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

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.

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.

Anti-tumor immunotherapy using engineered bacterial outer membrane vesicles fused to lysosome-targeting chimeras mediated by transferrin receptor.

The lysosome-targeting chimera (LYTAC) approach has shown promise for the targeted degradation of secreted and membrane proteins via lysosomes. However, there have been challenges in design, development, and targeting. Here, we have designed a genetically engineered transferrin receptor (TfR)-mediated lysosome-targeting chimera (TfR-LYTAC) that is efficiently internalized via TfR-mediate endocytosis and targets PD-L1 for lysosomal degradation in cultured cells but not in vivo due to short half-life and poor tumor targeting. A delivery platform was developed by fusing TfR-LYTAC to the surface of bacterial outer membrane vesicles (OMVs). The engineered OMV-LYTAC combines PD-1/PD-L1 pathway inhibition with LYTAC and immune activation by bacterial OMVs. OMV-LYTAC significantly reduced tumor growth in vivo. We have provided a modular and simple genetic strategy for lysosomal degradation as well as a delivery platform for in vivo tumor targeting. The study paves the way for the targeting and degradation of extracellular proteins using the TfR-LYTAC system.

The ULK3 kinase is a determinant of keratinocyte self-renewal and tumorigenesis targeting the arginine methylome.

Epigenetic mechanisms oversee epidermal homeostasis and oncogenesis. The identification of kinases controlling these processes has direct therapeutic implications. We show that ULK3 is a nuclear kinase with elevated expression levels in squamous cell carcinomas (SCCs) arising in multiple body sites, including skin and Head/Neck. ULK3 loss by gene silencing or deletion reduces proliferation and clonogenicity of human keratinocytes and SCC-derived cells and affects transcription impinging on stem cell-related and metabolism programs. Mechanistically, ULK3 directly binds and regulates the activity of two histone arginine methyltransferases, PRMT1 and PRMT5 (PRMT1/5), with ULK3 loss compromising PRMT1/5 chromatin association to specific genes and overall methylation of histone H4, a shared target of these enzymes. These findings are of translational significance, as downmodulating ULK3 by RNA interference or locked antisense nucleic acids (LNAs) blunts the proliferation and tumorigenic potential of SCC cells and promotes differentiation in two orthotopic models of skin cancer.

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

Histone deacetylases (HDACs) are important regulators of gene expression. Specific structural features and distinct regulative mechanisms rationalize the separation of the 18 different human HDACs into four classes. The class II comprises a heterogeneous group of nuclear and cytosolic HDACs involved in the regulation of several cellular functions, not just limited to transcriptional repression. In particular, HDAC4, 5, 7 and 9 belong to the subclass IIa and share many transcriptional partners, including members of the MEF2 family. Genetic studies in mice have disclosed the fundamental contribution of class IIa HDACs to specific developmental/differentiation pathways. In this review, we discuss about the recent literature, which hints a role of class IIa HDACs in the development, growth and aggressiveness of cancer cells.

Sustained androgen receptor signaling is a determinant of melanoma cell growth potential and tumorigenesis.

Melanoma susceptibility differs significantly in male versus female populations. Low levels of androgen receptor (AR) in melanocytes of the two sexes are accompanied by heterogeneous expression at various stages of the disease. Irrespective of expression levels, genetic and pharmacological suppression of AR activity in melanoma cells blunts proliferation and induces senescence, while increased AR expression or activation exert opposite effects. AR down-modulation elicits a shared gene expression signature associated with better patient survival, related to interferon and cytokine signaling and DNA damage/repair. AR loss leads to dsDNA breakage, cytoplasmic leakage, and STING activation, with AR anchoring the DNA repair proteins Ku70/Ku80 to RNA Pol II and preventing RNA Pol II-associated DNA damage. AR down-modulation or pharmacological inhibition suppresses melanomagenesis, with increased intratumoral infiltration of macrophages and, in an immune-competent mouse model, cytotoxic T cells. AR provides an attractive target for improved management of melanoma independent of patient sex.

NOTCH1 gene amplification promotes expansion of Cancer Associated Fibroblast populations in human skin.

Cancer associated fibroblasts (CAFs) are a key component of the tumor microenvironment. Genomic alterations in these cells remain a point of contention. We report that CAFs from skin squamous cell carcinomas (SCCs) display chromosomal alterations, with heterogeneous NOTCH1 gene amplification and overexpression that also occur, to a lesser extent, in dermal fibroblasts of apparently unaffected skin. The fraction of the latter cells harboring NOTCH1 amplification is expanded by chronic UVA exposure, to which CAFs are resistant. The advantage conferred by NOTCH1 amplification and overexpression can be explained by NOTCH1 ability to block the DNA damage response (DDR) and ensuing growth arrest through suppression of ATM-FOXO3a association and downstream signaling cascade. In an orthotopic model of skin SCC, genetic or pharmacological inhibition of NOTCH1 activity suppresses cancer/stromal cells expansion. Here we show that NOTCH1 gene amplification and increased expression in CAFs are an attractive target for stroma-focused anti-cancer intervention.

A role for stromal autophagy in cancer-associated fibroblast activation.

In the tumor stroma, cancer-associated fibroblasts (CAFs) affect all aspects of tumor evolution. Whereas several programs leading to CAF activation have been elucidated, little is known about the impact of the microenvironment on the turnover of key CAF regulators. RBPJ/CSL is a transcriptional repressor that mediates NOTCH signaling and its down-modulation activates the gene expression program(s) leading to stromal senescence and CAF activation. We overview our evidence that conditions increasing macroautophagy/autophagy, as often found in the stroma of tumors, cause the down-modulation of the RBPJ protein. This event requires the autophagic machinery and is functionally relevant because it is associated with an increase of CAF effector gene expression. The mechanism involves the direct association with the autophagy receptor SQSTM1/p62, which is required for RBPJ down-modulation. As a reflection of increased autophagy in the stroma, both the RBPJ and SQSTM1 proteins are down-modulated in Squamous Cell Carcinoma (SCC) patient-derived CAFs. Increasing RBPJ cellular levels stabilizes SQSTM1 and down-modulates the autophagic process. Our findings identify an autophagy-initiated mechanism for RBPJ down-modulation leading to increased CAF gene expression.

CSL controls telomere maintenance and genome stability in human dermal fibroblasts.

Genomic instability is a hallmark of cancer. Whether it also occurs in Cancer Associated Fibroblasts (CAFs) remains to be carefully investigated. Loss of CSL/RBP-Jκ, the effector of canonical NOTCH signaling with intrinsic transcription repressive function, causes conversion of dermal fibroblasts into CAFs. Here, we find that CSL down-modulation triggers DNA damage, telomere loss and chromosome end fusions that also occur in skin Squamous Cell Carcinoma (SCC)-associated CAFs, in which CSL is decreased. Separately from its role in transcription, we show that CSL is part of a multiprotein telomere protective complex, binding directly and with high affinity to telomeric DNA as well as to UPF1 and Ku70/Ku80 proteins and being required for their telomere association. Taken together, the findings point to a central role of CSL in telomere homeostasis with important implications for genomic instability of cancer stromal cells and beyond.

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

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.

Androgen receptor functions as transcriptional repressor of cancer-associated fibroblast activation.

The aging-associated increase of cancer risk is linked with stromal fibroblast senescence and concomitant cancer-associated fibroblast (CAF) activation. Surprisingly little is known about the role of androgen receptor (AR) signaling in this context. We have found downmodulated AR expression in dermal fibroblasts underlying premalignant skin cancer lesions (actinic keratoses and dysplastic nevi) as well as in CAFs from the 3 major skin cancer types, squamous cell carcinomas (SCCs), basal cell carcinomas, and melanomas. Functionally, decreased AR expression in primary human dermal fibroblasts (HDFs) from multiple individuals induced early steps of CAF activation, and in an orthotopic skin cancer model, AR loss in HDFs enhanced tumorigenicity of SCC and melanoma cells. Forming a complex, AR converged with CSL/RBP-Jκ in transcriptional repression of key CAF effector genes. AR and CSL were positive determinants of each other's expression, with BET inhibitors, which counteract the effects of decreased CSL, restoring AR expression and activity in CAFs. Increased AR expression in these cells overcame the consequences of CSL loss and was by itself sufficient to block the growth and tumor-enhancing effects of CAFs on neighboring cancer cells. As such, the findings establish AR as a target for stroma-focused cancer chemoprevention and treatment.

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.

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.

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 control operated by the cell cycle machinery on MEF2 stability contributes to the downregulation of CDKN1A and entry into S phase.

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.

Beside the MEF2 axis: unconventional functions of HDAC4.

The class IIa deacetylase HDAC4 is unequivocally known as a negative regulator of MEF2-dependent transcription. In the past years several works have allowed us to understand how different signals, mirroring specific environmental circumstances keep in check the repressive action of HDAC4 against MEF2s. At the same time, pieces of evidence have gradually accumulated about HDAC4 activities emancipated from MEF2s. The aim of this review is to discuss about these "unconventional functions" of HDAC4.

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

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.

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

HDAC4 (histone deacetylase 4) belongs to class IIa of histone deacetylases, which groups important regulators of gene expression, controlling pleiotropic cellular functions. Here we show that, in addition to the well-defined nuclear/cytoplasmic shuttling, HDAC4 activity is modulated by the ubiquitin-proteasome system. Serum starvation elicits the poly-ubiquitination and degradation of HDAC4 in nontransformed cells. Phosphorylation of serine 298 within the PEST1 sequence plays an important role in the control of HDAC4 stability. Serine 298 lies within a glycogen synthase kinase 3ß consensus sequence, and removal of growth factors fails to trigger HDAC4 degradation in cells deficient in this kinase. GSK3ß can phosphorylate HDAC4 in vitro, and phosphorylation of serine 302 seems to play the role of priming phosphate. We have also found that HDAC4 modulates random cell motility possibly through the regulation of KLF2 transcription. Apoptosis, autophagy, cell proliferation, and growth arrest were unaffected by HDAC4. Our data suggest a link between regulation of HDAC4 degradation and the control of cell motility as operated by growth factors.