Notch3 signaling promotes tumor cell adhesion and progression in a murine epithelial ovarian cancer model.

High grade serous ovarian cancer (HGSC) is the most common and deadly type of ovarian cancer, largely due to difficulties in early diagnosis and rapid metastasis throughout the peritoneal cavity. Previous studies have shown that expression of Notch3 correlates with worse prognosis and increased tumorigenic cell behaviors in HGSC. We investigated the mechanistic role of Notch3 in a model of metastatic ovarian cancer using the murine ovarian surface epithelial cell line, ID8 IP2. Notch3 was activated in ID8 IP2 cells via expression of the Notch3 intracellular domain (Notch3IC). Notch3IC ID8 IP2 cells injected intraperitoneally caused accelerated ascites and reduced survival compared to control ID8 IP2, particularly in early stages of disease. We interrogated downstream targets of Notch3IC in ID8 IP2 cells by RNA sequencing and found significant induction of genes that encode adhesion and extracellular matrix proteins. Notch3IC ID8 IP2 showed increased expression of ITGA1 mRNA and cell-surface protein. Notch3IC-mediated increase of ITGA1 was also seen in two human ovarian cancer cells. Notch3IC ID8 IP2 cells showed increased adhesion to collagens I and IV in vitro. We propose that Notch3 activation in ovarian cancer cells causes increased adherence to collagen-rich peritoneal surfaces. Thus, the correlation between increased Notch3 signaling and poor prognosis may be influenced by increased metastasis of HGSC via increased adherence of disseminating cells to new metastatic sites in the peritoneum.

The opposing effects of interferon-beta and oncostatin-M as regulators of cancer stem cell plasticity in triple-negative breast cancer.

BACKGROUND: Highly aggressive, metastatic and therapeutically resistant triple-negative breast cancers (TNBCs) are often enriched for cancer stem cells (CSC). Cytokines within the breast tumor microenvironment (TME) influence the CSC state by regulating tumor cell differentiation programs. Two prevalent breast TME cytokines are oncostatin-M (OSM) and interferon-ß (IFN-ß). OSM is a member of the IL-6 family of cytokines and can drive the de-differentiation of TNBC cells to a highly aggressive CSC state. Conversely, IFN-ß induces the differentiation of TNBC, resulting in the repression of CSC properties. Here, we assess how these breast TME cytokines influence CSC plasticity and clinical outcome. METHODS: Using transformed human mammary epithelial cell (HMEC) and TNBC cell models, we assessed the CSC markers and properties following exposure to OSM and/or IFN-ß. CSC markers included CD24, CD44, and SNAIL; CSC properties included tumor sphere formation, migratory capacity, and tumor initiation. RESULTS: There are three major findings from our study. First, exposure of purified, non-CSC to IFN-ß prevents OSM-mediated CD44 and SNAIL expression and represses tumor sphere formation and migratory capacity. Second, during OSM-induced de-differentiation, OSM represses endogenous IFN-ß mRNA expression and autocrine/paracrine IFN-ß signaling. Restoring IFN-ß signaling to OSM-driven CSC re-engages IFN-ß-mediated differentiation by repressing OSM/STAT3/SMAD3-mediated SNAIL expression, tumor initiation, and growth. Finally, the therapeutic use of IFN-ß to treat OSM-driven tumors significantly suppresses tumor growth. CONCLUSIONS: Our findings suggest that the levels of IFN-ß and OSM in TNBC dictate the abundance of cells with a CSC phenotype. Indeed, TNBCs with elevated IFN-ß signaling have repressed CSC properties and a better clinical outcome. Conversely, TNBCs with elevated OSM signaling have a worse clinical outcome. Likewise, since OSM suppresses IFN-ß expression and signaling, our studies suggest that strategies to limit OSM signaling or activate IFN-ß signaling will disengage the de-differentiation programs responsible for the aggressiveness of TNBCs.

Breaking the oncostatin M feed-forward loop to suppress metastasis and therapy failure.

Deciphering the complex milieu that makes up the tumor microenvironment (TME) and the signaling engaged by TME cytokines continues to provide novel targets for therapeutic intervention. The IL-6 family member oncostatin M (OSM) has recently emerged as a potent driver of tumorigenesis, metastasis, and therapy failure, molecular programs most frequently attributed to IL-6 itself. In a recent issue of The Journal of Pathology, Kucia-Tran et al describe how elevated oncostatin M receptor (OSMR) expression results in a feed-forward loop involving the de novo production of both OSM and OSMR to facilitate aggressive properties in squamous cell carcinoma (SCC). Here, we discuss how new findings implicating OSM in conferring aggressive cancer cell properties can be leveraged to suppress metastatic outgrowth and therapy failure in SCC as well as other cancers. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Magnetic resonance molecular imaging of metastatic breast cancer by targeting extradomain-B fibronectin in the tumor microenvironment.

PURPOSE: Non-invasive early accurate detection of malignant breast cancer is paramount to the clinical management of the life-threatening disease. Here, we aim to test a small peptide targeted MRI contrast agent, ZD2-Gd(HP-DO3A), specific to an oncoprotein, extradomain-B fibronectin (EDB-FN), in the tumor microenvironment for MR molecular imaging of breast cancer. METHOD: EDB-FN expression in 4T1 and MDA-MB-231 cancers was analyzed with quantitative real-time PCR and western blot. Primary and metastatic triple negative breast cancer mouse models were developed using 4T1 and MDA-MB-231 cells. Contrast-enhanced MRI was carried out to evaluate the use of ZD2-Gd(HP-DO3A) in detecting 4T1 and MDA-MB-231 primary and metastatic tumors. RESULTS: EDB-FN was abundantly expressed in the extracellular matrix (ECM) of both the primary and metastatic TNBC tumors. In T1 -weighted MRI, ZD2-Gd(HP-DO3A) generated superior contrast enhancement in primary TNBC tumors than a nonspecific clinical agent Gd(HP-DO3A), during 30 min after contrast injection. ZD2-Gd(HP-DO3A) also produced a significant increase in contrast-to-noise ratio (CNR) of TNBC metastases, enabling sensitive localization and delineation of metastases that occulted in non-contrast-enhanced or Gd(HP-DO3A)-enhanced MRI. CONCLUSIONS: These findings potentiate the use of ZD2-Gd(HP-DO3A) for MR molecular imaging of malignant breast cancers to improve the healthcare of breast cancer patients. Magn Reson Med 79:3135-3143, 2018. © 2017 International Society for Magnetic Resonance in Medicine.

Silencing the roadblocks to effective triple-negative breast cancer treatments by siRNA nanoparticles.

Over the past decade, RNA interference (RNAi) has been ubiquitously utilized to study biological function in vitro; however, limitations were associated with its utility in vivo More recently, small interfering RNA (siRNA) nanoparticles with improved biocompatibility have gained prevalence as a potential therapeutic option for the treatment of various diseases. The adaptability of siRNA nanoparticles enables the delivery of virtually any siRNA, which is especially advantageous for therapeutic applications in heterogeneous diseases that lack unifying molecular features, such as triple-negative breast cancer (TNBC). TNBC is an aggressive subtype of breast cancer that is stratified by the lack of estrogen receptor/progesterone receptor expression and HER2 amplification. There are currently no FDA-approved targeted therapies for the treatment of TNBCs, making cytotoxic chemotherapy the only treatment option available to these patients. In this review, we outline the current status of siRNA nanoparticles in clinical trials for cancer treatment and discuss the promising preclinical approaches that have utilized siRNA nanoparticles for TNBC treatment. Next, we address TNBC subtype-specific therapeutic interventions and highlight where and how siRNA nanoparticles fit into these strategies. Lastly, we point out ongoing challenges in the field of siRNA nanoparticle research that, if addressed, would significantly improve the efficacy of siRNA nanoparticles as a therapeutic option for cancer treatment.

Neoadjuvant therapy for early-stage breast cancer: the clinical utility of pertuzumab.

Approximately 20% of breast cancer patients harbor tumors that overexpress human epidermal growth factor receptor 2 (HER2; also known as ErbB2), a receptor tyrosine kinase that belongs to the epidermal growth factor receptor family of receptor tyrosine kinases. HER2 amplification and hyperactivation drive the growth and survival of breast cancers through the aberrant activation of proto-oncogenic signaling systems, particularly the Ras/MAP kinase and PI3K/AKT pathways. Although HER2-positive (HER2(+)) breast cancer was originally considered to be a highly aggressive form of the disease, the clinical landscape of HER2(+) breast cancers has literally been transformed by the approval of anti-HER2 agents for adjuvant and neoadjuvant settings. Indeed, pertuzumab is a novel monoclonal antibody that functions as an anti-HER2 agent by targeting the extracellular dimerization domain of the HER2 receptor; it is also the first drug to receive an accelerated approval by the US Food and Drug Administration for use in neoadjuvant settings in early-stage HER2(+) breast cancer. Here, we review the molecular and cellular factors that contribute to the pathophysiology of HER2 in breast cancer, as well as summarize the landmark preclinical and clinical findings underlying the approval and use of pertuzumab in the neoadjuvant setting. Finally, the molecular mechanisms operant in mediating resistance to anti-HER2 agents, and perhaps to pertuzumab as well, will be discussed, as will the anticipated clinical impact and future directions of pertuzumab in breast cancer patients.

Silencing ß3 Integrin by Targeted ECO/siRNA Nanoparticles Inhibits EMT and Metastasis of Triple-Negative Breast Cancer.

Metastatic breast cancer is the second leading cause of cancer-related deaths among women. Triple-negative breast cancer (TNBC) is a highly aggressive subcategory of breast cancer and currently lacks well-defined molecular targets for effective targeted therapies. Disease relapse, metastasis, and drug resistance render standard chemotherapy ineffective in the treatment of TNBC. Because previous studies coupled ß3 integrin (ITGB3) to epithelial-mesenchymal transition (EMT) and metastasis, we exploited ß3 integrin as a therapeutic target to treat TNBC by delivering ß3 integrin siRNA via lipid ECO-based nanoparticles (ECO/siß3). Treatment of TNBC cells with ECO/siß3 was sufficient to effectively silence ß3 integrin expression, attenuate TGFß-mediated EMT and invasion, restore TGFß-mediated cytostasis, and inhibit three-dimensional organoid growth. Modification of ECO/siß3 nanoparticles with an RGD peptide via a PEG spacer enhanced siRNA uptake by post-EMT cells. Intravenous injections of RGD-targeted ECO/siß3 nanoparticles in vivo alleviated primary tumor burden and, more importantly, significantly inhibited metastasis. In the span of 16 weeks of the experiments and observations, including primary tumor resection at week 9 and release from the treatment for 4 weeks, the mice bearing orthotopic, TGFß-prestimulated MDA-MB-231 tumors that were treated with RGD-targeted ECO/siß3 nanoparticles were free of metastases and relapse, in comparison with untreated mice. Collectively, these results highlight ECO/siß3 nanoparticles as a promising therapeutic regimen to combat TNBC.

Deptor enhances triple-negative breast cancer metastasis and chemoresistance through coupling to survivin expression.

Transforming growth factor-ß (TGF-ß) functions to suppress tumorigenesis in normal mammary tissues and early-stage breast cancers and, paradoxically, acts to promote the metastasis and chemoresistance in late-stage breast cancers, particularly triple-negative breast cancers (TNBCs). Precisely how TGF-ß acquires oncogenic characteristics in late-stage breast cancers remains unknown, as does the role of the endogenous mammalian target of rapamycin (mTOR) inhibitor, Dep domain-containing mTOR-interacting protein (Deptor), in coupling TGF-ß to TNBC development and metastatic progression. Here we demonstrate that Deptor expression was downregulated in basal-like/TNBCs relative to their luminal counterparts. Additionally, Deptor expression was 1) inversely correlated with the metastatic ability of human (MCF10A) and mouse (4T1) TNBC progression series and 2) robustly repressed by several inducers of epithelial-mesenchymal transition programs. Functional disruption of Deptor expression in 4T07 cells significantly inhibited their proliferation and organoid growth in vitro, as well as prevented their colonization and tumor formation in the lungs of mice. In stark contrast, elevated Deptor expression was significantly associated with poorer overall survival of patients harboring estrogen receptor α-negative breast cancers. Accordingly, enforced Deptor expression in MDA-MB-231 cells dramatically enhanced their 1) organoid growth in vitro, 2) pulmonary outgrowth in mice, and 3) resistance to chemotherapies, an event dependent on the coupling of Deptor to survivin expression. Collectively, our findings highlight the dichotomous functions of Deptor in modulating the proliferation and survival of TNBCs during metastasis; they also implicate Deptor and its stimulation of survivin as essential components of TNBC resistance to chemotherapies and apoptotic stimuli.

Upregulated WAVE3 expression is essential for TGF-ß-mediated EMT and metastasis of triple-negative breast cancer cells.

Breast cancer is the second leading cause of cancer death in women in the United States. Metastasis accounts for the death of ~90 % of these patients, yet the mechanisms underlying this event remain poorly defined. WAVE3 belongs to the WASP/WAVE family of actin-binding proteins that play essential roles in regulating cell morphology, actin polymerization, cytoskeleton remodeling, cell motility, and invasion. Accordingly, we demonstrated previously that WAVE3 promotes the acquisition of invasive and metastatic phenotypes by human breast cancers. Herein, we show that transforming growth factor-ß (TGF-ß) selectively and robustly induced the expression of WAVE3 in metastatic breast cancer cells, but not in their nonmetastatic counterparts. Moreover, the induction of WAVE3 expression in human and mouse triple-negative breast cancer cells (TNBCs) by TGF-ß likely reflects its coupling to microRNA expression via a Smad2- and ß3 integrin-dependent mechanism. We further demonstrate the requirement for WAVE3 expression in mediating the initiation of epithelial-mesenchymal transition (EMT) programs stimulated by TGF-ß. Indeed, stable depletion of WAVE3 expression in human TNBC cells prevented TGF-ß from inducing EMT programs and from stimulating the proliferation, migration, and the formation of lamellipodia in metastatic TNBC cells. Lastly, we observed WAVE3 deficiency to abrogate the outgrowth of TNBC cell organoids in 3-dimensional organotypic cultures as well as to decrease the growth and metastasis of 4T1 tumors produced in syngeneic Balb/C mice. Indeed, WAVE3 deficiency significantly reduced the presence of sarcomatoid morphologies indicative of EMT phenotypes in pulmonary TNBC tumors as compared to those detected in their parental counterparts. Collectively, these findings indicate the necessity for WAVE3 expression and activity during EMT programs stimulated by TGF-ß; they also suggest that measures capable of inactivating WAVE3 may play a role in alleviating metastasis stimulated by TGF-ß.

Targeted inactivation of ß1 integrin induces ß3 integrin switching, which drives breast cancer metastasis by TGF-ß.

Mammary tumorigenesis and epithelial-mesenchymal transition (EMT) programs cooperate in converting transforming growth factor-ß (TGF-ß) from a suppressor to a promoter of breast cancer metastasis. Although previous reports associated ß1 and ß3 integrins with TGF-ß stimulation of EMT and metastasis, the functional interplay and plasticity exhibited by these adhesion molecules in shaping the oncogenic activities of TGF-ß remain unknown. We demonstrate that inactivation of ß1 integrin impairs TGF-ß from stimulating the motility of normal and malignant mammary epithelial cells (MECs) and elicits robust compensatory expression of ß3 integrin solely in malignant MECs, but not in their normal counterparts. Compensatory ß3 integrin expression also 1) enhances the growth of malignant MECs in rigid and compliant three-dimensional organotypic cultures and 2) restores the induction of the EMT phenotypes by TGF-ß. Of importance, compensatory expression of ß3 integrin rescues the growth and pulmonary metastasis of ß1 integrin-deficient 4T1 tumors in mice, a process that is prevented by genetic depletion or functional inactivation of ß3 integrin. Collectively our findings demonstrate that inactivation of ß1 integrin elicits metastatic progression via a ß3 integrin-specific mechanism, indicating that dual ß1 and ß3 integrin targeting is necessary to alleviate metastatic disease in breast cancer patients.

Sox4, EMT programs, and the metastatic progression of breast cancers: mastering the masters of EMT.

Epithelial-mesenchymal transition (EMT) programs require the expression of a variety of so-called master regulators of EMT, including members of the Snail, Zeb, and Twist transcription factor families. Teleologically, the requirement for such a diverse group of 'master regulators' seems evolutionarily cumbersome, and emerging evidence indicates that these transcription factors do in fact mediate unique and specialized functions, suggesting the existence of higher-order 'masters' that truly direct and coordinate EMT programs. Accordingly, Tiwari and colleagues recently delineated an elegant pathway wherein transforming growth factor-beta stimulates Sox4 expression, which induces that of the histone methyltransferase, Ezh2, thereby reprogramming the epigenome to elicit EMT programs and metastasis of breast cancers. This viewpoint highlights Sox4 as a 'new' master of EMT programs and metastatic breast cancer.

The relevance of the TGF-ß Paradox to EMT-MET programs.

The role of transforming growth factor-ß (TGF-ß) during tumorigenesis is complex and paradoxical, reflecting its ability to function as a tumor suppressor in normal and early-stage cancers, and as a tumor promoter in their late-stage counterparts. The switch in TGF-ß function is known as the "TGF-ß Paradox," whose manifestations are intimately linked to the initiation of epithelial-mesenchymal transition (EMT) programs in developing and progressing carcinomas. Indeed, as carcinoma cells emerge from EMT programs stimulated by TGF-ß, they readily display a variety of acquired phenotypes that provide a selective advantage to growing carcinomas, including (i) enhanced cell migration and invasion; (ii) heightened resistance to cytotoxic agents, targeted chemotherapeutic, and radiation treatments; and (iv) boosted expansion of cancer-initiating and stem-like cell populations that underlie tumor metastasis and disease recurrence. At present, the molecular, cellular, and microenvironmental mechanisms that enable post-EMT and metastatic carcinoma cells to hijack the oncogenic activities of TGF-ß remain incompletely understood. Additionally, the molecular mechanisms that counter EMT programs and limit the aggressiveness of late-stage carcinomas, events that transpire via mesenchymal-epithelial transition (MET) reactions, also need to be further elucidated. Here we review recent advances that provide new insights into how TGF-ß promotes EMT programs in late-stage carcinoma cells, as well as how these events are balanced by MET programs during the development and metastatic progression of human carcinomas.

Noncanonical TGF-ß signaling during mammary tumorigenesis.

Breast cancer is a heterogeneous disease comprised of at least five major tumor subtypes that coalesce as the second leading cause of cancer death in women in the United States. Although metastasis clearly represents the most lethal characteristic of breast cancer, our understanding of the molecular mechanisms that govern this event remains inadequate. Clinically, ~30% of breast cancer patients diagnosed with early-stage disease undergo metastatic progression, an event that (a) severely limits treatment options, (b) typically results in chemoresistance and low response rates, and (c) greatly contributes to aggressive relapses and dismal survival rates. Transforming growth factor-ß (TGF-ß) is a pleiotropic cytokine that regulates all phases of postnatal mammary gland development, including branching morphogenesis, lactation, and involution. TGF-ß also plays a prominent role in suppressing mammary tumorigenesis by preventing mammary epithelial cell (MEC) proliferation, or by inducing MEC apoptosis. Genetic and epigenetic events that transpire during mammary tumorigenesis conspire to circumvent the tumor suppressing activities of TGF-ß, thereby permitting late-stage breast cancer cells to acquire invasive and metastatic phenotypes in response to TGF-ß. Metastatic progression stimulated by TGF-ß also relies on its ability to induce epithelial-mesenchymal transition (EMT) and the expansion of chemoresistant breast cancer stem cells. Precisely how this metamorphosis in TGF-ß function comes about remains incompletely understood; however, recent findings indicate that the initiation of oncogenic TGF-ß activity is contingent upon imbalances between its canonical and noncanonical signaling systems. Here we review the molecular and cellular contributions of noncanonical TGF-ß effectors to mammary tumorigenesis and metastatic progression.

The pathophysiology of epithelial-mesenchymal transition induced by transforming growth factor-beta in normal and malignant mammary epithelial cells.

Epithelial-mesenchymal transition (EMT) is an essential process that drives polarized, immotile mammary epithelial cells (MECs) to acquire apolar, highly migratory fibroblastoid-like features. EMT is an indispensable process that is associated with normal tissue development and organogenesis, as well as with tissue remodeling and wound healing. In stark contrast, inappropriate reactivation of EMT readily contributes to the development of a variety of human pathologies, particularly those associated with tissue fibrosis and cancer cell invasion and metastasis, including that by breast cancer cells. Although metastasis is unequivocally the most lethal aspect of breast cancer and the most prominent feature associated with disease recurrence, the molecular mechanisms whereby EMT mediates the initiation and resolution of breast cancer metastasis remains poorly understood. Transforming growth factor-beta (TGF-beta) is a multifunctional cytokine that is intimately involved in regulating numerous physiological processes, including cellular differentiation, homeostasis, and EMT. In addition, TGF-beta also functions as a powerful tumor suppressor in MECs, whose neoplastic development ultimately converts TGF-beta into an oncogenic cytokine in aggressive late-stage mammary tumors. Recent findings have implicated the process of EMT in mediating the functional conversion of TGF-beta during breast cancer progression, suggesting that the chemotherapeutic targeting of EMT induced by TGF-beta may offer new inroads in ameliorating metastatic disease in breast cancer patients. Here we review the molecular, cellular, and microenvironmental factors that contribute to the pathophysiological activities of TGF-beta during its regulation of EMT in normal and malignant MECs.

An oncogenic isoform of HER2 associated with locally disseminated breast cancer and trastuzumab resistance.

The HER2-targeted therapy trastuzumab is widely used for the treatment of patients with metastatic breast tumors overexpressing HER2. However, an objective response is observed in only 12% to 24% of patients treated with trastuzumab as a single agent and initial responders regress in <6 months (1-3). The reason for the clinical failure of trastuzumab in this setting remains unclear. Here we show that local lymph node-positive disease progression in 89% of breast cancer patients with HER2-positive tumors involves the HER2 oncogenic variant HER2Delta16. We further show that ectopic expression of HER2Delta16, but not wild-type HER2, promotes receptor dimerization, cell invasion, and trastuzumab resistance of NIH3T3 and MCF-7 tumor cell lines. The potentiated metastatic and oncogenic properties of HER2Delta16 were mediated through direct coupling of HER2Delta16 to Src kinase. Cotargeting of HER2Delta16 and Src kinase with the single-agent tyrosine kinase inhibitor dasatinib resulted in Src inactivation, destabilization of HER2Delta16, and suppressed tumorigenicity. Activated Src kinase was also observed in 44% of HER2Delta16-expressing breast carcinomas underscoring the potential clinical implications of coupled HER2Delta16 and Src signaling. Our results suggest that HER2Delta16 expression is an important genetic event driving trastuzumab-refractory breast cancer. We propose that successful targeted therapeutics for intervention of aggressive HER2-positive breast cancers will require a strategy to suppress HER2Delta16 oncogenic signaling. One possibility involves a therapeutic strategy employing single-agent tyrosine kinase inhibitors to disengage the functionally coupled oncogenic HER2Delta16 and Src tyrosine kinase pathways.

IL-21 induces apoptosis of antigen-specific CD8+ T lymphocytes.

IL-21, a member of the common gamma-chain family of cytokines, has pleiotropic effects on T, B, and NK cells. We found that IL-21 and the prototype common gamma-chain cytokine IL-2 can stimulate proliferation and cytokine secretion by Ag-specific rhesus monkey CD8+ T cells. However, unique among the members of this family of cytokines, we found that IL-21 drives these cells to apoptosis by down-regulation of Bcl-2. These findings suggest that IL-21 may play an important role in the contraction of CD8+ T cell responses.