A dataset of chromosomal instability gene signature scores in normal and cancer cells from the human breast.

These data show the relative amount of chromosomal instability (CIN) in a diverse array of human breast cell types, including non-transformed mammary epithelial cells as well as cancer cell lines. Additional data is also provided from human embryonic and mesenchymal stem cells. To produce this dataset, we compared a published chromosomal instability gene signature against publicly available datasets containing gene expression information for each cell. We then analyzed these data with the Python GSEAPY software package to provide a CIN enrichment score. These data are useful for comparing the relative amounts of CIN in different breast cell types. This includes cells representing the major clinical (ER/PR+, HER2+ & Triple-negative) as well as intrinsic breast cancer subtypes (Luminal B, HER2+, Basal-like and Claudin-low). Our dataset has a great potential for re-use given the recent surge in interest surrounding the role of CIN in breast cancer. The large size of the dataset, coupled with the diversity of the cell types represented, provides numerous possibilities for future comparisons.

Breast cancer stem cells tolerate chromosomal instability during tumor progression via c-Jun/AXL stress signaling.

Chromosomal instability (CIN) is critical for tumor evolution, yet its relationship with stemness is unclear. Here, we describe CIN as a key stress induced during tumor initiation that is uniquely tolerated by breast cancer stem cells in an activated signaling state (aCSCs). While we noted elevated CIN specifically in tumors from aCSCs, this was not intrinsic to these cells, as baseline levels were similar to non-stem cell types. This suggests that CIN is induced during tumor initiation, and that aCSCs can better tolerate this stress. Further, this increased CIN may be transient, as it was only in low-burden aCSC tumors, with levels diminishing in more established disease. Phospho-array profiling revealed specific activation of c-Jun stress signaling in aCSCs, which we hypothesized could induce genes responsible for CIN tolerance. Indeed, we identified AXL as a c-Jun dependent gene enriched in aCSCs that enhances resistance to this stress. Thus, CIN tolerance mediated by c-Jun/AXL signaling may be a defining feature of stemness, contributing to breast cancer progression.

Stem-like breast cancer cells in the activated state resist genetic stress via TGFBI-ZEB1.

Breast cancer cells with stem-like properties are critical for tumor progression, yet much about these cells remains unknown. Here, we characterize a population of stem-like breast cancer cells expressing the integrin αvß3 as transcriptionally related to activated stem/basal cells in the normal human mammary gland. An unbiased functional screen of genes unique to these cells identified the matrix protein TGFBI (BIG-H3) and the transcription factor ZEB1 as necessary for tumorsphere formation. Surprisingly, these genes were not required for cell proliferation or survival, but instead maintained chromosomal stability. Consistent with this finding, CRISPR deletion of either gene synergized with PARP inhibition to deplete αvß3+ stem-like cells, which are normally resistant to this therapy. Our findings highlight a critical role for TGFBI-ZEB1 protection against genetic stress as a key attribute of activated stem-like cells and suggest that disrupting this ability may enhance their "BRCAness" by increasing sensitivity to PARP inhibitors.

Combined Bcl-2/Src inhibition synergize to deplete stem-like breast cancer cells.

Breast cancer cells with stem cell properties play an important role in tumor progression and thus are key targets for therapy. Here, we show that combined Bcl-2/Src inhibition synergize to deplete stem-like cells. While Src inhibition increases pro-apoptotic PUMA, we find that a significant amount interacts with Bcl-2 and Bcl-xL, promoting resistance to cell death. Consistent with this, the clinically-approved Bcl-2 selective drug venetoclax was sufficient to overcome resistance by preventing PUMA/Bcl-2 binding, enhancing apoptosis. This effect was specific to stem-like breast cancer cells as there was no effect on luminal or basal-like cell types. In contrast, the Mcl-1 inhibitor S63845 potently targeted basal-like, but not stem-like cells, highlighting dependency on distinct sentinel Bcl-2 family members. Our findings reveal Bcl-2/Src inhibition as a superior therapy to target stemness, providing a foundation for a potential personalized strategy to reduce breast cancer progression.

Proapoptotic PUMA targets stem-like breast cancer cells to suppress metastasis.

Breast cancer cells with stem cell properties are key contributors to metastatic disease, and there remains a need to better understand and target these cells in human cancers. Here, we identified rare stem-like cells in patients' tumors characterized by low levels of the proapoptotic molecule p53-upregulated modulator of apoptosis (PUMA) and showed that these cells play a critical role in tumor progression that is independent of clinical subtype. A signaling axis consisting of the integrin αvß3, Src kinase, and the transcription factor Slug suppresses PUMA in these cells, promoting tumor stemness. We showed that genetic or pharmacological disruption of αvß3/Src signaling drives PUMA expression, specifically depleting these stem-like tumor cells; increases their sensitivity to apoptosis; and reduces pulmonary metastasis, with no effect on primary tumor growth. Taken together, these findings point to PUMA as a key vulnerability of stem-like cells and suggest that pharmacological upregulation of PUMA via Src inhibition may represent a strategy to selectively target these cells in a wide spectrum of aggressive breast cancers.

Galectin-3, a Druggable Vulnerability for KRAS-Addicted Cancers.

Identifying the molecular basis for cancer cell dependence on oncogenes such as KRAS can provide new opportunities to target these addictions. Here, we identify a novel role for the carbohydrate-binding protein galectin-3 as a lynchpin for KRAS dependence. By directly binding to the cell surface receptor integrin αvß3, galectin-3 gives rise to KRAS addiction by enabling multiple functions of KRAS in anchorage-independent cells, including formation of macropinosomes that facilitate nutrient uptake and ability to maintain redox balance. Disrupting αvß3/galectin-3 binding with a clinically active drug prevents their association with mutant KRAS, thereby suppressing macropinocytosis while increasing reactive oxygen species to eradicate αvß3-expressing KRAS-mutant lung and pancreatic cancer patient-derived xenografts and spontaneous tumors in mice. Our work reveals galectin-3 as a druggable target for KRAS-addicted lung and pancreas cancers, and indicates integrin αvß3 as a biomarker to identify susceptible tumors.Significance: There is a significant unmet need for therapies targeting KRAS-mutant cancers. Here, we identify integrin αvß3 as a biomarker to identify mutant KRAS-addicted tumors that are highly sensitive to inhibition of galectin-3, a glycoprotein that binds to integrin αvß3 to promote KRAS-mediated activation of AKT. Cancer Discov; 7(12); 1464-79. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1355.

Integrins and cancer: regulators of cancer stemness, metastasis, and drug resistance.

Interactions between cancer cells and their surroundings can trigger essential signaling cues that determine cell fate and influence the evolution of the malignant phenotype. As the primary receptors involved in cell-matrix adhesion, integrins present on the surface of tumor and stromal cells have a profound impact on the ability to survive in specific locations, but in some cases, these receptors can also function in the absence of ligand binding to promote stemness and survival in the presence of environmental and therapeutic stresses. Understanding how integrin expression and function is regulated in this context will enable the development of new therapeutic approaches to sensitize tumors to therapy and suppress their metastatic phenotype.

Integrin αvß3 drives slug activation and stemness in the pregnant and neoplastic mammary gland.

Although integrin αvß3 is linked to cancer progression, its role in epithelial development is unclear. Here, we show that αvß3 plays a critical role in adult mammary stem cells (MaSCs) during pregnancy. Whereas αvß3 is a luminal progenitor marker in the virgin gland, we noted increased αvß3 expression in MaSCs at midpregnancy. Accordingly, mice lacking αvß3 or expressing a signaling-deficient receptor showed defective mammary gland morphogenesis during pregnancy. This was associated with decreased MaSC expansion, clonogenicity, and expression of Slug, a master regulator of MaSCs. Surprisingly, αvß3-deficient mice displayed normal development of the virgin gland with no effect on luminal progenitors. Transforming growth factor ß2 (TGF-ß2) induced αvß3 expression, enhancing Slug nuclear accumulation and MaSC clonogenicity. In human breast cancer cells, αvß3 was necessary and sufficient for Slug activation, tumorsphere formation, and tumor initiation. Thus, pregnancy-associated MaSCs require a TGF-ß2/αvß3/Slug pathway, which may contribute to breast cancer progression and stemness.

An integrin ß3-KRAS-RalB complex drives tumour stemness and resistance to EGFR inhibition.

Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin α(v)ß3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, α(v)ß3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, α(v)ß3 expression and the resulting KRAS-RalB-NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify α(v)ß3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

PI3Kα activates integrin α4ß1 to establish a metastatic niche in lymph nodes.

Lymph nodes are initial sites of tumor metastasis, yet whether the lymph node microenvironment actively promotes tumor metastasis remains unknown. We show here that VEGF-C/PI3Kα-driven remodeling of lymph nodes promotes tumor metastasis by activating integrin α4ß1 on lymph node lymphatic endothelium. Activated integrin α4ß1 promotes expansion of the lymphatic endothelium in lymph nodes and serves as an adhesive ligand that captures vascular cell adhesion molecule 1 (VCAM-1)(+) metastatic tumor cells, thereby promoting lymph node metastasis. Experimental induction of α4ß1 expression in lymph nodes is sufficient to promote tumor cell adhesion to lymphatic endothelium and lymph node metastasis in vivo, whereas genetic or pharmacological blockade of integrin α4ß1 or VCAM-1 inhibits it. As lymph node metastases accurately predict poor disease outcome, and integrin α4ß1 is a biomarker of lymphatic endothelium in tumor-draining lymph nodes from animals and patients, these results indicate that targeting integrin α4ß1 or VCAM to inhibit the interactions of tumor cells with the lymph node microenvironment may be an effective strategy to suppress tumor metastasis.

EGFR-mediated carcinoma cell metastasis mediated by integrin αvß5 depends on activation of c-Src and cleavage of MUC1.

Receptor tyrosine kinases and integrins play an essential role in tumor cell invasion and metastasis. We previously showed that EGF and other growth factors induce human carcinoma cell invasion and metastasis mediated by integrin αvß5 that is prevented by Src blockade. MUC1, a transmembrane glycoprotein, is expressed in most epithelial tumors as a heterodimer consisting of an extracellular and a transmembrane subunit. The MUC1 cytoplasmic domain of the transmembrane subunit (MUC1.CD) translocates to the nucleus where it promotes the transcription of a metastatic gene signature associated with epithelial to mesenchymal transition. Here, we demonstrate a requirement for MUC1 in carcinoma cell metastasis dependent on EGFR and Src without affecting primary tumor growth. EGF stimulates Src-dependent MUC1 cleavage and nuclear localization leading to the expression of genes linked to metastasis. Moreover, expression of MUC1.CD results in its nuclear localization and is sufficient for transcription of the metastatic gene signature and tumor cell metastasis. These results demonstrate that EGFR and Src activity contribute to carcinoma cell invasion and metastasis mediated by integrin αvß5 in part by promoting proteolytic cleavage of MUC1 and highlight the ability of MUC1.CD to promote metastasis in a context-dependent manner. Our findings may have implications for the use and future design of targeted therapies in cancers known to express EGFR, Src, or MUC1.

RBBP9: a tumor-associated serine hydrolase activity required for pancreatic neoplasia.

Pancreatic cancer is one of the most lethal malignancies. To discover functionally relevant modulators of pancreatic neoplasia, we performed activity-based proteomic profiling on primary human ductal adenocarcinomas. Here, we identify retinoblastoma-binding protein 9 (RBBP9) as a tumor-associated serine hydrolase that displays elevated activity in pancreatic carcinomas. Whereas RBBP9 is expressed in normal and malignant tissues at similar levels, its elevated activity in tumor cells promotes anchorage-independent growth in vitro as well as pancreatic carcinogenesis in vivo. At the molecular level, RBBP9 activity overcomes TGF-beta-mediated antiproliferative signaling by reducing Smad2/3 phosphorylation, a previously unknown role for a serine hydrolase in cancer biology. Conversely, loss of endogenous RBBP9 or expression of mutationally inactive RBBP9 leads to elevated Smad2/3 phosphorylation, implicating this serine hydrolase as an essential suppressor of TGF-beta signaling. Finally, RBBP9-mediated suppression of TGF-beta signaling is required for E-cadherin expression as loss of the serine hydrolase activity leads to a reduction in E-cadherin levels and a concomitant decrease in the integrity of tumor cell-cell junctions. These data not only define a previously uncharacterized serine hydrolase activity associated with epithelial neoplasia, but also demonstrate the potential benefit of functional proteomics in the identification of new therapeutic targets.

Integrins in cancer: biological implications and therapeutic opportunities.

The integrin family of cell adhesion receptors regulates a diverse array of cellular functions crucial to the initiation, progression and metastasis of solid tumours. The importance of integrins in several cell types that affect tumour progression has made them an appealing target for cancer therapy. Integrin antagonists, including the alphavbeta3 and alphavbeta5 inhibitor cilengitide, have shown encouraging activity in Phase II clinical trials and cilengitide is currently being tested in a Phase III trial in patients with glioblastoma. These exciting clinical developments emphasize the need to identify how integrin antagonists influence the tumour and its microenvironment.

An integrin alpha(v)beta(3)-c-Src oncogenic unit promotes anchorage-independence and tumor progression.

Integrins regulate adhesion-dependent growth, survival and invasion of tumor cells. In particular, expression of integrin alpha(v)beta(3) is associated with progression of a variety of human tumors. Here we reveal a previously undescribed adhesion-independent role for integrin alpha(v)beta(3) in pancreatic cancer and other carcinomas. Specifically, alpha(v)beta(3) expressed in carcinoma cells enhanced anchorage-independent tumor growth in vitro and increased lymph node metastases in vivo. These effects required recruitment of c-Src to the beta(3) integrin cytoplasmic tail, leading to c-Src activation, Crk-associated substrate (CAS) phosphorylation and tumor cell survival that, unexpectedly, was independent of cell adhesion or focal adhesion kinase (FAK) activation. Pharmacological blockade of c-Src kinase activity or decreased expression of endogenous alpha(v)beta(3) integrin or c-Src not only inhibited anchorage-independent growth but also suppressed metastasis in vivo, yet these manipulations did not affect tumor cell migration or invasion. These data define an unexpected role for an integrin as a mediator of anchorage independence, suggesting that an alpha(v)beta(3)-c-Src signaling module may account for the aggressive behavior of integrin alpha(v)beta(3)-expressing tumors in humans.

Transforming growth factor beta regulates the expression of the M2 muscarinic receptor in atrial myocytes via an effect on RhoA and p190RhoGAP.

Transforming growth factor beta (TGFbeta) signaling is involved in the development and regulation of multiple organ systems and cellular signaling pathways. We recently demonstrated that TGFbeta regulates the response of atrial myocytes to parasympathetic stimulation. Here, TGFbeta(1) is shown to inhibit expression of the M(2) muscarinic receptor (M(2)), which plays a critical role in the parasympathetic response of the heart. This effect is mimicked by overexpression of a dominant negative mutant of RhoA and by the RhoA kinase inhibitor Y27632, whereas adenoviral expression of a dominant activating-RhoA reverses TGFbeta inhibition of M(2) expression. TGFbeta(1) also mediates a decrease in GTP-bound RhoA and a reciprocal increase in the expression of the RhoA GTPase-activating protein, p190RhoGAP, whereas total RhoA is unchanged. Inhibition of M(2) promoter activity by TGFbeta(1) is mimicked by overexpression of p190RhoGAP, whereas a dominant negative mutant of p190RhoGAP reverses this effect of TGFbeta(1). In contrast to atrial myocytes, in mink lung epithelial cells, in which TGFbeta signaling through activation of RhoA has been previously identified, TGFbeta(1) stimulated an increase in GTP-bound RhoA in association with a reciprocal decrease in the expression of p190RhoGAP. Both effects demonstrated a similar dose dependence on TGFbeta(1). Thus TGFbeta regulation of M(2) muscarinic receptor expression is dependent on RhoA, and TGFbeta regulation of p190RhoGAP expression may be a cell type-specific mechanism for TGFbeta signaling through RhoA.

Activin receptor-like kinase 2 and Smad6 regulate epithelial-mesenchymal transformation during cardiac valve formation.

Epithelial-mesenchymal transformation (EMT) occurs during both development and tumorigenesis. Transforming growth factor beta (TGFbeta) ligands signal EMT in the atrioventricular (AV) cushion of the developing heart, a critical step in valve formation. TGFbeta signals through a complex of type I and type II receptors. Several type I receptors exist although activin receptor-like kinase (ALK) 5 mediates the majority of TGFbeta signaling. Here, we demonstrate that ALK2 is sufficient to induce EMT in the heart. Both ALK2 and ALK5 are expressed throughout the heart with ALK2 expressed abundantly in endocardial cells of the outflow tract (OFT), ventricle, and AV cushion. Misexpression of constitutively active (ca) ALK2 in non-transforming ventricular endocardial cells induced EMT, while caALK5 did not, thus demonstrating that ALK2 activity alone is sufficient to stimulate EMT. Smad6, an inhibitor of Smad signaling downstream of ALK2, but not ALK5, inhibited EMT in AV cushion endocardial cells. These data suggest that ALK2 activation may stimulate EMT in the AV cushion and that Smad6 may act downstream of ALK2 to negatively regulate EMT.

Early events in valvulogenesis: a signaling perspective.

The proper formation and function of the vertebrate heart requires a multitude of specific cell and tissue interactions. These interactions drive the early specification and assembly of components of the cardiovascular system that lead to a functioning system before the attainment of the definitive cardiac and vascular structures seen in the adult. Many of these adult structures are hypothesized to require both proper molecular and physical cues to form correctly. Unlike any other organ system in the embryo, the cardiovascular system requires concurrent function and formation for the embryo to survive. An example of this complex interaction between molecular and physical cues is the formation of the valves of the heart. Both molecular cues that regulate cell transformation, migration, and extracellular matrix deposition, and physical cues emanating from the beating heart, as well as hemodynamic forces, are required for valvulogenesis. This review will focus on molecules and emerging pathways that guide early events in valvulogenesis.

Transforming growth factor beta (TGFbeta ) signaling via differential activation of activin receptor-like kinases 2 and 5 during cardiac development. Role in regulating parasympathetic responsiveness.

Little is known regarding factors that induce parasympathetic responsiveness during cardiac development. We demonstrated previously that in atrial cells cultured from chicks 14 days in ovo, transforming growth factor beta (TGFbeta) decreased parasympathetic inhibition of beat rate by the muscarinic agonist, carbamylcholine, by 5-fold and decreased expression of Galpha(i2). Here in atrial cells 5 days in ovo, TGFbeta increased carbamylcholine inhibition of beat rate 2.5-fold and increased expression of Galpha(i2). TGFbeta also stimulated Galpha(i2) mRNA expression and promoter activity at day 5 while inhibiting them at day 14 in ovo. Over the same time course expression of type I TGFbeta receptors, chick activin receptor-like kinase 2 and 5 increased with a 2.3-fold higher increase in activin receptor-like kinase 2. Constitutively active activin receptor-like kinase 2 inhibited Galpha(i2) promoter activity, whereas constitutively active activin receptor-like kinase 5 stimulated Galpha(i2) promoter activity independent of embryonic age. In 5-day atrial cells, TGFbeta stimulated the p3TP-lux reporter, which is downstream of activin receptor-like kinase 5 and had no effect on the activity of the pVent reporter, which is downstream of activin receptor-like kinase 2. In 14-day cells, TGFbeta stimulated both pVent and p3TP-lux. Thus TGFbeta exerts opposing effects on parasympathetic response and Galpha(i2) expression by activating different type I TGFbeta receptors at distinct stages during cardiac development.