ABSTRACT
Differentiation events contribute to phenotypic cellular heterogeneity within tumors and influence disease progression and response to therapy. Here, we dissect mechanisms controlling intratumoral heterogeneity within triple-negative basal-like breast cancers. Tumor cells expressing the cytokeratin K14 possess a differentiation state that is associated with that of normal luminal progenitors, and K14-negative cells are in a state closer to that of mature luminal cells. We show that cells can transition between these states through asymmetric divisions, which produce one K14+ and one K14- daughter cell, and that these asymmetric divisions contribute to the generation of cellular heterogeneity. We identified several regulators that control the proportion of K14+ cells in the population. EZH2 and Notch increase the numbers of K14+ cells and their rates of symmetric divisions, and FOXA1 has an opposing effect. Our findings demonstrate that asymmetric divisions generate differentiation transitions and heterogeneity, and identify pathways that control breast cancer cellular composition.
Subject(s)
Asymmetric Cell Division , Triple Negative Breast Neoplasms/pathology , Animals , Cell Line, Tumor , Cells, Cultured , Enhancer of Zeste Homolog 2 Protein/genetics , Enhancer of Zeste Homolog 2 Protein/metabolism , Female , Hepatocyte Nuclear Factor 3-alpha/genetics , Hepatocyte Nuclear Factor 3-alpha/metabolism , Humans , Keratins/genetics , Keratins/metabolism , Mice , Receptors, Notch/genetics , Receptors, Notch/metabolism , Triple Negative Breast Neoplasms/metabolismABSTRACT
Breast cancer subtypes display distinct biological traits that influence their clinical behavior and response to therapy. Recent studies have highlighted the importance of chromatin structure regulators in tumorigenesis. The RNF20-RNF40 E3 ubiquitin ligase complex monoubiquitylates histone H2B to generate H2Bub1, while the deubiquitinase (DUB) USP44 can remove this modification. We found that RNF20 and RNF40 expression and global H2Bub1 are relatively low, and USP44 expression is relatively high, in basal-like breast tumors compared with luminal tumors. Consistent with a tumor-suppressive role, silencing of RNF20 in basal-like breast cancer cells increased their proliferation and migration, and their tumorigenicity and metastatic capacity, partly through upregulation of inflammatory cytokines. In contrast, in luminal breast cancer cells, RNF20 silencing reduced proliferation, migration and tumorigenic and metastatic capacity, and compromised estrogen receptor transcriptional activity, indicating a tumor-promoting role. Notably, the effects of USP44 silencing on proliferation and migration in both cancer subtypes were opposite to those of RNF20 silencing. Hence, RNF20 and H2Bub1 have contrasting roles in distinct breast cancer subtypes, through differential regulation of key transcriptional programs underpinning the distinctive traits of each subtype.
Subject(s)
Breast Neoplasms/pathology , Histones/metabolism , Ubiquitin-Protein Ligases/metabolism , Animals , Breast Neoplasms/metabolism , Breast Neoplasms/mortality , Cell Line, Tumor , Cell Movement , Cell Proliferation , Cytokines/metabolism , Female , Humans , Interleukin-6/genetics , Interleukin-6/metabolism , MCF-7 Cells , Mice , NF-kappa B/metabolism , Receptors, Estrogen/genetics , Receptors, Estrogen/metabolism , Survival Rate , Transcription Factor RelA/antagonists & inhibitors , Transcription Factor RelA/genetics , Transcription Factor RelA/metabolism , Ubiquitin Thiolesterase , Ubiquitin-Protein Ligases/antagonists & inhibitors , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Specific Proteases/antagonists & inhibitors , Ubiquitin-Specific Proteases/genetics , Ubiquitin-Specific Proteases/metabolism , UbiquitinationABSTRACT
Genomic instability, a hallmark of cancer, is commonly caused by failures in the DNA damage response. Here we conducted a bioinformatical screen to reveal DNA damage response genes that are upregulated by estrogen and highly mutated in breast and ovarian cancers. This screen identified 53 estrogen-dependent cancer genes, some of which are novel. Notably, the screen retrieved 9 DNA helicases as well as 5 nucleases. DNA2, which functions as both a helicase and a nuclease and plays a role in DNA repair and replication, was retrieved in the screen. Mutations in DNA2, found in estrogen-dependent cancers, are clustered in the helicase and nuclease domains, suggesting activity impairment. Indeed, we show that mutations found in ovarian cancers impair DNA2 activity. Depletion of DNA2 in cells reduces their tumorogenicity in mice. In human, high expression of DNA2 correlates with poor survival of estrogen receptor-positive patients but not of estrogen receptor-negative patients. We also demonstrate that depletion of DNA2 in cells reduces proliferation, while addition of estrogen restores proliferation. These findings suggest that cells responding to estrogen will proliferate despite impaired in DNA2 activity, potentially promoting genomic instability and triggering cancer development.
