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.