1 transcription. Mechanistically, both HMGA2 and EZH2 displaced Groucho/TLE1 from TCF-4 and served as gatekeepers for K49 acetylation on β-CATENIN, which is essential for transcription. In addition, we discovered that HMGA2-EZH2 interacts with the PRC2 complex. Absence of HMGA2 or EZH2 expression or chemical inhibition of Wnt signaling in a chemoresistant patient-derived xenograft (PDX) model of TNBC abolished visceral metastasis, repressing AXIN2, MYC, EZH2, and HMGA2 expression in vivo. Combinatorial therapy of a WNT inhibitor with doxorubicin synergistically activated apoptosis in vitro, resensitized PDX-derived cells to doxorubicin, and repressed lung metastasis in vivo. We propose that targeting the WNT10B biomarker network will provide improved outcomes for TNBC. Significance: These findings reveal targeting the WNT signaling pathway as a potential therapeutic strategy in triple-negative breast cancer.

Triple negative breast cancer (TNBC) patients have poorer overall prognosis relative to patients diagnosed with other molecular subtypes due to rapid onset of drug resistance to conventional chemotherapies and increased risk of visceral metastases. The microtubule inhibitor paclitaxel (Taxol, a taxane) is a frontline therapy for advanced breast cancer. We evaluated in TNBC models the preclinical safety and efficacy of a novel, potent, and orally bioavailable tubulin inhibitor, VERU-111, a tubulin inhibitor targeting the colchicine binding site. VERU-111 showed potent anti-proliferative and anti-migratory activity against several taxane-sensitive and taxane-resistant TNBC breast cancer cell lines. Based on these observations, taxane-resistant HER2+ cell lines were generated, and were also found to be responsive to VERU-111 treatment. In vivo, orally administered VERU-111 inhibited MDA-MB-231 tumor growth in a dose-dependent manner with antitumor potency similar to paclitaxel, and repressed metastases originating from the mammary fat pad or following tail vein injection. In contrast, in a MDA-MB-231 paclitaxel-resistant (TxR) subline, tumor growth was refractory to paclitaxel whereas VERU-111 significantly inhibited primary tumor growth and reduced lung and liver metastases. VERU-111 was then tested in a luciferase-labeled, multidrug resistant patient-derived xenograft (PDX) TNBC model. VERU-111 significantly inhibited HCI-10 PDX tumor growth and suppressed the expansion of axillary lymph node metastases present prior to initiation of therapy while suppressing lung, liver, bone and kidney metastases at study endpoint. Moreover, in contrast to paclitaxel, VERU-111 therapy did not cause a significant decrease in mouse body weight during treatment. Evaluation of efficacy of VERU-111 in taxane-sensitive and -resistant HER2+ xenograft models is in progress. Overall, we conclude that VERU-111 is a new generation orally bioavailable tubulin inhibitor that potently inhibits the growth of taxane-sensitive and taxane-resistant breast cancers with reduced adverse side effects relative to paclitaxel. Importantly, VERU-111 is well-tolerated in patients as evaluated in phase I/II clinical trials for advanced prostate cancer patients (NCT03752099). We propose that VERU-111 will be an effective second line therapy for patients with advanced breast cancer who progress on taxane-based therapeutic regimens.

A body of epidemiological evidence implicates exposure to endocrine disrupting chemicals (EDCs) with increased susceptibility to breast cancer. To evaluate the physiological effects of a suspected EDC in vivo, we exposed MCF-7 breast cancer cells and a patient-derived xenograft (PDX, estrogen receptor positive) to physiological levels of methylparaben (mePB), which is commonly used in personal care products as a preservative. mePB pellets (4.4 μg per day) led to increased tumor size of MCF-7 xenografts and ER+ PDX tumors. mePB has been thought to be a xenoestrogen; however, in vitro exposure of 10 nM mePB failed to increase MCF-7 cell proliferation or induction of canonical estrogen-responsive genes (pS2 and progesterone receptor), in contrast to 17β-estradiol (E2) treatment. MCF-7 and PDX-derived mammospheres exhibited increased size and up-regulation of canonical stem cell markers ALDH1, NANOG, OCT4 and SOX2 when exposed to mePB; these effects were not observed for MDA-MB-231 (ER−) mammospheres. As tumor-initiating cells (TICs) are also believed to be responsible for chemoresistance, mammospheres were treated with either tamoxifen or the pure anti-estrogen fulvestrant in the presence of mePB. Blocking the estrogenic response was not sufficient to block NANOG expression in mammospheres, pointing to a non-classic estrogen response or an ER-independent mechanism of mePB promotion of mammosphere activity. Overall, these results suggest that mePB increases breast cancer tumor proliferation through enhanced TIC activity, in part via regulation of NANOG, and that mePB may play a direct role in chemoresistance by modulating stem cell activity. Copyright © 2016 John Wiley & Sons, Ltd.

Triple-negative breast cancer (TNBC) has aggressive clinical features strongly associated with poorer overall prognosis and higher mortality rates relative to other molecular subtypes. FDA-approved drugs, such as paclitaxel, are effective in treating TNBC. Yet, treatment failure is commonly observed due to the development of acquired chemoresistance, which remains a clinical challenge for TNBC therapy. We have shown that orally administered VERU-111 inhibits MDA-MB-231 xenograft growth with similar efficacy to paclitaxel, but without acute toxicity, and represses metastases originating from the mammary fat pad. To better understand the effect of VERU-111 on taxane-resistant TNBC, we generated a panel of paclitaxel-resistant TNBC sublines (MDA-MB-231/TxR, MDA-MB-468/TxR, SUM159/TxR and Hs578T/TxR) by gradually exposing paclitaxel to parental lines. MTS assays were used to confirm the chemoresistance of each subline to paclitaxel. VERU-111 maintained its potency against all four taxane-resistant sublines. We further investigated the underlying mechanisms of VERU-111 action and found that it inhibited taxane-resistant TNBC cell proliferation by inducing mitotic arrest and apoptosis via disruption of microtubule stability. In vivo, relative to paclitaxel, VERU-111 significantly inhibited tumor growth in an orthotopic MDA-MB-231/TxR model and reduced lung metastases originating from the mammary fat pad. VERU-111 induced tumor necrosis, anti-angiogenesis and apoptosis in both taxane-sensitive and taxane-resistant TNBC models in vivo. In addition to the MDA-MB-231/TxR model, we also tested the efficacy of VERU-111 in a multidrug-resistant PDX model, HCI-10. VERU-111 significantly inhibited HCI-10 primary tumor growth and suppressed the growth of lymph node metastases present prior to therapy and suppressed lung, liver and kidney metastases at the endpoint. In contrast, paclitaxel had no efficacy to repress tumor growth or metastasis in this model, and in fact, stimulated liver metastasis. Taken together, our data demonstrate that VERU-111 is a new generation of oral tubulin inhibitor that potently inhibits the growth of taxane-sensitive and taxane-resistant TNBC. Therefore, VERU-111 therapy is a promising strategy for the treatment of metastatic TNBC patients, including those patients who progress on taxanes. Citation Format: Shanshan Deng, Raisa I. Krutilina, Deanna N. Parke, Hao Chen, Duane D. Miller, Tiffany N. Seagroves, Wei Li. VERU-111, a novel orally bioavailable tubulin inhibitor, overcomes taxane resistance in metastatic 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 3071.

The metastatic cascade is a complex process that requires cancer cells to survive despite conditions of high physiologic stress. Previously, cooperation between the glucocorticoid receptor (GR) and hypoxia-inducible factors (HIF) was reported as a point of convergence for host and cellular stress signaling. These studies indicated p38 MAPK-dependent phosphorylation of GR on Ser134 and subsequent p-GR/HIF–dependent induction of breast tumor kinase (PTK6/Brk), as a mediator of aggressive cancer phenotypes. Herein, p-Ser134 GR was quantified in human primary breast tumors (n = 281) and the levels of p-GR were increased in triple-negative breast cancer (TNBC) relative to luminal breast cancer. Brk was robustly induced following exposure of TNBC model systems to chemotherapeutic agents (Taxol or 5-fluorouracil) and growth in suspension [ultra-low attachment (ULA)]. Notably, both Taxol and ULA resulted in upregulation of the Aryl hydrocarbon receptor (AhR), a known mediator of cancer prosurvival phenotypes. Mechanistically, AhR and GR copurified and following chemotherapy and ULA, these factors assembled at the Brk promoter and induced Brk expression in an HIF-dependent manner. Furthermore, Brk expression was upregulated in Taxol-resistant breast cancer (MCF-7) models. Ultimately, Brk was critical for TNBC cell proliferation and survival during Taxol treatment and in the context of ULA as well as for basal cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade. These studies nominate AhR as a p-GR binding partner and reveal ways to target epigenetic events such as adaptive and stress-induced acquisition of cancer skill sets required for metastatic cancer spread. Implication: Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. Mol Cancer Res; 16(11); 1761–72. ©2018 AACR.

Metastatic breast cancer is the leading cause of worldwide cancer-related deaths among women. Triple negative breast cancers (TNBC) are highly metastatic and are devoid of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) amplification. TNBCs are unresponsive to Herceptin and/or anti-estrogen therapies and too often become highly chemoresistant when exposed to standard chemotherapy. TNBCs frequently metastasize to the lung and brain. We have previously shown that TNBCs are active for oncogenic Wnt10b/β-catenin signaling and that WNT10B ligand and its downstream target HMGA2 are predictive of poorer outcomes and are strongly associated with chemoresistant TNBC metastatic disease. In search of new chemicals to target the oncogenic WNT10B/β-CATENIN/HMGA2 signaling axis, the anti-proliferative activity of the diterpene Jatrophone (JA), derived from the plant Jatropha isabelli, was tested on TNBC cells. JA interfered with the WNT TOPFLASH reporter at the level between receptor complex and β-catenin activation. JA efficacy was determined in various subtypes of TNBC conventional cell lines or in TNBC cell lines derived from TNBC PDX tumors. The differential IC50 (DCI50) responsiveness was compared among the TNBC models based on etiological-subtype and their cellular chemoresistance status. Elevated WNT10B expression also coincided with increased resistance to JA exposure in several metastatic cell lines. JA interfered with cell cycle progression, and induced loss of expression of the canonical Wnt-direct targets genes AXIN2, HMGA2, MYC, PCNA and CCND1. Mechanistically, JA reduced steady-state, non-phosphorylated (activated) β-catenin protein levels, but not total β-catenin levels. JA also caused the loss of expression of key EMT markers and significantly impaired wound healing in scratch assays, suggesting a direct role for JA inhibiting migration of TNBC cells. These results indicate that Jatrophone could be a powerful new chemotherapeutic agent against highly chemoresistant triple negative breast cancers by targeting the oncogenic Wnt10b/β-catenin signaling pathway.

Metastatic breast cancer is the leading cause of worldwide cancer-related deaths among women. Triple negative breast cancers (TNBC) are highly metastatic and are devoid of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2) amplification. TNBCs are unresponsive to Herceptin and/or anti-estrogen therapies and too often become highly chemoresistant when exposed to standard chemotherapy. TNBCs frequently metastasize to the lung and brain. We have previously shown that TNBCs are active for oncogenic Wnt10b/β-catenin signaling and that WNT10B ligand and its downstream target HMGA2 are predictive of poorer outcomes and are strongly associated with chemoresistant TNBC metastatic disease. In search of new chemicals to target the oncogenic WNT10B/β-CATENIN/HMGA2 signaling axis, the anti-proliferative activity of the diterpene Jatrophone (JA), derived from the plant Jatropha isabelli, was tested on TNBC cells. JA interfered with the WNT TOPFLASH reporter at the level between receptor complex and β-catenin activation. JA efficacy was determined in various subtypes of TNBC conventional cell lines or in TNBC cell lines derived from TNBC PDX tumors. The differential IC50 (DCI50) responsiveness was compared among the TNBC models based on etiological-subtype and their cellular chemoresistance status. Elevated WNT10B expression also coincided with increased resistance to JA exposure in several metastatic cell lines. JA interfered with cell cycle progression, and induced loss of expression of the canonical Wnt-direct targets genes AXIN2, HMGA2, MYC, PCNA and CCND1. Mechanistically, JA reduced steady-state, non-phosphorylated (activated) β-catenin protein levels, but not total β-catenin levels. JA also caused the loss of expression of key EMT markers and significantly impaired wound healing in scratch assays, suggesting a direct role for JA inhibiting migration of TNBC cells. These results indicate that Jatrophone could be a powerful new chemotherapeutic agent against highly chemoresistant triple negative breast cancers by targeting the oncogenic Wnt10b/β-catenin signaling pathway.

Triple-negative breast cancer (TNBC) cases account for about 15% of all breast cancers in the United States and have poorer overall prognosis relative to other molecular subtypes, partially due to the rapid development of drug resistance to chemotherapies and the increased risk of visceral metastasis. One of the standard treatment regimens for TNBC is the use of a taxane-based chemotherapy, such as paclitaxel, which stabilizes microtubules. However, drug resistance and neurotoxicities often limit the clinical efficacy of taxanes. Therefore, there are continuous needs to develop more effective therapies that could overcome resistance to taxanes. In this study, a novel series of structurally related pyridine analogs based on our previously reported lead compound ABI-274, was designed and synthesized to identify a molecule with improved antiproliferative potency. Most of these pyridine compounds exhibited potent cytotoxicity when tested in a panel of melanoma and breast cancer cell lines, with IC50 values in the low nanomolar range. Among them, CH-II-77 is the most potent compound with an IC50 value of 1−3 nM against these cancer cell lines, including paclitaxel-resistant sublines. The high-resolution X-ray crystal structure of CH-II-77 in complex with tubulin protein confirmed its direct binding to the colchicine-binding site. It strongly induced apoptosis and produced G2/M phase cell cycle arrest in TNBC cells in a dose-dependent manner in vitro. In vivo, CH-II-77 inhibited tumor growth in A375 melanoma xenografts and MDA-MB-231 TNBC xenografts in a dose-dependent manner. CH-II-77 was able to induce tumor necrosis and apoptosis in vivo. Collectively, these studies strongly suggest that CH-II-77 is a potent inhibitor of the growth of TNBC in vitro and in vivo. Thus, CH-II-77 and optimization of this analog are promising new generation of tubulin inhibitors for the treatment of TNBC and other types of cancers where tubulin inhibitors are currently being used clinically. Citation Format: Shanshan Deng, Hao Chen, Raisa Krutilina, Najah G. Albadari, Tiffany N. Seagroves, Duane D. Miller, Wei Li. Colchicine binding site agents as potent tubulin inhibitors suppressing triple negative 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 4608.

The metastatic cascade is a complex process that requires cancer cells to survive despite exposure to conditions of high physiologic stress. We previously showed that breast tumor kinase (Brk; also known as PTK6), a mediator of aggressive breast cancer phenotypes, is induced in breast cancer cells in response to a convergence of cellular and hormonal stress signals mediated by cross talk between hypoxia-inducible factors (HIFs) and glucocorticoid receptors (GR). Specifically, p38-MAPK dependent phosphorylation of GR-Ser134 (p-GR) and p-GR/HIF transcriptional complexes mediated heightened Brk gene expression in response to multiple inputs to stress pathway activation. Following studies in mice to demonstrate that Brk is a GR target gene in the mammary gland, immunohistochemistry (IHC) was performed on human primary breast tumor tissues using total and p-GR antibodies. To model stress-induced p-GR action relevant to tumor progression, molecular markers of stress signaling were measured in triple negative breast cancer (TNBC) cell lines treated with chemotherapy (Taxol and 5-flourouricil) and following growth in suspension (ultra-low attachment (ULA)). Co-immunoprecipitation and ChIP assays were used to demonstrate association of p-GR with novel co-regulatory factors in transcription complexes at the Brk promoter. Cell viability and migration assays were performed following Brk knock-out using CRISPR/Cas9 gene editing. Systemic Dex administration in mice confirmed that Brk is a GR target gene in vivo. In human breast tumor samples, phospho-GR was significantly associated with TNBC relative to luminal cancers. Chemotherapy and ULA induced activation of p38 MAPK, phosphorylation of GR, and upregulation of HIFs as well as the Aryl hydrocarbon receptor (AhR), a known mediator of cancer cell survival under cellular stress. Moreover, AhR and GR co-purified constitutively, and following chemotherapy or ULA culture, these factors assembled at the Brk promoter in a HIF-dependent manner. Brk induction was critical for TNBC cell survival during Taxol treatment or during ULA culture and for cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade.These studies define AhR as a novel p-GR binding partner and show that increased p-GR/AhR and Brk expression drive a migratory phenotype relevant to TNBC progression. Strategies to target p-GR signaling may ameliorate stress-induced acquisition of aggressive cancer phenotypes required for metastatic cancer spread that are linked to high breast cancer patient mortality. Citation Format: Tarah M. Regan Anderson, Shihong Ma, Carlos J. Perez Kerkvliet, Taylor M. Helle, Raisa Krutilina, Ganesh V. Raj, John A. Cidlowski, Kathryn L. Schwertfeger, Tiffany N. Seagroves, Carol A. Lange. Chemotherapy enables Brk/PTK6-dependent survival of triple-negative breast cancer cells via induction of an AhR/GR/HIF signaling axis [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 3457.

e13110Background: Triple negative breast cancer (TNBC) is highly aggressive that has poor prognosis due to the development of drug resistance. In this study, we evaluated the preclinical efficacy o...

Cancer cells use stress response pathways to sustain their pathogenic behavior. In breast cancer, stress response–associated phenotypes are mediated by the breast tumor kinase, Brk (PTK6), via the hypoxia-inducible factors HIF-1α and HIF-2α. Given that glucocorticoid receptor (GR) is highly expressed in triple-negative breast cancer (TNBC), we investigated cross-talk between stress hormone–driven GR signaling and HIF-regulated physiologic stress. Primary TNBC tumor explants or cell lines treated with the GR ligand dexamethasone exhibited robust induction of Brk mRNA and protein that was HIF1/2–dependent. HIF and GR coassembled on the BRK promoter in response to either hypoxia or dexamethasone, indicating that Brk is a direct GR/HIF target. Notably, HIF-2α, not HIF-1α, expression was induced by GR signaling, and the important steroid receptor coactivator PELP1 was also found to be induced in a HIF-dependent manner. Mechanistic investigations showed how PELP1 interacted with GR to activate Brk expression and demonstrated that physiologic cell stress, including hypoxia, promoted phosphorylation of GR serine 134, initiating a feed-forward signaling loop that contributed significantly to Brk upregulation. Collectively, our findings linked cellular stress (HIF) and stress hormone (cortisol) signaling in TNBC, identifying the phospho-GR/HIF/PELP1 complex as a potential therapeutic target to limit Brk-driven progression and metastasis in TNBC patients. Cancer Res; 76(6); 1653–63. ©2016 AACR.

Cancers exhibit altered metabolism characterized by increased glucose and glutamine uptake. The hexosamine biosynthetic pathway (HBP) uses glucose and glutamine, and directly contributes to O-linked-β-N-acetylglucosamine (O-GlcNAc) modifications on intracellular proteins. Multiple tumor types contain elevated total O-GlcNAcylation, in part, by increasing O-GlcNAc transferase (OGT) levels, the enzyme that catalyzes this modification. Although cancer cells require OGT for oncogenesis, it is not clear how tumor cells regulate OGT expression and O-GlcNAcylation. Here, it is shown that the PI3K–mTOR–MYC signaling pathway is required for elevation of OGT and O-GlcNAcylation in breast cancer cells. Treatment with PI3K and mTOR inhibitors reduced OGT protein expression and decreased levels of overall O-GlcNAcylation. In addition, both AKT and mTOR activation is sufficient to elevate OGT/O-GlcNAcylation. Downstream of mTOR, the oncogenic transcription factor c-MYC is required and sufficient for increased OGT protein expression in an RNA-independent manner and c-MYC regulation of OGT mechanistically requires the expression of c-MYC transcriptional target HSP90A. Finally, mammary tumor epithelial cells derived from MMTV-c-myc transgenic mice contain elevated OGT and O-GlcNAcylation and OGT inhibition in this model induces apoptosis. Thus, OGT and O-GlcNAcylation levels are elevated via activation of an mTOR/MYC cascade. Implications: Evidence indicates OGT as a therapeutic target in c-MYC–amplified cancers. Mol Cancer Res; 13(5); 923–33. ©2015 AACR.

Introduction In breast cancer, distinct expression profiles of microRNAs (miRNAs) have been associated with molecular subgroups and clinicopathological characteristics, implicating a diagnostic and prognostic role of miRNAs. However, the biological functions of deregulated miRNAs in tumor progression are not yet completely defined. In this study, we investigated the function of miR-18a in regulating breast cancer metastasis through the hypoxia-inducible factor 1α (HIF1A)–dependent hypoxic response. Methods An orthotopic metastatic breast cancer xenograft model (MDA-MB-231 cells) was used to identify miRNAs associated with spontaneous lung metastasis. The function of miR-18a in regulating HIF1A expression, as well as cellular responses to hypoxia and metastasis, were then studied in vitro and in vivo by assessing ectopic miR-18a expression or miR-18a inhibition. miRNA–mRNA interactions (AGO2 immunoprecipitation and 3′ untranslated region Luciferase reporter assays), gene expression (quantitative PCR and microarray), cell migration and invasion, and cell growth were assessed under normoxic or hypoxic conditions, complemented by orthotopic xenograft of tumor cells to the mammary fat pad to investigate the effect of modulating miR-18a expression on primary tumor growth and lung metastasis. Last, clinically relevant correlations between miR-18a, HIF1A, hypoxia-responsive gene expression and distant metastasis–free survival (DMFS) were assessed using published expression array breast tumors data sets. Results miRNAs encoded by the MIR17HG gene were downregulated in lung metastases compared to primary tumors. Ectopic expression of miR-18a, a MIR17HG family member, in a metastatic variant of MDA-MB-231 cells reduced primary tumor growth and lung metastasis, whereas miR-18a inhibition in the parental cells promoted tumor growth and lung metastasis. We identified HIF1A as a direct target of miR-18a. Modulating miR-18a expression significantly affected hypoxic gene expression, cell invasiveness and sensitivity to anoikis and hypoxia in vitro in a HIF1A-dependent manner. Analysis of previously published data revealed that higher expression of HIF1A and a panel of hypoxic genes is associated with shorter DMFS interval in patients with basal-like breast tumors, and that, within this subtype, miR-18a expression is inversely correlated with hypoxic gene expression. Together, these data support a role of miR-18a in repressing distant metastasis through a HIF1A-dependent pathway. Conclusions The results of this study reveal a novel role for miR-18a in targeting HIF1A and repressing metastasis of basal-like breast tumors. Electronic supplementary material The online version of this article (doi:10.1186/bcr3693) contains supplementary material, which is available to authorized users.

MicroRNAs (miRNAs) regulate mRNA stability and translation through the action of the RNAi-induced silencing complex. In this study, we systematically identified endogenous miRNA target genes by using AGO2 immunoprecipitation (AGO2-IP) and microarray analyses in two breast cancer cell lines, MCF7 and MDA-MB-231, representing luminal and basal-like breast cancer, respectively. The expression levels of ∼70% of the AGO2-IP mRNAs were increased by DROSHA or DICER1 knockdown. In addition, integrated analysis of miRNA expression profiles, mRNA-AGO2 interaction, and the 3′-UTR of mRNAs revealed that >60% of the AGO2-IP mRNAs were putative targets of the 50 most abundantly expressed miRNAs. Together, these results suggested that the majority of the AGO2-associated mRNAs were bona fide miRNA targets. Functional enrichment analysis uncovered that the AGO2-IP mRNAs were involved in regulation of cell cycle, apoptosis, adhesion/migration/invasion, stress responses (e.g. DNA damage and endoplasmic reticulum stress and hypoxia), and cell-cell communication (e.g. Notch and Ephrin signaling pathways). A role of miRNAs in regulating cell migration/invasion and stress response was further defined by examining the impact of DROSHA knockdown on cell behaviors. We demonstrated that DROSHA knockdown enhanced cell migration and invasion, whereas it sensitized cells to cell death induced by suspension culture, glucose depletion, and unfolding protein stress. Data from an orthotopic xenograft model showed that DROSHA knockdown resulted in reduced growth of primary tumors but enhanced lung metastasis. Taken together, these results suggest that miRNAs collectively function to promote survival of tumor cells under stress but suppress cell migration/invasion in breast cancer cells. Background: miRNA deregulation contributes to tumor progression. Results: Endogenous miRNA targets were identified in two breast cancer cell lines by integrated analysis of miRNA/mRNA expression and miRNA-mRNA interaction. Conclusion: miRNAs collectively function to promote survival but suppress cell migration/invasion. Significance: The defined endogenous miRNA targets will facilitate future studies to link miRNA deregulation with breast cancer cell properties.

Background Hypoxia-inducible factors (HIFs) are well-established mediators of tumor growth, the epithelial to mesenchymal transition (EMT) and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell (CSC) activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 (ITGA6; CD49f) is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix (ECM), but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion. Methods We used two models of metastatic breast cancer (MBC), polyoma middle T (MMTV-PyMT) and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout (KO) or knockdown cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f + cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49fneg or low cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients. Results We report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes. Conclusions Our data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f + cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes. Electronic supplementary material The online version of this article (doi:10.1186/s12943-016-0510-x) contains supplementary material, which is available to authorized users.