Brian Bierie

Tumour microenvironment: TGFβ: the molecular Jekyll and Hyde of cancer

Transforming growth factor-β (TGFβ) signalling regulates cancer through mechanisms that function either within the tumour cell itself or through host–tumour cell interactions. Studies of tumour-cell-autonomous TGFβ effects show clearly that TGFβ signalling has a mechanistic role in tumour suppression and tumour promotion. In addition, factors in the tumour microenvironment, such as fibroblasts, immune cells and the extracellular matrix, influence the ability of TGFβ to promote or suppress carcinoma progression and metastasis. The complex nature of TGFβ signalling and crosstalk in the tumour microenvironment presents a unique challenge, and an opportunity to develop therapeutic intervention strategies for targeting cancer.

Normal and neoplastic nonstem cells can spontaneously convert to a stem-like state

Current models of stem cell biology assume that normal and neoplastic stem cells reside at the apices of hierarchies and differentiate into nonstem progeny in a unidirectional manner. Here we identify a subpopulation of basal-like human mammary epithelial cells that departs from that assumption, spontaneously dedifferentiating into stem-like cells. Moreover, oncogenic transformation enhances the spontaneous conversion, so that nonstem cancer cells give rise to cancer stem cell (CSC)-like cells in vitro and in vivo. We further show that the differentiation state of normal cells-of-origin is a strong determinant of posttransformation behavior. These findings demonstrate that normal and CSC-like cells can arise de novo from more differentiated cell types and that hierarchical models of mammary stem cell biology should encompass bidirectional interconversions between stem and nonstem compartments. The observed plasticity may allow derivation of patient-specific adult stem cells without genetic manipulation and holds important implications for therapeutic strategies to eradicate cancer.

Effect of conditional knockout of the type II TGF-beta receptor gene in mammary epithelia on mammary gland development and polyomavirus middle T antigen induced tumor formation and metastasis.

Transforming growth factor-beta (TGF-beta) isoforms are growth factors that function physiologically to regulate development, cellular proliferation, and immune responses. The role of TGF-beta signaling in mammary tumorigenesis is complex, as TGF-beta has been reported to function as both a tumor suppressor and tumor promoter. To elucidate the role of TGF-beta signaling in mammary gland development, tumorigenesis, and metastasis, the gene encoding type II TGF-beta receptor, Tgfbr2, was conditionally deleted in the mammary epithelium (Tgfbr2MGKO). Loss of Tgfbr2 in the mammary epithelium results in lobular-alveolar hyperplasia in the developing mammary gland and increased apoptosis. Tgfbr2MGKO mice were mated to the mouse mammary tumor virus-polyomavirus middle T antigen (PyVmT) transgenic mouse model of metastatic breast cancer. Loss of Tgfbr2 in the context of PyVmT expression results in a shortened median tumor latency and an increased formation of pulmonary metastases. Thus, our studies support a tumor-suppressive role for epithelial TGF-beta signaling in mammary gland tumorigenesis and show that pulmonary metastases can occur and are even enhanced in the absence of TGF-beta signaling in the carcinoma cells.

Inhibiting Cxcr2 disrupts tumor-stromal interactions and improves survival in a mouse model of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC), one of the most lethal neoplasms, is characterized by an expanded stroma with marked fibrosis (desmoplasia). We previously generated pancreas epithelium-specific TGF-β receptor type II (Tgfbr2) knockout mice in the context of Kras activation (mice referred to herein as Kras+Tgfbr2KO mice) and found that they developed aggressive PDAC that recapitulated the histological manifestations of the human disease. The mouse PDAC tissue showed strong expression of connective tissue growth factor (Ctgf), a profibrotic and tumor-promoting factor, especially in the tumor-stromal border area, suggesting an active tumor-stromal interaction. Here we show that the PDAC cells in Kras+Tgfbr2KO mice secreted much higher levels of several Cxc chemokines compared with mouse pancreatic intraepithelial neoplasia cells, which are preinvasive. The Cxc chemokines induced Ctgf expression in the pancreatic stromal fibroblasts, not in the PDAC cells themselves. Subcutaneous grafting studies revealed that the fibroblasts enhanced growth of PDAC cell allografts, which was attenuated by Cxcr2 inhibition. Moreover, treating the Kras+Tgfbr2KO mice with the CXCR2 inhibitor reduced tumor progression. The decreased tumor progression correlated with reduced Ctgf expression and angiogenesis and increased overall survival. Taken together, our data indicate that tumor-stromal interactions via a Cxcr2-dependent chemokine and Ctgf axis can regulate PDAC progression. Further, our results suggest that inhibiting tumor-stromal interactions might be a promising therapeutic strategy for PDAC.

Protein Kinase C α Is a Central Signaling Node and Therapeutic Target for Breast Cancer Stem Cells

The epithelial-mesenchymal transition program becomes activated during malignant progression and can enrich for cancer stem cells (CSCs). We report that inhibition of protein kinase C α (PKCα) specifically targets CSCs but has little effect on non-CSCs. The formation of CSCs from non-stem cells involves a shift from EGFR to PDGFR signaling and results in the PKCα-dependent activation of FRA1. We identified an AP-1 molecular switch in which c-FOS and FRA1 are preferentially utilized in non-CSCs and CSCs, respectively. PKCα and FRA1 expression is associated with the aggressive triple-negative breast cancers, and the depletion of FRA1 results in a mesenchymal-epithelial transition. Hence, identifying molecular features that shift between cell states can be exploited to target signaling components critical to CSCs.

A delicate balance: TGF‐β and the tumor microenvironment

The activated form of TGF‐β is a known regulator of epithelial cell autonomous tumor initiation, progression, and metastasis. Recent studies have also indicated that TGF‐β mediates interactions between cancer cells and their local tumor microenvironment. Specifically, the loss of TGF‐β signaling in stromal components including fibroblasts and T‐cells can result in an “activated” microenvironment that supports and even initiates transformation of adjacent epithelial cells. TGF‐β signaling in cancer can be regulated through mechanisms involving ligand activation and expression of essential components within the pathway including the receptors and downstream effectors. TGF‐β signaling in the tumor microenvironment significantly impacts carcinoma initiation, progression, and metastasis via epithelial cell autonomous and interdependent stromal–epithelial interactions in vivo. J. Cell. Biochem. 101: 851–861, 2007.

Activation of PKA leads to mesenchymal-to-epithelial transition and loss of tumor-initiating ability

Have cancer stem cells MET their match? Solid tumors have been hypothesized to contain a subset of highly aggressive cells that fuel tumor growth and metastasis. The search is on for drugs that selectively kill or diminish the malignant properties of these tumor-initiating cells (TICs; previously called “cancer stem cells”). Pattabiraman et al. hypothesized that compounds that induce TICs to undergo a phenotypic change called the mesenchymal-to-epithelial transition (MET) would therefore cause TICs to lose their tumor-initiating ability. Indeed, drugs activating the protein kinase A signaling pathway triggered an epigenetic reprogramming of TICs that resulted in the cells acquiring a more benign epithelial-like phenotype. Science, this issue p. 10.1126/science.aad3680 Tumor-initiating cells differentiate to a more benign state when treated with drugs that activate protein kinase A. INTRODUCTION Tumor-initiating cells (TICs) have emerged in recent years as important targets for cancer therapy owing to their elevated resistance to conventional chemotherapy and their tumor-initiating ability. Although their mode of generation and biological properties have been explored in a diverse array of cancer types, our understanding of the biology of TICs remains superficial. The epithelial-to-mesenchymal transition (EMT) is a cell-biological program that confers mesenchymal traits on both normal and neoplastic epithelial cells, which enables both to acquire stemlike properties. In the case of carcinoma cells, entrance into a more mesenchymal state is associated with elevated resistance to a variety of conventional chemotherapeutics. This association between the EMT program and the TIC state has presented an attractive opportunity for drug development using agents that preferentially target more mesenchymal carcinoma cells, rather than their epithelial counterparts, in an effort to eliminate TICs. Adenosine 3′,5′-monophosphate (cAMP) is a second messenger that transmits intracellular signals through multiple downstream effectors; the most well studied of these is protein kinase A (PKA). In this study, we explore the role of PKA in determining the epithelial versus mesenchymal properties of mammary epithelial cells and how this signaling pathway affects the tumor-initiating ability of transformed cells. RATIONALE At least two approaches might be taken to target mesenchymal TICs. One strategy that has been used previously is the development of agents that show specific or preferential cytotoxicity toward TICs. In the current study, we have embraced an alternative strategy that is designed to induce TICs to undergo a mesenchymal-to-epithelial transition (MET). This “induced differentiation” approach would trigger cells to exit the more mesenchymal tumor-initiating state and enter into an epithelial non-stemlike state. In principle, this transition would make the cells more vulnerable to conventional cytotoxic treatments and thereby reduce the likelihood of metastasis and clinical relapse. RESULTS To identify agents that might induce an MET in mesenchymal mammary epithelial cells, we performed a screen for compounds that stimulate transcription of CDH1, which encodes E-cadherin, a key epithelial protein. Through this screen, compounds that activate adenylate cyclase (cholera toxin, CTx; and forskolin, Fsk) were identified as key inducers of the epithelial state. We found that mesenchymal cells treated with either CTx or Fsk differentiated into benign epithelial derivatives that had lost their ability to effectively initiate tumors and that were more susceptible to conventional chemotherapeutic agents in vitro. Further interrogation revealed that these agents elevated the intracellular levels of cAMP, which in turn activates PKA. PHF2, a histone H3 with acetylated lysine 9 (H3K9) histone demethylase and PKA substrate, was found to be essential for the cAMP-induced MET. By studying the genome occupancy of PHF2 and the epigenomic state of the cells before and after PKA activation, we determined that PHF2 promotes the demethylation and derepression of epithelial genes that ultimately contribute to acquisition of an epithelial state. CONCLUSION We conclude that PKA participates in the differentiation of TICs by enforcing residence in the epithelial state and preventing or reversing the EMT program. Our study reveals a new direction for targeting the TIC population. We propose that pharmacological induction of epigenetic reprogramming of these cells could promote their differentiation to a more epithelial state and increase their susceptibility to conventional chemotherapeutic drugs. Induction of the MET as a potential cancer therapy. TICs have mesenchymal attributes that contribute to their ability to seed new tumors. Treatment of TICs with compounds that increase cAMP levels (e.g., CTx and Fsk) activates PKA. This leads to epigenetic reprogramming through subsequent activation of the histone demethylase PHF2, a PKA substrate, which in turn promotes differentiation of the cells into a more epithelial state, accompanied by a loss of their tumor-initiating ability. Drugs targeting various steps of this signaling pathway might be developed into a differentiation-based cancer therapy for certain breast cancers. Cite this article as D. R. Pattabiraman et al., Science 351, aad3680 (2016). DOI: 10.1126/science.aad3680 The epithelial-to-mesenchymal transition enables carcinoma cells to acquire malignancy-associated traits and the properties of tumor-initiating cells (TICs). TICs have emerged in recent years as important targets for cancer therapy, owing to their ability to drive clinical relapse and enable metastasis. Here, we propose a strategy to eliminate mesenchymal TICs by inducing their conversion to more epithelial counterparts that have lost tumor-initiating ability. We report that increases in intracellular levels of the second messenger, adenosine 3′,5′-monophosphate, and the subsequent activation of protein kinase A (PKA) induce a mesenchymal-to-epithelial transition (MET) in mesenchymal human mammary epithelial cells. PKA activation triggers epigenetic reprogramming of TICs by the histone demethylase PHF2, which promotes their differentiation and loss of tumor-initiating ability. This study provides proof-of-principle for inducing an MET as differentiation therapy for TICs and uncovers a role for PKA in enforcing and maintaining the epithelial state.

Gain or loss of TGF-β signaling in mammary carcinoma cells can promote metastasis

The transforming growth factor beta (TGF-β) is a potent regulator of tumor initiation, progression and metastasis. It has been known for many years that TGF-β signaling in the carcinoma cell can suppress or promote tumor progression depending on the context of stimulation. While the impact of TGF-β on the carcinoma cell is significant, it is now generally accepted that primary and metastatic carcinoma progression is regulated by an intricate network of host-tumor cell interactions. Interestingly, recent results have revealed that gain or loss of TGF-β signaling in carcinoma cells can promote metastasis through carcinoma cell derived TGF-β dependent host-tumor cell interactions in vivo. Further, gain or complete abrogation of TGF-β signaling was shown to result in gene expression signatures that correlated with poor patient prognosis in breast cancer. Specifically, the TGF-β responsive gene expression signature correlated with poor prognosis for estrogen receptor negative (ER-) breast cancer while complete abrogation of TGF-β signaling resulted in a correlation with poor outcome in lymph node positive (LN+) and ER+ breast cancers. Importantly, in both cases the correlation with poor prognosis was linked to carcinoma cell derived interactions with the adjacent microenvironment. Together the current results suggest that, in addition to intrinsic carcinoma cell signaling, TGF-β dependent host-tumor cell interactions should be considered when designing therapeutic strategies to manage carcinoma progression.

GFAP-Cre-Mediated Activation of Oncogenic K-ras Results in Expansion of the Subventricular Zone and Infiltrating Glioma

A subset of neoplastic cells within human high-grade gliomas has features associated with stem cells. These cells may sustain glioma growth, and their stem-like properties may confer resistance to standard glioma treatments. Whether glioma stem cells derive from indigenous neural stem cells (NSC), or from tumor cells that have reacquired stem cell-like properties, is unknown. However, signaling pathways that are tightly regulated and central to NSC biology, including the Ras/Raf/Erk pathway, are hyperactive and pathogenic in gliomagenesis. Furthermore, data in animal models suggests that, in some cases, tumors are initiated in the subventricular zone (SVZ), a stem/progenitor cell niche in the mature brain. We activated oncogenic K-ras in mouse glioneuronal precursor cells and adult SVZ cells using GFAP-Cre. GFAP-Cre+/K-rasG12D mice showed a marked expansion of glial fibriallary acidic protein (GFAP)- and TUJ1–expressing cell populations in the SVZ. In addition, mice developed intermediate grade, infiltrating glioma with 100% penetrance. Tumors were consistently located in the amygdalohippocampal region and nearby cortex, often in association with the lateral ventricle and expanded SVZ. Tumor cells expressed markers associated with neural progenitor cells, including Olig2, Bmi-1, and PDGFR-α. These data suggest that infiltrating tumor cells may arise from NSC transformed by activation of oncogenic K-ras in vivo. (Mol Cancer Res 2009;7(5):645–53)

The role of adipocyte XBP1 in metabolic regulation during lactation.

The adipocyte is central to organismal metabolism and exhibits significant functional and morphological plasticity during its formation and lifespan. Remarkable transformations of this cell occur during obesity and lactation, and thus it is essential to gain a better understanding of adipocyte function in these two metabolic processes. Considering the critical importance of the cellular organelle endoplasmic reticulum (ER) in adapting to fluctuations in synthetic processes, we explored the role of XBP1, a central regulator of ER adaptive responses, in adipocyte formation and function. Unexpectedly, deletion of adipocyte-XBP1 in vivo in mice (XBP1ΔAd) had no effect on adipocyte formation or on systemic homeostatic metabolism in mice fed a a regular or high-fat diet. However, during lactation, XBP1ΔAd dams displayed increased adiposity, decreased milk production, and decreased litter growth as compared with control dams. Moreover, we demonstrate that XBP1 is regulated during lactation and responds to prolactin to alter lipogenic gene expression. These results demonstrate a role for adipocyte-XBP1 in the regulation of lactational metabolism.