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SMYD3 overexpression in several human cancers highlights its crucial role in carcinogenesis. Nonetheless, SMYD3 specific activity in cancer development and progression is currently under debate. Taking advantage of a library of rare tripeptides, which we first tested for their

Colorectal tumorigenesis is driven by alterations in genes and proteins responsible for cancer initiation, progression, and invasion. This multistage process is based on a dense network of protein–protein interactions (PPIs) that become dysregulated as a result of changes in various cell signaling effectors. PPIs in signaling and regulatory networks are known to be mediated by short linear motifs (SLiMs), which are conserved contiguous regions of 3–10 amino acids within interacting protein domains. SLiMs are the minimum sequences required for modulating cellular PPI networks. Thus, several in silico approaches have been developed to predict and analyze SLiM-mediated PPIs. In this review, we focus on emerging evidence supporting a crucial role for SLiMs in driver pathways that are disrupted in colorectal cancer (CRC) tumorigenesis and related PPI network alterations. As a result, SLiMs, along with short peptides, are attracting the interest of researchers to devise small molecules amenable to be used as novel anti-CRC targeted therapies. Overall, the characterization of SLiMs mediating crucial PPIs in CRC may foster the development of more specific combined pharmacological approaches.

SMYD3 is a multifunctional epigenetic enzyme with lysine methyltransferase activity and various interaction partners. It is implicated in the pathophysiology of cancers but with an unclear mechanism. To discover tool compounds for clarifying its biochemistry and potential as a therapeutic target, a set of drug‐like compounds was screened in a biosensor‐based competition assay. Diperodon was identified as an allosteric ligand; its R and S enantiomers were isolated, and their affinities to SMYD3 were determined (K D=42 and 84 μM, respectively). Co‐crystallization revealed that both enantiomers bind to a previously unidentified allosteric site in the C‐terminal protein binding domain, consistent with its weak inhibitory effect. No competition between diperodon and HSP90 (a known SMYD3 interaction partner) was observed although SMYD3–HSP90 binding was confirmed (K D=13 μM). Diperodon clearly represents a novel starting point for the design of tool compounds interacting with a druggable allosteric site, suitable for the exploration of noncatalytic SMYD3 functions and therapeutics with new mechanisms of action., From a distance: SMYD3 lysine methyltransferase is an epigenetic enzyme with multiple cellular functions and the ability to recognize broad range of substrates from histones to cytosolic proteins. An SPR biosensors‐based biophysical strategy for screening SMYD3 ligands was developed and revealed an allosteric binding site. Subsequent crystallographic studies provided a structural description of the novel binding site of SMYD3.

Colorectal cancer (CRC) is one of the deadliest forms of cancer worldwide. CRC development occurs mainly through the adenoma-carcinoma sequence, which can last decades, giving the opportunity for primary prevention and early detection. CRC prevention involves different approaches, ranging from fecal occult blood testing and colonoscopy screening to chemoprevention. In this review, we discuss the main findings gathered in the field of CRC chemoprevention, focusing on different target populations and on various precancerous lesions that can be used as efficacy evaluation endpoints for chemoprevention. The ideal chemopreventive agent should be well tolerated and easy to administer, with low side effects. Moreover, it should be readily available at a low cost. These properties are crucial because these compounds are meant to be used for a long time in populations with different CRC risk profiles. Several agents have been investigated so far, some of which are currently used in clinical practice. However, further investigation is needed to devise a comprehensive and effective chemoprevention strategy for CRC.

Recent findings support the hypothesis that inhibition of SMYD3 methyltransferase may be a therapeutic avenue for some of the deadliest cancer types. Herein, active site-selective covalent SMYD3 inhibitors were designed by introducing an appropriate reactive cysteine trap into reversible first-generation SMYD3 inhibitors. The 4-aminopiperidine derivative EM127 (11C) bearing a 2-chloroethanoyl group as reactive warhead showed selectivity for Cys186, located in the substrate/histone binding pocket. Selectivity towards Cys186 was retained even at high inhibitor/enzyme ratio, as shown by mass spectrometry. The mode of interaction with the SMYD3 substrate/histone binding pocket was revealed by crystallographic studies. In enzymatic assays, 11C showed a stronger SMYD3 inhibitory effect compared to the reference inhibitor EPZ031686. Remarkably, 11C attenuated the proliferation of MDA-MB-231 breast cancer cell line at the same low micromolar range of concentrations that reduced SMYD3 mediated ERK signaling in HCT116 colorectal cancer and MDA-MB-231 breast cancer cells. Furthermore, 11C (5 μM) strongly decreased the steady-state mRNA levels of genes important for tumor biology such as cyclin dependent kinase 2, c-MET, N-cadherin and fibronectin 1, all known to be regulated, at least in part, by SMYD3. Thus, 11C is as a first example of second generation SMYD3 inhibitors; this agent represents a covalent and a site specific SMYD3 binder capable of potent and prolonged attenuation of methyltransferase activity.

Background Intrahepatic Cholangiocarcinoma (iCCA) is characterized by a strong stromal reaction playing a role in tumor progression. Thymus cell antigen 1 (THY1), also called Cluster of Differentiation 90 (CD90), is a key regulator of cell–cell and cell–matrix interaction. In iCCA, CD90 has been reported to be associated with a poor prognosis. In an iCCA PDX model, we recently found that CD90 was downregulated in mice treated with the Notch γ-secretase inhibitor Crenigacestat. The study aims to investigate the role of CD90 in relation to the NOTCH pathway. Methods THY1/CD90 gene and protein expression was evaluated in human iCCA tissues and xenograft models by qRT-PCR, immunohistochemistry, and immunofluorescence. Notch1 inhibition was achieved by siRNA. THY1/CD90 functions were investigated in xenograft models built with HuCCT1 and KKU-M213 cell lines, engineered to overexpress or knockdown THY1, respectively. Results CD90 co-localized with EPCAM, showing its epithelial origin. In vitro, NOTCH1 silencing triggered HES1 and THY1 down-regulation. RBPJ, a critical transcriptional regulator of NOTCH signaling, exhibited putative binding sites on the THY1 promoter and bound to the latter, implying CD90 as a downstream NOTCH pathway effector. In vivo, Crenigacestat suppressed iCCA growth and reduced CD90 expression in the PDX model. In the xenograft model, Crenigacestat inhibited tumor growth of HuCCT1 cells transfected to overexpress CD90 and KKU-M213 cells constitutively expressing high levels of CD90, while not affecting the growth of HuCCT1 control cells and KKU-M213 depleted of CD90. In an iCCA cohort, patients with higher expression levels of NOTCH1/HES1/THY1 displayed a significantly shorter survival. Conclusions iCCA patients with higher NOTCH1/HES1/THY1 expression have the worst prognosis, but they are more likely to benefit from Notch signaling inhibition. These findings represent the scientific rationale for testing NOTCH1 inhibitors in clinical trials, taking the first step toward precision medicine for iCCA.

Genetic variants located in non-coding regions can affect processes that regulate protein expression, functionally contributing to human disease. Germline heterozygous mutations in the non-coding region of the

Germline mutations of the APC gene, which encodes a multidomain protein of 2843 amino acid residues, cause familial adenomatous polyposis (FAP). Three FAP clinical variants are correlated with the location of APC mutations: (1) classic FAP with profuse polyposis (>1000 adenomas), associated with mutations from codon 1250 to 1424; (2) attenuated FAP (APC extremities (before codon 157 and after codon 1595); (3) classic FAP with intermediate colonic polyposis (100–1000 adenomas), associated with mutations located in the remaining part of APC. In an effort to decipher the clinical phenotype associated with APC C-terminal germline truncating mutations in patients with FAP, after screening APC mutations in one family whose members (n=4) developed gastric polyposis, colon oligo-polyposis and desmoid tumours, we performed a literature meta-analysis of clinically characterised patients (n=97) harbouring truncating mutations in APC C-terminus. The APC distal mutations identified in this study cluster with a phenotype characterised by colon oligo-polyposis, diffuse gastric polyposis and desmoid tumours. In conclusion, we describe a novel FAP clinical variant, which we propose to refer to as Gastric Polyposis and Desmoid FAP, that may require tailored management.

Lynch syndrome is a hereditary cancer-predisposing syndrome caused by germline defects in DNA mismatch repair (MMR) genes such as MLH1, MSH2, MSH6, and PMS2. Carriers of pathogenic mutations in these genes have an increased lifetime risk of developing colorectal cancer (CRC) and other malignancies. Despite intensive surveillance, Lynch patients typically develop CRC after 10 years of follow-up, regardless of the screening interval. Recently, three different molecular models of colorectal carcinogenesis were identified in Lynch patients based on when MMR deficiency is acquired. In the first pathway, adenoma formation occurs in an MMR-proficient background, and carcinogenesis is characterized by APC and/or KRAS mutation and IGF2, NEUROG1, CDK2A, and/or CRABP1 hypermethylation. In the second pathway, deficiency in the MMR pathway is an early event arising in macroscopically normal gut surface before adenoma formation. In the third pathway, which is associated with mutations in CTNNB1 and/or TP53, the adenoma step is skipped, with fast and invasive tumor growth occurring in an MMR-deficient context. Here, we describe the association between molecular and histological features in these three routes of colorectal carcinogenesis in Lynch patients. The findings summarized in this review may guide the use of individualized surveillance guidelines based on a patient’s carcinogenesis subtype.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature me-ta-analysis of APC splicing mutations. We found that 123 unique APC splice site mutations, in-cluding the one described here, have been reported in FAP patients, 69 of which have been char-acterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with 4 mutations causing skipping of exon 12 and/or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and 5 mutations leading to skipping of exon 5, 7, 8, and (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations considered in this study cluster with the AFAP phenotype and delineate a novel molecular mechanism of pathogenesis in FAP disease.

mTOR dysregulation has been described in pathological conditions, such as cardiovascular and overgrowth disorders. Here we report on the first case of a patient with a complex congenital heart disease and an interstitial duplication in the short arm of chromosome 1, encompassing part of the mTOR gene. Our results suggest that an intragenic mTOR microduplication might play a role in the pathogenesis of non-syndromic congenital heart defects (CHDs) due to an upregulation of mTOR/Rictor and consequently an increased phosphorylation of PI3K/AKT and MEK/ERK signaling pathways in patient-derived amniocytes. This is the first report which shows a causative role of intragenic mTOR microduplication in the etiology of an isolated complex CHD.

Genetic variants located in non-coding regions can affect processes that regulate protein expression, functionally contributing to human disease. Germline heterozygous mutations in the non-coding region of the PTEN gene have been previously identified in patients with PTEN hamartoma tumor syndrome (PHTS) diagnosed with breast, thyroid, and/or endometrial cancer. In this study, we report a PTEN promoter variant (rs34149102 A allele) that was identified by direct sequencing in an Italian family with a history of gastroesophageal junction (GEJ) adenocarcinoma and breast cancer. In order to investigate the putative functional role of the rs34149102 A allele variant, we evaluated the status of PTEN alterations at the somatic level. We found that PTEN protein expression was absent in the GEJ adenocarcinoma tissue of the index case. Moreover, we detected the occurrence of copy number loss involving the PTEN rs34149102 major C allele in tumor tissue, revealing that the second allele was somatically inactivated. This variant is located within an active regulatory region of the PTEN core promoter, and in silico analysis suggests that it may affect the binding of the nuclear transcription factor MAZ and hence PTEN expression. Overall, these results reveal the functional role of the PTEN promoter rs34149102 A allele variant in the modulation of PTEN protein expression and highlight its contribution to hereditary cancer risk.

The HSF and FOXO families of transcription factors play evolutionarily conserved roles in stress resistance and lifespan. In humans, the rs2802292 G-allele at FOXO3 locus has been associated with longevity in all human populations tested; moreover, its copy number correlated with reduced frequency of age-related diseases in centenarians. At the molecular level, the intronic rs2802292 G-allele correlated with increased expression of FOXO3, suggesting that FOXO3 intron 2 may represent a regulatory region. Here we show that the 90-bp sequence around the intronic single nucleotide polymorphism rs2802292 has enhancer functions, and that the rs2802292 G-allele creates a novel HSE binding site for HSF1, which induces FOXO3 expression in response to diverse stress stimuli. At the molecular level, HSF1 mediates the occurrence of a promoter–enhancer interaction at FOXO3 locus involving the 5′UTR and the rs2802292 region. These data were confirmed in various cellular models including human HAP1 isogenic cell lines (G/T). Our functional studies highlighted the importance of the HSF1-FOXO3-SOD2/CAT/GADD45A cascade in cellular stress response and survival by promoting ROS detoxification, redox balance and DNA repair. Our findings suggest the existence of an HSF1-FOXO3 axis in human cells that could be involved in stress response pathways functionally regulating lifespan and disease susceptibility.

Postzygotic mutations of the PIK3CA [phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit alpha] gene constitutively activate the PI3K/AKT/mTOR pathway in PIK3CA-related overgrowth spectrum (PROS) patients, causing congenital mosaic tissue overgrowth that even multiple surgeries cannot solve. mTOR inhibitors are empirically tested and given for compassionate use in these patients. PROS patients could be ideal candidates for enrolment in trials with PI3K/AKT pathway inhibitors, considering the “clean” cellular setting in which a unique driver, a PIK3CA mutation, is present. We aimed to assess the effects of blocking the upstream pathway of mTOR on PROS patient-derived cells by using ARQ 092, a potent, selective, allosteric, and experimental orally bioavailable and highly selective AKT-inhibitor with activity and long-term tolerability, currently under clinical development for treatment of cancer and Proteus syndrome. Cell samples (i.e., primary fibroblasts) were derived from cultured tissues obtained from six PROS patients [3 boys, 3 girls; aged 2 to 17 years] whose spectrum of PIK3A-related overgrowth included HHML [hemihyperplasia multiple lipomatosis; n = 1], CLOVES [congenital lipomatosis, overgrowth, vascular malformations, epidermal nevi, spinal/skeletal anomalies, scoliosis; n = 1], and MCAP [megalencephaly capillary malformation syndrome; n = 4]. We performed the following: (a) a deep sequencing assay of PI3K/AKT pathway genes in the six PROS patients’ derived cells to identify the causative mutations and (b) a pathway analysis to assess the phosphorylation status of AKT [Ser473 and Thr308] and its downstream targets [pAKTS1 (Thr246), pRPS6 (Ser235/236), and pRPS6Kβ1 (Ser371)]. The anti-proliferative effect of ARQ 092 was tested and compared to other PI3K/AKT/mTOR inhibitors [i.e., wortmannin, LY249002, and rapamycin] in the six PROS patient-derived cells. Using ARQ 092 to target AKT, a critical node connecting PI3K and mTOR pathways, we observed the following: (1) strong anti-proliferative activity [ARQ 092 at 0.5, 1, and 2.5 μM blunted phosphorylation of AKT and its downstream targets (in the presence or absence of serum) and inhibited proliferation after 72 h; rapamycin at 100 nM did not decrease AKT phosphorylation] and (2) less cytotoxicity as compared to rapamycin and wortmannin. We demonstrated the following: (a) that PROS cells are dependent on AKT; (b) the advantage of inhibiting the pathway immediately downstream of PI3K to circumventing problems depending on multiple classes a PI3K kinases; and (c) that PROS patients benefit from inhibition of AKT rather than mTOR. Clinical development of ARQ 092 in PROS patients is on going in these patients. Electronic supplementary material The online version of this article (10.1007/s10048-018-0540-1) contains supplementary material, which is available to authorized users.

Simple Summary BRCA1 and BRCA2 are two major high-penetrance breast/ovarian cancer predisposition genes, whose mutations can lead to high risk and early onset of breast and ovarian cancer. Numerous studies are focused on spectrum and prevalence of BRCA1/2 mutations worldwide. This is the first study that exclusively focused on native Apulian probands. We found that ten recurrent BRCA1/2 pathogenic variants account for more than half of the patients with proven HBOC syndrome from Apulia. Besides BRCA1 c.5266dupC, which is present in significant numbers in every Apulian province, the other PVs occur at a high frequency in some areas and not others. In-depth knowledge of the mutation spectrum of the target population and of the relatively small number of recurrent mutations is crucial to develop a specific cost-effective strategy for mutation screening and a program for breast–ovarian cancer control and prevention through more liberal, yet rational, genetic testing and counseling. Abstract BRCA1/2-associated hereditary breast and ovarian cancer is the most common form of hereditary breast and ovarian cancer and occurs in all ethnicities and racial populations. Different BRCA1/BRCA2 pathogenic variants (PVs) have been reported with a wide variety among populations. In this study, we retrospectively analyzed prevalence and geographic distribution of pathogenic germline BRCA1/2 variants in families from Apulia in southern Italy and evaluated the genotype–phenotype correlations. Data were collected from Oncogenetic Services present in Apulian hospitals and a shared database was built containing Apulian native probands (n = 2026) that had undergone genetic testing from 2004 to 2019. PVs were detected in 499 of 2026 (24.6%) probands and 68.5% of them (342 of 499) were in the BRCA1 gene. We found 65 different PVs in BRCA1 and 46 in BRCA2. There were 10 most recurrent PVs and their geographical distribution appears to be significantly specific for each province. We have assumed that these PVs are related to the historical and geopolitical changes that occurred in Apulia over time and/or to a “founder effect”. Broader knowledge of BRCA1/2 prevalence and recurring PVs in specific geographic areas could help establish more flexible genetic testing strategies that may enhance our ability to detect high-risk subjects.

SMYD3 is frequently overexpressed in a wide variety of cancers. Indeed, its inactivation reduces tumor growth in preclinical in vivo animal models. However, extensive characterization in vitro failed to clarify SMYD3 function in cancer cells, although confirming its importance in carcinogenesis. Taking advantage of a SMYD3 mutant variant identified in a high-risk breast cancer family, here we show that SMYD3 phosphorylation by ATM enables the formation of a multiprotein complex including ATM, SMYD3, CHK2, and BRCA2, which is required for the final loading of RAD51 at DNA double-strand break sites and completion of homologous recombination (HR). Remarkably, SMYD3 pharmacological inhibition sensitizes HR-proficient cancer cells to PARP inhibitors, thereby extending the potential of the synthetic lethality approach in human tumors.

Background Activating pathogenic variants in PIK3CA gene usually occur at a mosaic status and underlie a variety of segmental overgrowth phenotypes. Germline variants in PIK3CA have been rarely reported, described in a total of 12 patients with macrocephaly to date. Clinical and prognostic features of these germline variants have not been described in detail yet. Methods Targeted deep sequencing by custom panel of the 21 genes involved in the PI3K/AKT/mTOR pathway was performed in a 13‐year‐old boy with macrocephaly and physical overgrowth. PI3K/AKT/mTOR pathway analysis was performed in fibroblasts by Western blot. The effects of miransertib (AKT inhibitor) and rapamycin (mTOR inhibitor) were assessed. Results A de novo pathogenic variant (c.1090G>C; p.Gly364Arg) in PIK3CA gene was detected in a non‐mosaic status in peripheral blood cells, buccal smears, and skin fibroblasts. Increased levels of phosphorylated AKT residues were observed in fibroblasts, rescued by miransertib. Conclusion Germline variants in PIK3CA are associated to a mild phenotype characterized by overgrowth, severe macrocephaly, mild intellectual disability, and few dysmorphic features. Investigations of PI3K/AKT/mTOR pathway should be performed in patients with severe macrocephaly and unspecific physical overgrowth. Longitudinal studies to assess prognosis and cancer predisposition are recommended.

Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature meta-analysis of APC splicing mutations. We found that 119 unique APC splicing mutations, including the one described here, have been reported in FAP patients, 69 of which have been characterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with four mutations causing skipping of exon 12 or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and five mutations leading to skipping of exon 5, 7, 8, or (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations causing skipping of exon 12 or 13 considered in this study cluster with the AFAP phenotype and reveal a potential molecular mechanism of pathogenesis in FAP disease.

Colorectal cancer (CRC) poses a formidable challenge in terms of molecular heterogeneity, as it involves a variety of cancer-related pathways and molecular changes unique to an individual’s tumor. On the other hand, recent advances in DNA sequencing technologies provide an unprecedented capacity to comprehensively identify the genetic alterations resulting in tumorigenesis, raising the hope that new therapeutic approaches based on molecularly targeted drugs may prevent the occurrence of chemoresistance. Regulation of the transcription factor FOXO3a in response to extracellular cues plays a fundamental role in cellular homeostasis, being part of the molecular machinery that drives cells towards survival or death. Indeed, FOXO3a is controlled by a range of external stimuli, which not only influence its transcriptional activity, but also affect its subcellular localization. These regulation mechanisms are mediated by cancer-related signaling pathways that eventually drive changes in FOXO3a post-translational modifications (e.g., phosphorylation). Recent results showed that FOXO3a is imported into the mitochondria in tumor cells and tissues subjected to metabolic stress and cancer therapeutics, where it induces expression of the mitochondrial genome to support mitochondrial metabolism and cell survival. The current review discusses the potential clinical relevance of multidrug therapies that drive cancer cell fate by regulating critical pathways converging on FOXO3a.

Introduction: Lynch syndrome is caused by germline mutations in one of the mismatch repair genes ( MLH1, MSH2, MSH6, and PMS2) or in the EPCAM gene. Lynch syndrome is defined on the basis of clinical, pathological, and genetic findings. Accordingly, the identification of predisposing genes allows for accurate risk assessment and tailored screening protocols. Case Description: Here, we report a family case with three family members manifesting the Lynch syndrome phenotype, all of which harbor the rare variant c.2635-2A>G affecting the splice site consensus sequence of intron 15 of the MSH2 gene. This mutation was previously described only in one family with Lynch syndrome, in which mismatch repair protein expression in tumor tissues was not assessed. In this study, we report for the first time the molecular characterization of the MSH2 c.2635-2A>G variant through in silico prediction analysis, microsatellite instability, and mismatch repair protein expression experiments on tumor tissues of Lynch syndrome patients. The potential effect of the splice site variant was revealed by three splicing prediction bioinformatics tools, which suggested the generation of a new cryptic splicing site. The potential pathogenic role of this variant was also revealed by the presence of microsatellite instability and the absence of MSH2/MSH6 heterodimer protein expression in the tumor cells of cancer tissues of the affected family members. Conclusions: We provide compelling evidence in favor of the pathogenic role of the MSH2 variant c.2635-2A>G, which could induce an alteration of the canonical splice site and consequently an aberrant form of the protein product (MSH2).

SMYD3 is a histone lysine methyltransferase that plays an important role in transcriptional activation as a member of an RNA polymerase complex, and its oncogenic role has been described in different cancer types. We studied the expression and activity of SMYD3 in a preclinical model of colorectal cancer (CRC) and found that it is strongly upregulated throughout tumorigenesis both at the mRNA and protein level. Our results also showed that RNAi-mediated SMYD3 ablation impairs CRC cell proliferation indicating that SMYD3 is required for proper cancer cell growth. These data, together with the importance of lysine methyltransferases as a target for drug discovery, prompted us to carry out a virtual screening to identify new SMYD3 inhibitors by testing several candidate small molecules. Here we report that one of these compounds (BCI-121) induces a significant reduction in SMYD3 activity both in vitro and in CRC cells, as suggested by the analysis of global H3K4me2/3 and H4K5me levels. Of note, the extent of cell growth inhibition by BCI-121 was similar to that observed upon SMYD3 genetic ablation. Most of the results described above were obtained in CRC; however, when we extended our observations to tumor cell lines of different origin, we found that SMYD3 inhibitors are also effective in other cancer types, such as lung, pancreatic, prostate, and ovarian. These results represent the proof of principle that SMYD3 is a druggable target and suggest that new compounds capable of inhibiting its activity may prove useful as novel therapeutic agents in cancer treatment.

While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response to metabolic stress and cancer therapeutics. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK, which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism. Using FoxO3A−/− cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin. Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel therapeutic strategies to selectively disable FoxO3A prosurvival activity.

Introduction FoxO3A is a well-known tumour suppressor transcription factor involved in the regulation of various metabolic and cell-death/survival genes. Its activity is finely modulated through specific post-translational modifications functioning as a ‘molecular FoxO code’. Recently, we described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Here, we show that the MEK/ERK and AMPK pathways induce FoxO3A mitochondrial accumulation in cancer cells upon metabolic stress or chemotherapy treatment. Material and methods We performed an extensive in vitro characterisation of the cleaved intra-mitochondrial form of FoxO3A, by analysing mitoplasts purified from several cancer cell lines and tumours. Then, after an in silico preliminary analysis, we generated FoxO3A mutants to identify the key residues required for its mitochondrial accumulation and we extended our in vitro analysis to define the involved kinases. Therefore, to dissect the impact of the MEK/ERK and AMPK pathways on FoxO3A mitochondrial import and functions, we expressed the previously generated mutants in FoxO3A-knockout cancer cell lines obtained by using the CRISPR-Cas9 genome editing system. Results and discussions In metabolically stressed cancer cells, activation of the MEK/ERK and AMPK pathways is required to phosphorylate, respectively, S12 and S30 on FoxO3A N-terminal domain, and promote FoxO3A mitochondrial translocation. Once into the mitochondria, FoxO3A is cleaved by MPPs (mitochondrial processing peptidases) to reach and bind to mitochondrial DNA in complex with TFAM, SIRT3 and mtRNAPol, activating its expression and supporting mitochondrial metabolism and cancer cell survival. Intriguingly, cancer cells treated with chemotherapeutic drugs only require the MEK/ERK pathway to accumulate FoxO3A into the mitochondria, through S12 phosphorylation, and promote resistance and cell survival. Finally, mitochondrial FoxO3A recruitment is necessary for metformin-induced apoptosis. Conclusion The interplay between the MEK/ERK and AMPK pathways, which converge on the N-terminal domain of FoxO3A to eventually increase the expression of mitochondrial-encoded core subunits of the OXPHOS machinery, drives cancer cells towards survival or death. Further elucidation of the FoxO3A ‘mitochondrial code’ will be instrumental to devise personalised therapeutic strategies to selectively disable FoxO3A pro-survival activity.

// Yuichiro Yoshioka 1, 6 , Takehiro Suzuki 2 , Yo Matsuo 3 , Makoto Nakakido 1 , Giichiro Tsurita 4 , Cristiano Simone 5 , Toshiaki Watanabe 6 , Naoshi Dohmae 2 , Yusuke Nakamura 1 , Ryuji Hamamoto 1 1 Section of Hematology/Oncology, Department of Medicine, The University of Chicago, MC2115 Chicago, IL 60637, USA 2 Biomolecular Characterization Unit, RIKEN Center for Sustainable Resource Science, Wako, Saitama 351-0198, Japan 3 OncoTherapy Science, Inc., Takatsu-ku, Kawasaki, Kanagawa 213-0012, Japan 4 Department of Surgery, IMSUT Hospital, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan 5 Division of Medical Genetics, Department of Biomedical Science and Human Oncology (DIMO), University of Bari ‘Aldo Moro’, Bari 70124, Italy 6 Department of Surgical Oncology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-8654, Japan Correspondence to: Ryuji Hamamoto, email: rhamamoto@medicine.bsd.uchicago.edu Keywords: SMYD3, AKT1, lysine methylation, PH domain, human cancer Received: April 08, 2016 Accepted: August 24, 2016 Published: September 08, 2016 ABSTRACT AKT1 is a cytosolic serine/threonine kinase that is overexpressed in various types of cancer and has a central role in human tumorigenesis. Although it is known that AKT1 is post-translationally modified in various ways including phosphorylation and ubiquitination, methylation has not been reported so far. Here we demonstrate that the protein lysine methyltransferase SMYD3 methylates lysine 14 in the PH domain of AKT1 both in vitro and in vivo . Lysine 14-substituted AKT1 shows significantly lower levels of phosphorylation at threonine 308 than wild-type AKT1, and knockdown of SMYD3 as well as treatment with a SMYD3 inhibitor significantly attenuates this phosphorylation in cancer cells. Furthermore, substitution of lysine 14 diminishes the plasma membrane accumulation of AKT1, and cancer cells overexpressing lysine 14-substiuted AKT1 shows lower growth rate than those overexpressing wild-type AKT1. These results imply that SMYD3-mediated methylation of AKT1 at lysine 14 is essential for AKT1 activation and that SMYD3-mediated AKT1 methylation appears to be a good target for development of anti-cancer therapy.

In the search for new strategies to efficiently fight colorectal cancer, efforts are being increasingly focused on targeting regulatory signaling pathways involved in cancer-specific features. As a result, several studies have recently addressed the therapeutic potential of molecularly-targeted drugs capable of inhibiting the activity of protein kinases involved in relevant signaling cascades. Here we show that simultaneous inhibition of the DFG-in and DFG-out conformations of p38α by means of type-I and type-II inhibitors is beneficial to impair more efficiently its kinase activity. Moreover, we found that SB202190 (type-I) and sorafenib (type-II) synergize at the molecular and biological level, as co-treatment with these compounds enhances tumor growth inhibition and induction of apoptosis both in colorectal cancer cell lines and animal models. These results support the need to reconsider sorafenib as a therapeutic agent against colorectal cancer and provide new insights that underline the importance to elucidate the activity of protein kinase inhibitors for the treatment of colorectal carcinoma.

Introduction One of the most commonly deregulated signalling pathways in colorectal cancer (CRC) is the Wnt cascade, which is controlled by APC. APC regulates β-catenin levels, thereby modulating the transcriptional activity of the TCF/LEF transcription factors. High levels of nuclear β-catenin lead to constitutive activation of the Wnt pathway, loss of normal cellular architecture and neoplastic transformation. Previous reports indicate that Wnts are capable of activating p38 MAPKs. A few older studies carried out in other tissues provided evidence that Wnt3a could activate p38 suggesting that the p38 pathway may feed into the canonical Wnt/β-catenin pathway at least at the level of GSK3β. We recently showed that p38α is required to maintain CRC metabolism and survival, as its inhibition leads to activation of FoxO3A, autophagy, cell death and tumour growth reduction both in vitro and in vivo. Material and methods We performed extensive characterisation of the functional interaction between p38 and the APC/β-catenin/GSK3β complex (co-localization analysis by confocal microscopy and co-immunoprecipitation studies) in several cell lines in vitro and in the APCMin/+ mouse preclinical model in vivo. Results and discussions Our data showed that CRC cells have higher levels of activated p38 than their normal counterparts, and experiments using kinase-specific inhibitors revealed that these cells are ‘addicted’ to p38 activity. Interestingly, p38α blockade reduced the size and number of adenomas in the small bowel of APCMin/+ mice. Significant results were obtained in vivo by co-immunoprecipitation analysis of tissues from normal mice and APCMin/+ mice treated or not with AOM. Our findings confirmed the presence of p38α in APC/β-catenin/GSK3β complexes in CRC cells. Importantly, p38α co-localised with β-catenin in both normal and cancer cells; however, these proteins were confined to the cytoplasm in colonocytes, while they occupied discrete nuclear regions in CRC cells. These data were further corroborated by the inhibitory effect of p38α blockade on β-catenin-responsive genes (i.e. c-Myc, cyclin D1/2). Characterisation of this novel functional interaction was also extended with chromatin immunoprecipitation experiments. Conclusion Identification of p38α as a novel member of the APC/β-catenin/GSK3β complex could help elucidate mechanisms contributing to human colon tumour pathogenesis and allow for the development of new strategies for CRC treatment.

Introduction Human cancers arise from a combination of genetic and epigenetic changes. Epigenetic factors regulate chromatin structure, affecting biological processes and promoting cancer. Drugs that target epigenetic modifiers are a new therapeutic challenge, due to the reversibility of epi-modifications. Indeed, epigenetic drugs might sensitise cancer resistant cells to chemotherapy. The SMYD3 histone methyltransferase has an oncogenic role in several cancer types. It is overexpressed in various cancers and promotes cell proliferation, making it a potential target for drug discovery. Material and methods We performed a virtual screening to identify new compounds able to inhibit SMYD3 and then evaluated phenotypic and molecular changes in cells treated with the selected molecule 4- (aminocarbonyl)-N-(4-bromophenyl)−1-piperidineacetamide (BCI-121). Its inhibitory action was assessed by in vitro methylation and surface plasmon resonance assays. To characterise SMYD3 role in cancer response to therapy, we tested potential changes in the sensitivity of cancer cells treated with a combination of BCI-121 and S-phase-specific drugs. Finally, we investigated SMYD3 contribution in DNA repair by evaluating 53 BP1 nuclear foci formation. Results and discussions We observed that SMYD3 is overexpressed in several cancer cell lines, with cells expressing high levels of SMYD3 being highly sensitive to its genetic depletion or pharmacological inhibition by BCI-121. BCI-121 reduces proliferation by arresting cancer cell cycle at the S/G2 boundary. Of note, cell cycle plays a key role in chemosensitivity, particularly for drugs displaying targeted cell cycle effects. Our results showed that pre-treatment with BCI-121 significantly increased cytotoxicity of S-phase agents. Breast cancer cells exposed to DNA damaging agents showed increased levels of nuclear SMYD3 following activation of the repair signals, and an accumulation of unrepaired DNA lesions after SMYD3 genetic ablation. We also evaluated the potential of combined treatment with BCI-121 and S-phase drugs in Triple Negative Breast Cancer (TNBC), which does not usually respond to common therapies. TNBC cells overexpressing SMYD3 confirmed the efficacy of the combined treatment. Conclusion New therapeutic strategies focused on SMYD3 targeting might overcome cancer resistance to existing drugs, thus allowing not only to reduce dose and side effects, but also to treat cancers not usually responding to common therapies.

Introduction c-Myc plays a central role in cellular proliferation, differentiation, and apoptosis. Therefore its deregulation represents a powerful trigger of tumorigenesis, particularly in colorectal cancer (CRC). It has been shown that the MEK/ERK pathway phosphorylates c-Myc on serine 62, which stabilises c-Myc by preventing ubiquitin/proteasomal degradation. We recently reported that MEK/ERK inhibition is counteracted by over-activation of p38α MAPK. Here, we identified cellular mechanisms that lead to c-Myc deregulation, which is a crucial issue for improving CRC treatment and survival. Material and methods The cross-talk between p38α and ERK was assessed in CRC cell lines and in APC Min/+ mice, a murine model of familial adenomatous polyposis. To this aim, animals were treated with the p38α inhibitor 4-(4-Fluorophenyl)−2-(4-hydroxyphenyl)−5-(4-pyridyl)−1H-imidazole (SB202190) alone or in combination with the MEK1 inhibitor N-[(2R)−2,3-Dihydroxypropoxy]−3,4-difluoro-2-[(2-fluoro-4-iodophenyl)amino]-benzamide (PD0325901). In order to evaluate the role of p38α and ERK in c-Myc regulation, we used pharmacological inhibitors of these two kinases alone or in combination with inhibitors of the transcriptional mechanism, translational process and proteasome in CRC cell lines. Moreover, the function of p38α and ERK in Myc stabilisation was assessed by genetic ablation. Results and discussions Here we show that concomitant inhibition of the p38α and MEK/ERK pathways significantly increases the survival of APC Min/+ mice in which tumorigenesis is driven by c-Myc deregulation. Genetic ablation of p38α and ERK revealed that these two MAPKs do not regulate c-Myc expression, nor do they affect c-Myc protein translational process. We found that p38α and ERK collaborate in c-Myc stabilisation by inhibiting its proteasomal degradation in CRC cell lines. These results were also confirmed by using the p38α and ERK pharmacological inhibitors LY2228820 (Ralimetinib) and GSK1120212 (Mekinist), respectively, which are currently in clinical trials for inflammatory diseases and cancer. Conclusion Since c-MYC supports the processes required for normal growth and homeostasis, its ablation is less attractive than modulation of its expression or function. Our results confirmed the essential role of the MAPK/c-Myc axis in intestinal tumorigenesis regulation, suggesting MAPK manipulation as a potential therapeutic approach to counteract c-Myc dependent carcinogenesis.

Introduction While aberrant cancer cell growth is frequently associated with altered biochemical metabolism, normal mitochondrial functions are usually preserved and necessary for full malignant transformation. The transcription factor FoxO3A is a key determinant of cancer cell homeostasis, playing a dual role in survival/death response. We recently described a novel mitochondrial arm of the AMPK-FoxO3A axis in normal cells upon nutrient shortage. Material and methods After extensive characterisation of mitochondrial FoxO3A function in vitro in several cell lines and tumours, we generated FoxO3A-knockout cancer cells with the CRISPR/Cas9 system and reconstituted FoxO3A expression with wild-type or mutant vectors. Results and discussions Here we show that in metabolically stressed cancer cells, FoxO3A is recruited to the mitochondria through activation of MEK/ERK and AMPK which phosphorylate serine 12 and 30, respectively, on FoxO3A N-terminal domain. Subsequently, FoxO3A is imported and cleaved to reach mitochondrial DNA, where it activates expression of the mitochondrial genome to support mitochondrial metabolism and cell survival. Using FoxO3A-/- cancer cells generated with the CRISPR/Cas9 genome editing system and reconstituted with FoxO3A mutants being impaired in their nuclear or mitochondrial subcellular localization, we show that mitochondrial FoxO3A promotes survival in response to metabolic stress. In cancer cells treated with chemotherapeutic agents, accumulation of FoxO3A into the mitochondria promoted survival in a MEK/ERK-dependent manner, while mitochondrial FoxO3A was required for apoptosis induction by metformin. Conclusion Elucidation of FoxO3A mitochondrial vs. nuclear functions in cancer cell homeostasis might help devise novel personalised therapeutic strategies to selectively disable FoxO3A pro-survival activity and manipulate cellular metabolism to counteract cancer initiation and progression.

This paper introduces a new substrate for reverse-phase protein microarray applications based on macroporous silicon. A key feature of the microarray substrate is the vastly surface enlarging properties of the porous silicon, which simultaneously offers highly confined microarray spots. The proof of principle of the reverse array concept was demonstrated in the detection of different levels of cyclin E, a possible cancer biomarker candidate which regulates G1-S transition and correlates with poor prognosis in different types of human cancers. The substrate properties were studied performing analysis of total cyclin E expression in human colon cancer cell lines Hct116 and SW480. The absence of unspecific binding and good microarray quality was demonstrated. In order to verify the performance of the 3-D textured macroporous surface for complex biological samples, lysates of the human tissue spiked to different levels with cell extract overproducing cyclin E (Hct116) were arrayed on the chip surface. The samples were spotted in a noncontact mode in 100 pL droplets with spots sizes ranged between 50 and 70 m and spot-to-spot center distances 100 m, allowing microarray spot densities up to 14 000 spots per cm2. The different sample types of increasing complexities did not have any impact on the spot intensities recorded and the protein spots showed good homogeneity and reproducibility over the recorded microarrays. The data demonstrate the potential use of macroporous silicon as a substrate for quantitative determination of a cancer biomarker cyclin E in tissue lysates. (Less)

Autophagy is a vacuolar process leading to the degradation of long-lived proteins and cytoplasmic organelles in eukaryotes. This process has an important role in normal and cancer cells during adaptation to changing environmental conditions, cellular and tissue remodeling, and cell death. To date, several signaling cascades have been described to regulate autophagy in a cell type-specific and signal-dependent manner. We found that pharmacological blockade of the p38 pathway in colorectal cancer cells, either by the inhibitor SB202190 or by genetic ablation of p38 alpha kinase, causes cell cycle arrest and autophagic cell death. In these cells, a complex network of intracellular kinase cascades controls autophagy and survival since the effect of p38 alpha blockade is differentially affected by the pharmacological inhibition of MEK1, PI(3)K class I and III, and mTOR or by the differentiation status. Collectively, our results suggest an opportunity for exploiting the pharmacological manipulation of the p38 alpha pathway in the treatment of colorectal cancer. Given the number of drugs, currently available or under development, that target the p38 pathway, it stands to reason that elucidating the molecular mechanisms that link p38 and autophagy might have an impact on the clinical translation of these drugs.

Metabolomic profiling offers a powerful methodology for understanding the perturbations of biochemical systems occurring during a disease process. During neoplastic transformation, prostate cells undergo metabolic reprogramming to satisfy the demands of growth and proliferation. An early event in prostate cell transformation is the loss of capacity to accumulate zinc. This change is associated with a higher energy efficiency and increased lipid biosynthesis for cellular proliferation, membrane formation and cell signaling. Moreover, recent studies have shown that sarcosine, an N-methyl derivative of glycine, was significantly increased during disease progression from normal to localized to metastatic prostate cancer. Mapping the metabolomic profiles to their respective biochemical pathways showed an upregulation of androgen-induced protein synthesis, an increased amino acid metabolism and a perturbation of nitrogen breakdown pathways, along with high total choline-containing compounds and phosphocholine levels. In this review, the role of emerging biomarkers is summarized, based on the current understanding of the prostate cancer metabolome.

Cyclin-dependent kinases (cdks) are the catalytic subunits of a large family of serine/threonine protein kinases whose best-characterized members are key regulators of eukaryotic cell cycle progression. They are activated by binding to regulatory subunits generally termed as cyclins. Cdk10 is a cdc2-related kinase that contains the canonical regulatory Tyr and Thr residues present in all protein kinases and a PSTAIRE-like motif named PISSLRE. Although little is known about this protein, human cdk10 has been shown to encode two different isoforms, each having a distinct function. They differ at both the carboxy- and amino-terminals, although most of the amino acid sequence is predicted to be identical for the two isoforms. A role at the G2/M transition has been suggested for an isoform of cdk10, while the alternative splicing form interacts with the N-terminus of the Ets2 transcription factor. Here we report the cloning and the functional characterization of a cDNA encoding the murine homologue of cdk10. Unlike its human counterpart, only one murine cdk10 protein has been identified, and this unique murine cdk10 cDNA encodes a putative protein of 360 amino acids. Comparison of the amino acid sequences of murine and human cdk10 shows high homology. Murine cdk10 binds Ets2 transcription factors in vitro, does not show a direct involvement in the G2/M transition and, therefore, does not affect the proliferation rate of the cell lines analyzed.

Roberts syndrome (RS) is a rare disorder characterized by tetraphocomelia and several other clinical features. Cells from RS patients exhibit characteristic premature separation of heterochromatic region of many chromosomes and abnormalities in cell cycle. Mutations in the ESCO2 gene have recently been identified in 20 RS families. We performed mutational analysis of the ESCO2 gene in two fetuses diagnosed with RS and their normal parents. In both fetuses, we identified homozygosity for the c. 745_746delGT mutation, while the non-consanguineous parents were both heterozygous for the same mutation. Considering the position of the mutation identified, we carried out qualitative and quantitative real-time ESCO2 cDNA analysis on RNA isolated from CVS-stromal cells in one fetus, amniocytes in the second fetus, and lymphocytes from the heterozygous parents. The results of this analysis showed that despite the presence of a premature termination codon (PTC) 112 nucleotides upstream of the next exon3-exon4 junction, the mutant ESCO2 mRNA was present in both fetuses, albeit at low levels, indicating a partial resistance to nonsense mediated decay (NMD). Interestingly, when cells derived from the two fetuses were treated with an inhibitor of translation, they revealed the presence of tissue and individual variability in NMD efficiency, despite the identical mutational status. The existence of such a variation in the NMD efficiency could explain the broad intrafamilial and interfamilial variability in the clinical presentation of RS patients, and in other genetic diseases where nonsense mutations are responsible for most of the mutation load. Moreover, considering that a mutated full length mRNA was produced in both fetuses, we used Western blot analysis to demonstrate the absence of the ESCO2-truncated protein in cells derived from both fetuses and in a lymphoblastoid cell line derived from the parents.

During skeletal myogenesis, muscle-regulatory factors bHLH and MEF2 promote the expression of muscle-specific genes by recruiting several chromatin-modifying complexes on specific DNA regulatory sequences. A number of MyoD-interacting proteins have been reported, but whether they are recruited to the chromatin of myogenic loci, and the relationship with other chromatin bound proteins is unknown. We show that MyoD recruits cdk9/cyclin T2, together with the histone acetyltransferases p300 and PCAF, and the chromatin remodeling complex SWI/SNF, on promoters and enhancers of muscle-specific genes, and that this event correlates with the acetylation of histone tails, remodeling of chromatin, and phosphorylation of the C-terminal domain (CTD) of the RNA polymerase II at these elements.

DNA damage induces cell cycle arrest and DNA repair or apoptosis in proliferating cells. Terminally differentiated cells are permanently withdrawn from the cell cycle and partly resistant to apoptosis. To investigate the effects of genotoxic agents in postmitotic cells, we compared DNA damage-activated responses in mouse and human proliferating myoblasts and their differentiated counterparts, the myotubes. DNA double-strand breaks caused by ionizing radiation (IR) induced rapid activating autophosphorylation of ataxia-teleangiectasia-mutated (ATM), phosphorylation of histone H2AX, recruitment of repair-associated proteins MRE11 and Nbs1, and activation of Chk2 in both myoblasts and myotubes. However, IR-activated, ATM-mediated phosphorylation of p53 at serine 15 (human) or 18 (mouse) [Ser15(h)/18(m)], and apoptosis occurred in myoblasts but was impaired in myotubes. This phosphorylation could be enforced in myotubes by the anthracycline derivative doxorubicin, leading to selective activation of proapoptotic genes. Unexpectedly, the abundance of autophosphorylated ATM was indistinguishable after exposure of myotubes to IR (10 Gy) or doxorubicin (1 microM/24 h) despite efficient phosphorylation of p53 Ser15(h)/18(m), and apoptosis occurred only in response to doxorubicin. These results suggest that radioresistance in myotubes might reflect a differentiation-associated, pathway-selective blockade of DNA damage signaling downstream of ATM. This mechanism appears to preserve IR-induced activation of the ATM-H2AX-MRE11/Rad50/Nbs1 lesion processing and repair pathway yet restrain ATM-p53-mediated apoptosis, thereby contributing to life-long maintenance of differentiated muscle tissues.

During skeletal myogenesis, genomic reprogramming toward terminal differentiation is achieved by recruiting chromatin-modifying enzymes to muscle-specific loci. The relative contribution of extracellular signaling cascades in targeting these enzymes to individual genes is unknown. Here we show that the differentiation-activated p38 pathway targets the SWI-SNF chromatin-remodeling complex to myogenic loci. Upon differentiation, p38 kinases were recruited to the chromatin of muscle-regulatory elements. Blockade of p38 alpha/beta repressed the transcription of muscle genes by preventing recruitment of the SWI-SNF complex at these elements without affecting chromatin binding of muscle-regulatory factors and acetyltransferases. The SWI-SNF subunit BAF60 could be phosphorylated by p38 alpha-beta in vitro, and forced activation of p38 alpha/beta in myoblasts by expression of a constitutively active MKK6 (refs. 5,6,7) promoted unscheduled SWI-SNF recruitment to the myogenin promoter. Conversely, inactivation of SWI-SNF enzymatic subunits abrogated MKK6-dependent induction of muscle gene expression. These results identify an unexpected function of differentiation-activated p38 in converting external cues into chromatin modifications at discrete loci, by selectively targeting SWI-SNF to muscle-regulatory elements.

Myogenic transcription is repressed in myoblasts by serum-activated cyclin-dependent kinases, such as cdk2 and cdk4. Serum withdrawal promotes muscle-specific gene expression at least in part by down-regulating the activity of these cdks. Unlike the other cdks, cdk9 is not serum- or cell cycle-regulated and is instead involved in the regulation of transcriptional elongation by phosphorylating the carboxyl-terminal domain (CTD) of RNA polymerase II. While ectopic expression of cdk2 together with its regulatory subunits (cyclins E and A) inhibits myogenic transcription, overproduction of cdk9 and its associated cyclin (cyclin T2a) strengthens MyoD-dependent transcription and stimulates myogenic differentiation in both MyoD-converted fibroblasts and C2C12 muscle cells. Conversely, inhibition of cdk9 activity by a dominant negative form (cdk9-dn) represses the myogenic program. Cdk9, cyclinT2 and MyoD can be detected in a multimeric complex in C2C12 cells, with the minimal cdk9-binding region of MyoD mapping within 101-161 aa of the bHLH region. Finally, cdk9 can phosphorylate MyoD in vitro, suggesting the possibility that cdk9/cycT2a regulation of muscle differentiation includes the direct enzymatic activity of the kinase on MyoD.

So far, somatic mutations of the PTEN gene have been found in several different neoplasms but not in colorectal tumours. As exons 7 and 8 of the PTEN coding sequence contain an (A)(6)repeat and mononucleotide repeat sequences are targets for mutations in tumours with microsatellite instability (MI), we screened a panel of sporadic colorectal tumours exhibiting MI to test whether PTEN gene repeats are frequently mutated in MI(+)colorectal cancers. Of 32 cases studied, seven mutations were found in six (18.75%) patients, as a PTEN biallelic frameshift mutation was observed in one case, with consequent loss of function of the gene. Loss of heterozygosity, evaluated in the remaining five cases using the microsatellite marker D10S541, was detected in two of three informative samples. To further address the role of the PTEN gene in MI(+)colorectal cancer, in the six patients with mutated PTEN, we analysed the mononucleotide repeats of six other genes: BAX, hMSH3, hMSH6, TGFbRII, IGFIIR and APC. In two of these six patients, mutations of the TGFbRII gene only were present, indicating that PTEN may have a role in the mutator pathway of colorectal tumorigenesis. Overall, these results indicate that PTEN mutations are selected for during tumorigenesis in MI(+)colorectal tumours. The mutation of both PTEN alleles and evidence that the PTEN protein is expressed in normal colon suggest that loss of function of this gene could play a direct role in tumorigenesis.

Summary Lynch syndrome is caused by germline mutations in 1 of the 4 DNA mismatch repair genes ( MLH1 , MSH2 , MSH6 , and PMS2 ). Mutations in MSH2 cause concomitant loss of hMSH6, whereas MLH1 mutations lead to concurrent loss of PMS2. Much less frequent mutations in MSH6 or PMS2 are associated with the isolated loss of the corresponding proteins. We here demonstrate the causative role of the first germline mutation of MSH2 , c.1249-1251 dupGTT (p.417V-418I dupV), associated with normal hMSH2 expression and lack of hMSH6 protein despite a normal MSH 6 gene sequence. hMSH6 protein was completely lost only in advanced cancer stages due to 2 different "second hits": a whole MSH2 gene deletion and a frame-shifting insertion in the MSH6 (C) 8 repeat in the coding sequence.

Chemoresistance is a major obstacle to effective therapy against colorectal cancer (CRC) and may lead to deadly consequences. The metabolism of CRC cells depends highly on the p38 MAPK pathway, whose involvement in maintaining a chemoresistant behavior is currently being investigated. Our previous studies revealed that p38α is the main p38 isoform in CRC cells. Here we show that p38α pharmacological inhibition combined with cisplatin administration decreases colony formation and viability of cancer cells and strongly increases Bax-dependent apoptotic cell death by activating the tumor suppressor protein FoxO3A. Our results indicate that FoxO3A activation up-regulates transcription of its target genes (p21, PTEN, Bim and GADD45), which forces both chemosensitive and chemoresistant CRC cells to undergo apoptosis. Additionally, we found that FoxO3A is required for apoptotic cell death induction, as confirmed by RNA interference experiments. In animal models xenografted with chemoresistant HT29 cells, we further confirmed that the p38-targeted dual therapy strategy produced an increase in apoptosis in cancer tissue leading to tumor regression. Our study uncovers a major role for the p38-FoxO3A axis in chemoresistance, thereby suggesting a new therapeutic approach for CRC treatment; moreover, our results indicate that Bax status may be used as a predictive biomarker.

Reduction of nutrient intake without malnutrition positively influences lifespan and healthspan from yeast to mice and exerts some beneficial effects also in humans. The AMPK-FoxO axis is one of the evolutionarily conserved nutrient-sensing pathways, and the FOXO3A locus is associated with human longevity. Interestingly, FoxO3A has been reported to be also a mitochondrial protein in mammalian cells and tissues. Here we report that glucose restriction triggers FoxO3A accumulation into mitochondria of fibroblasts and skeletal myotubes in an AMPK-dependent manner. A low-glucose regimen induces the formation of a protein complex containing FoxO3A, SIRT3, and mitochondrial RNA polymerase (mtRNAPol) at mitochondrial DNA-regulatory regions causing activation of the mitochondrial genome and a subsequent increase in mitochondrial respiration. Consistently, mitochondrial transcription increases in skeletal muscle of fasted mice, with a mitochondrial DNA-bound FoxO3A/SIRT3/mtRNAPol complex detectable also in vivo. Our results unveil a mitochondrial arm of the AMPK-FoxO3A axis acting as a recovery mechanism to sustain energy metabolism upon nutrient restriction.

Introduction: Ovarian cancer is highly sensitive to chemotherapy but also shows a high rate of recurrence and drug resistance. These negative outcomes mostly depend on altered apoptotic pathways, making the design of new therapeutic strategies based on the induction of other types of cell death highly desirable. Several lines of research are now addressing cancer-specific features to specifically target tumor cells, thus reducing adverse effects. In this light, a great deal of attention has been devoted to the metabolic reprogramming occurring in cancer cells, which display increased levels of glycolysis compared with their normal counterparts. We recently showed that inhibition of p38α impairs key metabolic functions of colorectal cancer cells, inducing growth arrest, autophagy, and cell death both in vivo and in vitro. These effects are mediated by a switch from hypoxia-inducible factor 1α (HIF1α) to forkhead transcription factor O (FoxO)-dependent transcription. Methods: We first characterized p38 expression in OVCAR-3, A2780, and SKOV-3 ovarian cancer cell lines. Then, we treated these cells with the p38α/p38β-specific inhibitor SB202190 and performed a morphological, proliferation, and survival analyses. Finally, we studied HIF1α and FoxO3A expressions and signaling pathways to evaluate their role in SB202190-induced effects. Results: p38α blockade induces the formation of intracellular autophagic vacuoles and reduces growth and viability of ovarian cancer cells. As in colorectal cancer, the underlying molecular mechanism seems to rely on a shift from HIF1α- to FoxO3A-dependent transcription, which is promoted by the activation of the adenosine monophosphate-activated protein kinase pathway. Conclusions: These data corroborate the hypothesis that pharmacological modulation of genes involved in cancer-specific homeostasis, such as p38α, might be exploited to design new therapeutic approaches to cancer treatment.

Autophagy is an essential process for the maintenance of cellular and metabolic homeostasis. Indeed, it is required for the recovery of ATP-generating substrates in cells subjected to different types of stress insults. Thus, the activity of the autophagic machinery strongly depends on the metabolic status of the cell.(1) It has been proposed that this principle applies not only to normal, but also to cancer cells,(2) despite the profound differences in their metabolism. Cancer cells predominantly produce ATP through the constitutive activation of aerobic glycolysis, a process that generally relies on the stabilization and activation of the transcription factor HIF1alpha, which regulates the expression of glycolytic genes.(3) We recently showed that p38alpha is required to sustain the expression of HIF1alpha target genes, and that its inhibition causes a rapid drop in ATP levels in colorectal cancer cells (CRCs). This acute energy need triggers AMPK-dependent nuclear accumulation of FoxO3A and subsequent activation of its transcriptional program, leading to sequential induction of autophagy, cell cycle arrest and cell death. In vivo, pharmacological blockade of p38alpha has both a cytostatic and cytotoxic effect on colorectal neoplasms, associated with nuclear enrichment of FoxO3A and expression of its target genes p21 and PTEN.(4) Our data suggest that CRCs impaired in their glycolytic metabolism trigger autophagy as a reversible recovery mechanism and undergo cell cycle arrest; however, the persistence of the stress insults inevitably leads to cell death.