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.

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.

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.

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 …