Diwakar R. Pattabiraman

Tackling the cancer stem cells — what challenges do they pose?

Since their identification in 1994, cancer stem cells (CSCs) have been objects of intensive study. Their properties and mechanisms of formation have become a major focus of current cancer research, in part because of their enhanced ability to initiate and drive tumour growth and their intrinsic resistance to conventional therapeutics. The discovery that activation of the epithelial-to-mesenchymal transition (EMT) programme in carcinoma cells can give rise to cells with stem-like properties has provided one possible mechanism explaining how CSCs arise and presents a possible avenue for their therapeutic manipulation. Here we address recent developments in CSC research, focusing on carcinomas that are able to undergo EMT. We discuss the signalling pathways that create these cells, cell-intrinsic mechanisms that could be exploited for selective elimination or induction of their differentiation, and the role of the tumour microenvironment in sustaining them. Finally, we propose ways to use our current knowledge of the complex biology of CSCs to design novel therapies to eliminate them.

Emerging biological principles of metastasis

Metastases account for the great majority of cancer-associated deaths, yet this complex process remains the least understood aspect of cancer biology. As the body of research concerning metastasis continues to grow at a rapid rate, the biological programs that underlie the dissemination and metastatic outgrowth of cancer cells are beginning to come into view. In this review we summarize the cellular and molecular mechanisms involved in metastasis, with a focus on carcinomas where the most is known, and we highlight the general principles of metastasis that have begun to emerge.

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.

miR-139-5p is a regulator of metastatic pathways in breast cancer

Metastasis is a complex, multistep process involved in the progression of cancer from a localized primary tissue to distant sites, often characteristic of the more aggressive forms of this disease. Despite being studied in great detail in recent years, the mechanisms that govern this process remain poorly understood. In this study, we identify a novel role for miR-139-5p in the inhibition of breast cancer progression. We highlight its clinical relevance by reviewing miR-139-5p expression across a wide variety of breast cancer subtypes using in-house generated and online data sets to show that it is most frequently lost in invasive tumors. A biotin pull-down approach was then used to identify the mRNA targets of miR-139-5p in the breast cancer cell line MCF7. Functional enrichment analysis of the pulled-down targets showed significant enrichment of genes in pathways previously implicated in breast cancer metastasis (P < 0.05). Further bioinformatic analysis revealed a predicted disruption to the TGFβ, Wnt, Rho, and MAPK/PI3K signaling cascades, implying a potential role for miR-139-5p in regulating the ability of cells to invade and migrate. To corroborate this finding, using the MDA-MB-231 breast cancer cell line, we show that overexpression of miR-139-5p results in suppression of these cellular phenotypes. Furthermore, we validate the interaction between miR-139-5p and predicted targets involved in these pathways. Collectively, these results suggest a significant functional role for miR-139-5p in breast cancer cell motility and invasion and its potential to be used as a prognostic marker for the aggressive forms of breast cancer.

Integrated genome-wide chromatin occupancy and expression analyses identify key myeloid pro-differentiation transcription factors repressed by Myb

To gain insight into the mechanisms by which the Myb transcription factor controls normal hematopoiesis and particularly, how it contributes to leukemogenesis, we mapped the genome-wide occupancy of Myb by chromatin immunoprecipitation followed by massively parallel sequencing (ChIP-Seq) in ERMYB myeloid progenitor cells. By integrating the genome occupancy data with whole genome expression profiling data, we identified a Myb-regulated transcriptional program. Gene signatures for leukemia stem cells, normal hematopoietic stem/progenitor cells and myeloid development were overrepresented in 2368 Myb regulated genes. Of these, Myb bound directly near or within 793 genes. Myb directly activates some genes known critical in maintaining hematopoietic stem cells, such as Gfi1 and Cited2. Importantly, we also show that, despite being usually considered as a transactivator, Myb also functions to repress approximately half of its direct targets, including several key regulators of myeloid differentiation, such as Sfpi1 (also known as Pu.1), Runx1, Junb and Cebpb. Furthermore, our results demonstrate that interaction with p300, an established coactivator for Myb, is unexpectedly required for Myb-mediated transcriptional repression. We propose that the repression of the above mentioned key pro-differentiation factors may contribute essentially to Myb’s ability to suppress differentiation and promote self-renewal, thus maintaining progenitor cells in an undifferentiated state and promoting leukemic transformation.

Interaction of c-Myb with p300 is required for the induction of acute myeloid leukemia (AML) by human AML oncogenes

The MYB oncogene is widely expressed in acute leukemias and is important for the continued proliferation of leukemia cells, suggesting that MYB may be a therapeutic target in these diseases. However, realization of this potential requires a significant therapeutic window for MYB inhibition, given its essential role in normal hematopoiesis, and an approach for developing an effective therapeutic. We previously showed that the interaction of c-Myb with the coactivator CBP/p300 is essential for its transforming activity. Here, by using cells from Booreana mice which carry a mutant allele of c-Myb, we show that this interaction is essential for in vitro transformation by the myeloid leukemia oncogenes AML1-ETO, AML1-ETO9a, MLL-ENL, and MLL-AF9. We further show that unlike cells from wild-type mice, Booreana cells transduced with AML1-ETO9a or MLL-AF9 retroviruses fail to generate leukemia upon transplantation into irradiated recipients. Finally, we have begun to explore the molecular mechanisms underlying these observations by gene expression profiling. This identified several genes previously implicated in myeloid leukemogenesis and HSC function as being regulated in a c-Myb-p300-dependent manner. These data highlight the importance of the c-Myb-p300 interaction in myeloid leukemogenesis and suggest disruption of this interaction as a potential therapeutic strategy for acute myeloid leukemia.

Role and potential for therapeutic targeting of MYB in leukemia

The Myb protein was first identified as an oncogene that causes leukemia in chickens. Since then, it has been widely associated with different types of cancers and studied in detail in myeloid leukemias. However, despite these studies, its role in the induction, pathogenesis and maintenance of AML, and other blood disorders, is still not well understood. Recent efforts to uncover its plethora of transcriptional targets have provided key insights into understanding its mechanism of action. This review evaluates our current knowledge of the role of Myb in leukemia, with a particular focus on AML, from the vast literature spanning three decades, highlighting key studies that have influenced our understanding. We discuss recent insights into its role in leukemogenesis and how these could be exploited for the therapeutic targeting of Myb, its associated co-regulators or its target genes, in order to improve outcomes in the treatment of a wide range of hematopoietic malignancies.

A recessive screen for genes regulating hematopoietic stem cells

Identification of genes that regulate the development, self-renewal, and differentiation of stem cells is of vital importance for understanding normal organogenesis and cancer; such knowledge also underpins regenerative medicine. Here we demonstrate that chemical mutagenesis of mice combined with advances in hematopoietic stem cell reagents and genome resources can efficiently recover recessive mutations and identify genes essential for generation and proliferation of definitive hematopoietic stem cells and/or their progeny. We used high-throughput fluorescence-activated cell sorter to analyze 9 subsets of blood stem cells, progenitor cells, circulating red cells, and platelets in more than 1300 mouse embryos at embryonic day (E) 14.5. From 45 pedigrees, we recovered 6 strains with defects in definitive hematopoiesis. We demonstrate rapid identification of a novel mutation in the c-Myb transcription factor that results in thrombocythemia and myelofibrosis as proof of principal of the utility of our fluorescence-activated cell sorter-based screen. Such phenotype-driven approaches will provide new knowledge of the genes, protein interactions, and regulatory networks that underpin stem cell biology.

Integrin-β4 identifies cancer stem cell-enriched populations of partially mesenchymal carcinoma cells

Significance It is widely appreciated that carcinoma cells exhibiting certain mesenchymal traits are enriched for cancer stem cells (CSCs) and can give rise to tumors with aggressive features. Whereas it has been proposed that mesenchymal carcinoma cell populations are internally heterogeneous, the field has made little progress in resolving the specific subtypes of mesenchymal carcinoma cells that pose the greatest risk for patients. We demonstrate the utility of integrin-β4 (ITGB4) in segregating these cells into distinct subpopulations with differing tumor-initiating abilities and pathological behaviors. In addition, we identified mechanistic links between ZEB1 (zinc finger E-box binding homeobox 1) and TAp63α (tumor protein 63 isoform 1) as regulators of ITGB4 expression and demonstrate that ITGB4 can be used as a marker to determine which patients are more likely to relapse after treatment. Neoplastic cells within individual carcinomas often exhibit considerable phenotypic heterogeneity in their epithelial versus mesenchymal-like cell states. Because carcinoma cells with mesenchymal features are often more resistant to therapy and may serve as a source of relapse, we sought to determine whether such cells could be further stratified into functionally distinct subtypes. Indeed, we find that a basal epithelial marker, integrin-β4 (ITGB4), can be used to enable stratification of mesenchymal-like triple-negative breast cancer (TNBC) cells that differ from one another in their relative tumorigenic abilities. Notably, we demonstrate that ITGB4+ cancer stem cell (CSC)-enriched mesenchymal cells reside in an intermediate epithelial/mesenchymal phenotypic state. Among patients with TNBC who received chemotherapy, elevated ITGB4 expression was associated with a worse 5-year probability of relapse-free survival. Mechanistically, we find that the ZEB1 (zinc finger E-box binding homeobox 1) transcription factor activity in highly mesenchymal SUM159 TNBC cells can repress expression of the epithelial transcription factor TAp63α (tumor protein 63 isoform 1), a protein that promotes ITGB4 expression. In addition, we demonstrate that ZEB1 and ITGB4 are important in modulating the histopathological phenotypes of tumors derived from mesenchymal TNBC cells. Hence, mesenchymal carcinoma cell populations are internally heterogeneous, and ITGB4 is a mechanistically driven prognostic biomarker that can be used to identify the more aggressive subtypes of mesenchymal carcinoma cells in TNBC. The ability to rapidly isolate and mechanistically interrogate the CSC-enriched, partially mesenchymal carcinoma cells should further enable identification of novel therapeutic opportunities to improve the prognosis for high-risk patients with TNBC.

Targeting the Epithelial-to-Mesenchymal Transition: The Case for Differentiation-Based Therapy.

Although important strides have been made in targeted therapy for certain leukemias and subtypes of breast cancer, the standard of care for most carcinomas still involves chemotherapy, radiotherapy, surgery, or a combination of these. Two processes serve as obstacles to the successful treatment of carcinomas. First, a majority of deaths from these types of cancers occurs as a result of distant metastases and not the primary tumors themselves. Second, subsets of cells that are able to survive conventional therapy drive the aggressive relapse of the tumors, often in forms that are resistant to treatment. A frequently observed feature of malignant carcinomas is the loss of epithelial traits and the gain of certain mesenchymal ones that are programmed by the cell-biological program termed the epithelial-to-mesenchymal transition (EMT). The EMT program can confer (i) an ability to disseminate, (ii) an ability to become stem-like tumor-initiating cells, (iii) an ability to found new tumor colonies at distant anatomical sites, and (iv) an elevated resistance to therapy. These multiple powers of the EMT program explain why it has become an attractive target for therapeutic intervention. Recent work has revealed the variable nature of the EMT, with multiple versions of the program being observed depending on the tissue context and the stage of tumor progression. In this review, we attempt to crystallize emerging concepts in the research on EMT and stemness and discuss the benefits of using a differentiation-based therapeutic strategy for the eradication of stem-like populations that have adopted various versions of the EMT program.