From genetics to histomolecular characterization: An insight into colorectal carcinogenesis in lynch syndrome

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, NEU‐ ROG1, 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 for-mation. 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

Apc splicing mutations leading to in-frame exon 12 or exon 13 skipping are rare events in fap pathogenesis and define the clinical outcome

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

po 006 the mapk c myc axis in crc new pathogenic mechanisms and therapeutic approaches

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

po 203 a novel member in the β catenin destruction complex may mapk14 p38α foster new therapeutic approaches in colorectal cancer

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 APC Min/+ 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 APC Min/+ mice. Significant results were obtained in vivo by co-immunoprecipitation analysis of tissues from normal mice and APC Min/+ 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

po 493 targeting the drug resistance epigenetic driver smyd3 as a new strategy to potentiate chemotherapeutic effects

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

po 161 the ampk and mek erk signalling pathways regulate mitochondrial foxo3a import through phosphorylation of serine 12 and serine 30

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

po 243 uncoupling foxo3a mitochondrial and nuclear functions in cancer cells undergoing metabolic stress and chemotherapy

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

Targeting SMYD3 to sensitize homologous recombination-proficient tumors to PARP-mediated synthetic lethality

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

The MAPK/C-Myc Axis in CRC: new pathogenic mechanisms and therapeutic approaches

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 stabilizes 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. Materials and Methods The cross-talk between p38α and ERK was assessed in CRC cell lines and in APCMin/+ 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 stabilization was assessed by genetic ablation. Results and Discussion Here we show that concomitant inhibition of the p38α and MEK/ERK pathways significantly increases the survival of APCMin/+ 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 stabilization 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