Andrea Clocchiatti

Class IIa HDACs: from important roles in differentiation to possible implications in tumourigenesis.

•  Introduction •  General concepts on class IIa HDACs ‐  Regulation of subcellular localization ‐  Binding partners ‐  Catalytic activity ‐  Class IIa HDAC orthologues in model organisms •  HDAC4 ‐  HDAC4 and differentiation ‐  HDAC4 and cancer •  HDAC5 ‐  HDAC5 and differentiation ‐  HDAC5 and cancer •  HDAC7 ‐  HDAC7 and vascular endothelium ‐  Is HDAC7 a negative regulator of cell proliferation? ‐  HDAC7 and cancer •  HDAC9 ‐  HDAC9 and differentiation ‐  HDAC9 and cancer •  Conclusions

Ubiquitin-dependent degradation of HDAC4, a new regulator of random cell motility

Histone deacetylase 4 (HDAC4) controls several cellular responses and is subjected to multiple levels of regulation. Here it is shown that HDAC4 is under the regulation of the proteasome, in a growth factor- and GSK3β-dependent manner. Degradation of HDAC4 could contribute to the attenuation of random cell motility observed in cells in the G0 phase of the cell cycle.

Sexual dimorphism in cancer

The incidence of many types of cancer arising in organs with non-reproductive functions is significantly higher in male populations than in female populations, with associated differences in survival. Occupational and/or behavioural factors are well-known underlying determinants. However, cellular and molecular differences between the two sexes are also likely to be important. In this Opinion article, we focus on the complex interplay that sex hormones and sex chromosomes can have in intrinsic control of cancer-initiating cell populations, the tumour microenvironment and systemic determinants of cancer development, such as the immune system and metabolism. A better appreciation of these differences between the two sexes could be of substantial value for cancer prevention as well as treatment.

The Control Operated by the Cell Cycle Machinery on MEF2 Stability Contributes to the Downregulation of CDKN1A and Entry into S Phase

ABSTRACT MEF2s are pleiotropic transcription factors (TFs) which supervise multiple cellular activities. During the cell cycle, MEF2s are activated at the G0/G1 transition to orchestrate the expression of the immediate early genes in response to growth factor stimulation. Here we show that, in human and murine fibroblasts, MEF2 activities are downregulated during late G1. MEF2C and MEF2D interact with the E3 ligase F-box protein SKP2, which mediates their subsequent degradation through the ubiquitin-proteasome system. The cyclin-dependent kinase 4 (CDK4)/cyclin D1 complex phosphorylates MEF2D on serine residues 98 and 110, and phosphorylation of these residues is an important determinant for SKP2 binding. Unscheduled MEF2 transcription during the cell cycle reduces cell proliferation, whereas its containment sustains DNA replication. The CDK inhibitor p21/CDKN1A gene is a MEF2 target gene required to exert this antiproliferative influence. MEF2C and MEF2D bind a region within the first intron of CDKN1A, presenting epigenetic markers of open chromatin. Importantly, H3K27 acetylation within this regulative region depends on the presence of MEF2D. We propose that following the initial engagement in the G0/G1 transition, MEF2C and MEF2D must be polyubiquitylated and degraded during G1 progression to diminish the transcription of the CDKN1A gene, thus favoring entry into S phase.

MEF2 is a converging hub for histone deacetylase 4 and phosphatidylinositol 3-kinase/Akt-induced transformation.

ABSTRACT The MEF2-class IIa histone deacetylase (HDAC) axis operates in several differentiation pathways and in numerous adaptive responses. We show here that nuclear active HDAC4 and HDAC7 display transforming capability. HDAC4 oncogenic potential depends on the repression of a limited set of genes, most of which are MEF2 targets. Genes verified as targets of the MEF2-HDAC axis are also under the influence of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway that affects MEF2 protein stability. A signature of MEF2 target genes identified by this study is recurrently repressed in soft tissue sarcomas. Correlation studies depicted two distinct groups of soft tissue sarcomas: one in which MEF2 repression correlates with PTEN downregulation and a second group in which MEF2 repression correlates with HDAC4 levels. Finally, simultaneous pharmacological inhibition of the PI3K/Akt pathway and of MEF2-HDAC interaction shows additive effects on the transcription of MEF2 target genes and on sarcoma cells proliferation. Overall, our work pinpoints an important role of the MEF2-HDAC class IIa axis in tumorigenesis.

Class IIa HDACs repressive activities on MEF2-depedent transcription are associated with poor prognosis of ER+ breast tumors

MEF2s transcription factors and class IIa HDACs compose a fundamental axis for several differentiation pathways. Functional relationships between this axis and cancer are largely unexplored. We have found that class IIa HDACs are heterogeneously expressed and display redundant activities in breast cancer cells. Applying gene set enrichment analysis to compare the expression profile of a list of putative MEF2 target genes, we have discovered a correlation between the down‐regulation of the MEF2 signature and the aggressiveness of ER+ breast tumors. Kaplan‐Meier analysis in ER+ breast tumors evidenced an association between increased class IIa HDACs expression and reduced survival. The important role of the MEF2‐HDAC axis in ER+ breast cancer was confirmed in cultured cells. MCF7 ER+ cells were susceptible to silencing of class IIa HDACs in terms of both MEF2‐dependent transcription and apoptosis. Conversely, in ER– MDA‐MB‐231 cells, the repressive influence of class IIa HDACs was dispensable. Similarly, a class IIa HDAC‐specific inhibitor preferentially promoted the up‐regulation of several MEF2 target genes and apoptosis in ER+ cell lines. The prosurvival function of class IIa HDACs could be explained by the repression of NR4A1/Nur77, a proapoptotic MEF2 target. In summary, our studies underscore a contribution of class IIa HDACs to aggressiveness of ER+ tumors.—Clocchiatti, A., Di Giorgio, E., Ingrao, S., Meyer‐Almes, F.‐J., Tripodo, C., Brancolini, C. Class IIa HDACs repressive activities on MEF2‐depedent transcription are associated with poor prognosis of ER+ breast tumors. FASEB J. 27, 942–954 (2013). www.fasebj.org

The MEF2-HDAC axis controls proliferation of mammary epithelial cells and acini formation in vitro

ABSTRACT The myocyte enhancer factor 2 and histone deacetylase (MEF2–HDAC) axis is a master regulator of different developmental programs and adaptive responses in adults. In this paper, we have investigated the contribution of the axis to the regulation of epithelial morphogenesis, using 3D organotypic cultures of MCF10A cells as a model. We have demonstrated that MEF2 transcriptional activity is upregulated during acini formation, which coincides with exit from the proliferative phase. Upregulation of the transcription of MEF2 proteins is coupled to downregulation of HDAC7, which occurs independently from changes in mRNA levels, and proteasome- or autophagy-mediated degradation. During acini formation, the MEF2–HDAC axis contributes to the promotion of cell cycle exit, through the engagement of the CDK inhibitor CDKN1A. Only in proliferating cells can HDAC7 bind to the first intron of the CDKN1A gene, a region characterized by epigenetic markers of active promoters and enhancers. In cells transformed by the oncogene HER2 (ERBB2), acini morphogenesis is altered, MEF2 transcription is repressed and HDAC7 is continuously expressed. Importantly, reactivation of MEF2 transcriptional activity in these cells, through the use of a HER2 inhibitor or by enhancing MEF2 function, corrected the proliferative defect and re-established normal acini morphogenesis. Summary: The HER2 oncogene alters the epithelial morphogenetic program and inhibits MEF2 transcriptional programs. Reactivation of MEF2s re-established normal acini morphogenesis.

PDCD4 is a CSL associated protein with a transcription repressive function in cancer associated fibroblast activation

The Notch/CSL pathway plays an important role in skin homeostasis and carcinogenesis. CSL, the key effector of canonical Notch signaling endowed with an intrinsic transcription repressive function, suppresses stromal fibroblast senescence and Cancer Associated Fibroblast (CAF) activation through direct down-modulation of key effector genes. Interacting proteins that participate with CSL in this context are as yet to be identified. We report here that Programmed Cell Death 4 (PDCD4), a nuclear/cytoplasmic shuttling protein with multiple functions, associates with CSL and plays a similar role in suppressing dermal fibroblast senescence and CAF activation. Like CSL, PDCD4 is down-regulated in stromal fibroblasts of premalignant skin actinic keratosis (AKs) lesions and squamous cell carcinoma (SCC). While devoid of intrinsic DNA binding capability, PDCD4 is present at CSL binding sites of CAF marker genes as well as canonical Notch/CSL targets and suppresses expression of these genes in a fibroblast-specific manner. Thus, we propose that PDCD4 is part of the CSL repressive complex involved in negative control of stromal fibroblasts conversion into CAFs.

The ULK3 Kinase Is Critical for Convergent Control of Cancer-Associated Fibroblast Activation by CSL and GLI

The connection between signaling pathways activating cancer-associated fibroblasts (CAFs) remains to be determined. Metabolic alterations linked to autophagy have also been implicated in CAF activation. CSL/RBPJ, a transcriptional repressor that mediates Notch signaling, suppresses the gene expression program(s), leading to stromal senescence and CAF activation. Deregulated GLI signaling can also contribute to CAF conversion. Here, we report that compromised CSL function depends on GLI activation for conversion of human dermal fibroblasts into CAFs, separately from cellular senescence. Decreased CSL upregulates the expression of the ULK3 kinase, which binds and activates GLI2. Increased ULK3 also induces autophagy, which is unlinked from GLI and CAF activation. ULK3 upregulation occurs in the CAFs of several tumor types, and ULK3 silencing suppresses the tumor-enhancing properties of these cells. Thus, ULK3 links two key signaling pathways involved in CAF conversion and is an attractive target for stroma-focused anti-cancer intervention.

Autophagy Controls CSL/RBPJκ Stability through a p62/SQSTM1-Dependent Mechanism

SUMMARY Cancer-associated fibroblasts (CAFs) are important at all tumor stages. CSL/RBPJκ suppresses the gene expression program leading to CAF activation and associated metabolic reprogramming, as well as autophagy. Little is known about CSL protein turnover, especially in the tumor microenvironment. We report that, in human dermal fibroblasts (HDFs), conditions inducing autophagy—often found in tumor stroma—down-regulate CSL protein levels but do not affect its mRNA levels. Genetic or pharmacologic targeting of the autophagic machinery blocks CSL down-modulation. Mechanistically, endogenous CSL associates with the autophagy and signaling adaptor p62/SQSTM1, which is required for CSL down-modulation by autophagy. This is functionally significant, because both CSL and p62 levels are lower in skin cancer-derived CAFs, in which autophagy is increased. Increasing cellular CSL levels stabilizes p62 and down-modulates the autophagic process. We reveal here an autophagy-initiated mechanism for CSL down-modulation, which could be targeted for stroma-focused cancer prevention and treatment.