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An essential function of the epidermis is to provide a physical barrier that prevents the loss of water. Essential mediators of this barrier function include ceramides, cholesterol, and very long chain fatty acids, and their alteration causes human pathologies, including psoriasis and atopic dermatitis. A frameshift mutation in the human ZNF750 gene, which encodes a zinc finger transcription factor, has been shown to cause a seborrhea-like dermatitis. Here, we show that genetic deletion of the mouse homolog ZFP750 results in loss of epidermal barrier function, which is associated with a substantial reduction of ceramides, nonpolar lipids. The alteration of epidermal lipid homeostasis is directly linked to the transcriptional activity of ZFP750. ZFP750 directly and/or indirectly regulates the expression of crucial enzymes primarily involved in the biosynthesis of ceramides. Overall, our study identifies the transcription factor ZFP750 as a master regulator epidermal homeostasis through lipid biosynthesis and thus contributing to our understanding of the pathogenesis of several human skin diseases.

Oral squamous cell carcinoma (OSCC) is a rapidly progressive cancer that often develops resistance against DNA damage inducers, such as radiotherapy and chemotherapy, which are still the standard of care regimens for this tumor. Thus, the identification of biomarkers capable of monitoring the clinical progression of OSCC and its responsiveness to therapy is strongly required. To meet this need, here we have employed Whole Genome Sequencing and RNA-seq data from a cohort of 316 patients retrieved from the TCGA Pan-Cancer Atlas to analyze the genomic and transcriptomic status of the DNA damage response (DDR) genes in OSCC. Then, we correlated the transcriptomic data with the clinical parameters of each patient. Finally, we relied on transcriptomic and drug sensitivity data from the CTRP v2 portal, performing Pearson’s correlation analysis to identify putative vulnerabilities of OSCC cell lines correlated with DDR gene expression. Our results indicate that several DDR genes show a high frequency of genomic and transcriptomic alterations and that the expression of some of them correlates with OSCC grading and infection by the human papilloma virus. In addition, we have identified a signature of eight DDR genes (namely CCNB1, CCNB2, CDK2, CDK4, CHECK1, E2F1, FANCD2, and PRKDC) that could be predictive for OSCC response to the novel antitumor compounds sorafenib and tipifarnib-P1. Altogether, our data demonstrate that alterations in DDR genes could have an impact on the biology of OSCC. Moreover, here we propose a DDR gene signature whose expression could be predictive of OSCC responsiveness to therapy.

LSD1 is a histone lysine demethylase proposed as therapeutic target in cancer. Chemical modifications applied at C2, C4 and/or C7 positions of the quinazoline core of the previously reported dual LSD1/G9a inhibitor 1 led to a series of non-covalent, highly active, and selective LSD1 inhibitors (2-4 and 6-30) and to the dual LSD1/G9a inhibitor 5 that was more potent than 1 against LSD1. In THP-1 and MV4-11 leukemic cells, the most potent compounds (7, 8, and 29) showed antiproliferative effects at sub-micromolar level without significant toxicity at 1μM in non-cancer AHH-1cells. In MV4-11cells, the new derivatives increased the levels of the LSD1 histone mark H3K4me2 and induced the re-expression of the CD86 gene silenced by LSD1, thereby confirming the inhibition of LSD1 at cellular level. In breast MDA-MB-231 as well as in rhabdomyosarcoma RD and RH30cells, taken as examples of solid tumors, the same compounds displayed cell growth arrest in the same IC50 range, highlighting a crucial anticancer role for LSD1 inhibition and suggesting no added value for the simultaneous G9a inhibition in these tumor cell lines.

Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase over-expressed and activated in both adult and pediatric cancers, where it plays important roles in the regulation of pathogenesis and progression of the malignant phenotype. FAK exerts its functions in cancer by two different ways: a kinase activity in the cytoplasm, mainly dependent on the integrin signaling, and a scaffolding activity into the nucleus by networking with different gene expression regulators. For this reason, FAK has to be considered a target with high therapeutic values. Indeed, evidence suggests that FAK targeting could be effective, either alone or in combination, with other already available treatments. Here, we propose an overview of the novel insights about FAK’s structure and nuclear functions, with a special focus on the recent findings concerning the roles of this protein in cancer. Additionally, we provide a recent update on FAK inhibitors that are currently in clinical trials for patients with cancer, and discuss the challenge and future directions of drug-based anti-FAK targeted therapies.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. About 25% of RMS expresses fusion oncoproteins such as PAX3/PAX7-FOXO1 (fusion-positive, FP) while fusion-negative (FN)-RMS harbors RAS mutations. Radiotherapy (RT) plays a crucial role in local control but metastatic RMS is often radio-resistant. HDAC inhibitors (HDACi) radio-sensitize different cancer cells types. Thus, we evaluated MS-275 (Entinostat), a Class I and IV HDACi, in combination with RT on RMS cells in vitro and in vivo. MS-275 reversibly hampered cell survival in vitro in FN-RMS RD (RASmut) and irreversibly in FP-RMS RH30 cell lines down-regulating cyclin A, B, and D1, up-regulating p21 and p27 and reducing ERKs activity, and c-Myc expression in RD and PI3K/Akt/mTOR activity and N-Myc expression in RH30 cells. Further, MS-275 and RT combination reduced colony formation ability of RH30 cells. In both cell lines, co-treatment increased DNA damage repair inhibition and reactive oxygen species formation, down-regulated NRF2, SOD, CAT and GPx4 anti-oxidant genes and improved RT ability to induce G2 growth arrest. MS-275 inhibited in vivo growth of RH30 cells and completely prevented the growth of RT-unresponsive RH30 xenografts when combined with radiation. Thus, MS-275 could be considered as a radio-sensitizing agent for the treatment of intrinsically radio-resistant PAX3-FOXO1 RMS.

Rhabdomyosarcoma (RMS) is a pediatric soft tissue sarcoma characterized by an impaired myogenic differentiation despite the expression of myogenic master genes MYOD and MYOG. Therefore, the restoration of differentiation is considered an anti-cancer therapy. SKP2 is an oncogenic E3-ubiquitin ligase that promotes cell proliferation by targeting the CDKi p21Cip1 and p27Kip1. Previous works showed that SKP2 overexpression is induced by the fusion oncoprotein PAX3-FOXO1 expressed in fusion positive (FP)-RMS cells, and promotes tumor cell proliferation through p27kip1 degradation. However, the role of SKP2 in fusion negative (FN)-RMS cells, devoid of any fusion gene, remains unclear. We report here that SKP2 transcript and protein levels are up-regulated in RMS patients and cell lines compared to normal tissue. Accordingly, we observed increased acetylation of H3K27 histone mark in RMS patients and cell lines compared to myoblasts and muscle tissue. We then show that in RMS cell lines SKP2 expression is induced by MYOD, which binds two SKP2 regulatory regions, an intronic and a distal enhancers, identified by Hi-C and 3C experiments. SKP2 knockdown in FN-RMS cells leads to p21Cip1 and p27Kip1 protein levels up-regulation coupled with G1/S cell cycle arrest. Rescue experiments showed that SKP2 promotes cell proliferation directly targeting p27Kip1. Moreover, SKP2 binds and promotes degradation of p57Kip2 and its silencing restores myogenic differentiation associated to MYOG and de novo MyHC expression in FN-RMS cells. SKP2 depletion also induces cell senescence and prevents anchorage-independent growth and stemness in vitro, and tumor growth in vivo. In turn, SKP2 forced expression partially rescued the anti-cancer effects preventing the increase of p21Cip1, p27Kip1, p57Kip2 and MYOG, promoting re-entry into cell cycle, inhibiting human myoblasts cell differentiation and restoring the tumorigenic potential in FN-RMS. Since neddylation is an essential step for the activity of SKP2, we used MLN4924, an inhibitor of the Nedd8 Activating Enzyme (NAE), under clinical investigation, to resume SKP2 knockdown features. MLN4924 induces p21Cip1 and p27Kip2 expression, promotes senescence and apoptosis, and hampers cell growth in vitro and in vivo both in FP- and FN-RMS. These results unveil an unprecedented role for SKP2 in governing both proliferation and myogenic differentiation in RMS, suggesting that targeting SKP2 functions through MLN4924 treatment might have clinical relevance in FP- and FN-RMS. The study has been founded by AIRC and 5xmille 2021/Ministero della Salute to RR. Citation Format: Silvia Pomella, Matteo Cassandri, Doris Phelps, Clara Perrone, Michele Pezzella, Marco Wachtel, Benjamin Sunkel, Antonella Cardinale, Zoe Walters, Cristina Cossetti, Sonia Rodriguez, Nadia Carlesso, Janet Shipley, Lucio Miele, Beat Schafer, Enrico Velardi, Peter Houghton, Berkley Gryder, Benjamin Stanton, Concetta Quintarelli, Biagio De Angelis, Franco Locatelli, Rossella Rota. A MYOD-SKP2 axis boosts oncogenic properties of fusion negative rhabdomyosarcoma and is counteracted by neddylation inhibition in vitro and in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 668.

Rhabdomyosarcoma (RMS) is an aggressive pediatric malignancy of the muscle, that includes Fusion Positive (FP)-RMS harboring PAX3/7-FOXO1 and Fusion Negative (FN)-RMS commonly with RAS pathway mutations. RMS express myogenic master transcription factors MYOD and MYOG yet are unable to terminally differentiate. Here, we report that SNAI2 is highly expressed in FN-RMS, is oncogenic, blocks myogenic differentiation, and promotes growth. MYOD activates SNAI2 transcription via super enhancers with striped 3D contact architecture. Genome wide chromatin binding analysis demonstrates that SNAI2 preferentially binds enhancer elements and competes with MYOD at a subset of myogenic enhancers required for terminal differentiation. SNAI2 also suppresses expression of a muscle differentiation program modulated by MYOG, MEF2, and CDKN1A. Further, RAS/MEK-signaling modulates SNAI2 levels and binding to chromatin, suggesting that the differentiation blockade by oncogenic RAS is mediated in part by SNAI2. Thus, an interplay between SNAI2, MYOD, and RAS prevents myogenic differentiation and promotes tumorigenesis., Rhabdomyosarcomas are tumours blocked in myogenic differentiation, which despite the expression of master muscle regulatory factors, including MYOD, are unable to differentiate. Here, the authors show that SNAI2 is upregulated by MYOD through super enhancers, binds to MYOD target enhancers, and arrests differentiation.

PURPOSE Herein we describe the in vitro and in vivo activity of FK228 (Romidepsin), an inhibitor of class I HDACs, in counteracting and radiosensitizing embryonal (ERMS, fusion-negative) and alveolar (ARMS, fusion-positive) rhabdomyosarcoma (RMS). METHODS RH30 (ARMS, fusion-positive) and RD (ERMS, fusion-negative) cell lines and human multipotent mesenchymal stromal cells (HMSC) were used. Flow cytometry analysis, RT-qPCR, western blotting and enzymatic assays were performed. Irradiation was delivered by using an x-6 MV photon linear accelerator. FK228 (1.2 mg/kg) in vivo activity, combined or not with radiation therapy (2 Gy), was assessed in murine xenografts. RESULTS Compared to HMSC, RMS expressed low levels of class I HDACs. In vitro, FK228, as single agents, reversibly downregulated class I HDACs expression and activity and induced oxidative stress, DNA damage and a concomitant growth arrest associated with PARP-1-mediated transient non-apoptotic cell death. Surviving cells upregulated the expression of cyclin A, B, D1, p27, Myc and activated PI3K/Akt/mTOR and MAPK signaling, known to be differently involved in cancer chemoresistance. Interestingly, while no radiosensitizing effects were detected, in vitro or in vivo, on RD cells, FK228 markedly radiosensitized RH30 cells by impairing antioxidant and DSBs repair pathways in vitro. Further, FK228 when combined with RT in vivo significantly reduced tumor mass in mouse RH30 xenografts. CONCLUSION FK228 did not show antitumor activity as a single agent whilst its combination with RT resulted in radiosensitization of fusion-positive RMS cells, thus representing a possible strategy for the treatment of the most aggressive RMS subtype.

The human zinc finger (C2H2-type) protein ZNF750 is a transcription factor regulated by p63 that plays a critical role in epithelial tissues homoeostasis, as well as being involved in the pathogenesis of cancer. Indeed, missense mutations, truncation and genomic deletion have been found in oesophageal squamous cell carcinoma. In keeping, we showed that ZNF750 negatively regulates cell migration and invasion in breast cancer cells; in particular, ZNF750 binds and recruits KDM1A and HDAC1 on the LAMB3 and CTNNAL1 promoters. This interaction, in turn, represses the transcription of LAMB3 and CTNNAL1 genes, which are involved in cell migration and invasion. Given that ZNF750 is emerging as a crucial transcription factor that acts as tumour suppressor gene, here, we show that ZNF750 represses the expression of the small GTPase, Ras-related C3 botulinum toxin substrate 1 (RAC1) in breast cancer cell lines, by directly binding its promoter region. In keeping with ZNF750 controlling RAC1 expression, we found an inverse correlation between ZNF750 and RAC1 in human breast cancer datasets. More importantly, we found a significant upregulation of RAC1 in human breast cancer datasets and we identified a direct correlation between RAC1 expression and the survival rate of breast cancer patient. Overall, our findings provide a novel molecular mechanism by which ZNF750 acts as tumour suppressor gene. Hence, we report a potential clinical relevance of ZNF750/RAC1 axis in breast cancer.

Cyclin-Dependent Kinases (CDKs) are well-known reliable targets for cancer treatment being often deregulated. Among them, since the transcription-associated CDK9 represents the sentry of cell transcriptional homeostasis, it can be a valuable target for managing cancers in which the transcriptional machinery is dysregulated by tumor-driver oncogenes. Here we give an overview of some natural compounds identified as CDK inhibitors with reported activity also against CDK9, that were taken as a model for the development of highly active synthetic anti-CDK9 agents. After, we summarize the data on CDK9 inhibition in a group of rare pediatric solid tumors such as rhabdomyosarcoma, Ewing's sarcoma, synovial sarcoma and malignant rhabdoid tumors (soft tissue sarcomas), highlighting the more recent results in this field. Finally, we discuss the perspective and challenge of CDK9 modulation in cancer.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.

Breast cancer is the second leading cause of cancer-related deaths among women, largely due to the progression of a significant fraction of primary tumours to the metastatic stage. Here, we show that zinc-finger protein 750 (ZNF750) opposes the migration and invasion of breast cancer cells by repressing a prometastatic transcriptional programme, which includes genes involved in focal adhesion and extracellular matrix interactions, such as LAMB3 and CTNNAL1. Mechanistically, ZNF750 recruits the epigenetic modifiers KDM1A and HDAC1 to the promoter regions of LAMB3 and CTNNAL1, influencing histone marks and transactivating these genomic sites. Gene expression analysis in cancer patient datasets indicated that ZNF750 and its targets were negative prognostic factors in breast cancer. Together, our findings shed light on the molecular mechanism by which ZNF750 regulates cell migration and invasion, suggesting a role in breast cancer metastasis.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of children and adolescents. The fusion-positive (FP)-RMS variant expressing chimeric oncoproteins such as PAX3-FOXO1 and PAX7-FOXO1 is at high risk. The fusion negative subgroup, FN-RMS, has a good prognosis when non-metastatic. Despite a multimodal therapeutic approach, FP-RMS and metastatic FN-RMS often show a dismal prognosis with 5-year survival of less than 30%. Therefore, novel targets need to be discovered to develop therapies that halt tumor progression, reducing long-term side effects in young patients. Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that regulates focal contacts at the cellular edges. It plays a role in cell motility, survival, and proliferation in response to integrin and growth factor receptors’ activation. FAK is often dysregulated in cancer, being upregulated and/or overactivated in several adult and pediatric tumor types. In RMS, both in vitro and preclinical studies point to a role of FAK in tumor cell motility/invasion and proliferation, which is inhibited by FAK inhibitors. In this review, we summarize the data on FAK expression and modulation in RMS. Moreover, we give an overview of the approaches to inhibit FAK in both preclinical and clinical cancer settings.

Rhabdomyosarcoma (RMS) is a pediatric malignancy of the muscle, and patients with high-risk fusion-negative RMS (FN-RMS), the major subtype of this disease, are associated with RAS pathway activating mutations and have a poor survival rate of Citation Format: Silvia Pomella, Prethish Sreenivas, Berkley E. Gryder, Long Wang, Matteo Cassandri, Kunal Baxi, Nicole R. Hensch, Elena Carcarino, Young Song, Marielle Yohe, Bruno Amadio, Ignazio Caruana, Cristiano De Stefanis, Rita De Vito, Franco Locatelli, Yidong Chen, Eleanor Y. Chen, Peter Houghton, Javed Khan, Rossella Rota, Myron S. Ignatius. Liaison between SNAI2 and MYOD enhances oncogenesis and suppresses differentiation in fusion-negative rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B35.

Zinc-finger proteins (ZNFs) are one of the most abundant groups of proteins and have a wide range of molecular functions. Given the wide variety of zinc-finger domains, ZNFs are able to interact with DNA, RNA, PAR (poly-ADP-ribose) and other proteins. Thus, ZNFs are involved in the regulation of several cellular processes. In fact, ZNFs are implicated in transcriptional regulation, ubiquitin-mediated protein degradation, signal transduction, actin targeting, DNA repair, cell migration, and numerous other processes. The aim of this review is to provide a comprehensive summary of the current state of knowledge of this class of proteins. Firstly, we describe the actual classification of ZNFs, their structure and functions. Secondly, we focus on the biological role of ZNFs in the development of organisms under normal physiological and pathological conditions.