Mutational signatures of colorectal cancers according to distinct computational workflows.

Tumor mutational signatures have gained prominence in cancer research, yet the lack of standardized methods hinders reproducibility and robustness. Leveraging colorectal cancer (CRC) as a model, we explored the influence of computational parameters on mutational signature analyses across 230 CRC cell lines and 152 CRC patients. Results were validated in three independent datasets: 483 endometrial cancer patients stratified by mismatch repair (MMR) status, 35 lung cancer patients by smoking status and 12 patient-derived organoids (PDOs) annotated for colibactin exposure. Assessing various bioinformatic tools, reference datasets and input data sizes including whole genome sequencing, whole exome sequencing and a pan-cancer gene panel, we demonstrated significant variability in the results. We report that the use of distinct algorithms and references led to statistically different results, highlighting how arbitrary choices may induce variability in the mutational signature contributions. Furthermore, we found a differential contribution of mutational signatures between coding and intergenic regions and defined the minimum number of somatic variants required for reliable mutational signature assignment. To facilitate the identification of the most suitable workflows, we developed Comparative Mutational Signature analysis on Coding and Extragenic Regions (CoMSCER), a bioinformatic tool which allows researchers to easily perform comparative mutational signature analysis by coupling the results from several tools and public reference datasets and to assess mutational signature contributions in coding and non-coding genomic regions. In conclusion, our study provides a comparative framework to elucidate the impact of distinct computational workflows on mutational signatures.

Transcriptome-wide gene expression outlier analysis pinpoints therapeutic vulnerabilities in colorectal cancer.

Multiple strategies are continuously being explored to expand the drug target repertoire in solid tumors. We devised a novel computational workflow for transcriptome-wide gene expression outlier analysis that allows the systematic identification of both overexpression and underexpression events in cancer cells. Here, it was applied to expression values obtained through RNA sequencing in 226 colorectal cancer (CRC) cell lines that were also characterized by whole-exome sequencing and microarray-based DNA methylation profiling. We found cell models displaying an abnormally high or low expression level for 3533 and 965 genes, respectively. Gene expression abnormalities that have been previously associated with clinically relevant features of CRC cell lines were confirmed. Moreover, by integrating multi-omics data, we identified both genetic and epigenetic alternations underlying outlier expression values. Importantly, our atlas of CRC gene expression outliers can guide the discovery of novel drug targets and biomarkers. As a proof of concept, we found that CRC cell lines lacking expression of the MTAP gene are sensitive to treatment with a PRMT5-MTA inhibitor (MRTX1719). Finally, other tumor types may also benefit from this approach.

Tolerance to colibactin correlates with homologous recombination proficiency and resistance to irinotecan in colorectal cancer cells.

The bacterial genotoxin colibactin promotes colorectal cancer (CRC) tumorigenesis, but systematic assessment of its impact on DNA repair is lacking, and its effect on response to DNA-damaging chemotherapeutics is unknown. We find that CRC cell lines display differential response to colibactin on the basis of homologous recombination (HR) proficiency. Sensitivity to colibactin is induced by inhibition of ATM, which regulates DNA double-strand break repair, and blunted by HR reconstitution. Conversely, CRC cells chronically infected with colibactin develop a tolerant phenotype characterized by restored HR activity. Notably, sensitivity to colibactin correlates with response to irinotecan active metabolite SN38, in both cell lines and patient-derived organoids. Moreover, CRC cells that acquire colibactin tolerance develop cross-resistance to SN38, and a trend toward poorer response to irinotecan is observed in a retrospective cohort of CRCs harboring colibactin genomic island. Our results shed insight into colibactin activity and provide translational evidence on its chemoresistance-promoting role in CRC.

Colorectal cancer patient-derived organoids and cell lines harboring ATRX and/or DAXX mutations lack Alternative Lengthening of Telomeres (ALT).

Telomere maintenance is necessary to maintain cancer cell unlimited viability. However, the mechanisms maintaining telomere length in colorectal cancer (CRC) have not been extensively investigated. Telomere maintenance mechanisms (TMM) include the re-expression of telomerase or alternative lengthening of telomeres (ALT). ALT is genetically associated with somatic alterations in alpha-thalassemia/mental retardation X-linked (ATRX) and death domain-associated protein (DAXX) genes. Cells displaying ALT present distinctive features including C-circles made of telomeric DNA, long and heterogenous telomeric tracts, and telomeric DNA co-localized with promyelocytic leukemia (PML) bodies forming so-called ALT-associated PML bodies (APBs). Here, we identified mutations in ATRX and/or DAXX genes in an extensive collection of CRC samples including 119 patient-derived organoids (PDOs) and 232 established CRC cell lines. C-circles measured in CRC PDOs and cell lines showed low levels overall. We also observed that CRC PDOs and cell lines did not display a significant accumulation of APBs or long telomeres with no appreciable differences between wild-type and mutated ATRX/DAXX samples. Overall, our extensive analyses indicate that CRC is not prone to engage ALT, even when carrying genetic lesions in ATRX and/or DAXX, and support the notion that ATRX/DAXX genomic footprints are not reliable predictors of ALT.

Targeting the DNA Damage Response Pathways and Replication Stress in Colorectal Cancer.

PURPOSE: Genomic instability is a hallmark of cancer and targeting DNA damage response (DDR) is emerging as a promising therapeutic strategy in different solid tumors. The effectiveness of targeting DDR in colorectal cancer has not been extensively explored. EXPERIMENTAL DESIGN: We challenged 112 cell models recapitulating the genomic landscape of metastatic colorectal cancer with ATM, ATR, CHK1, WEE1, and DNA-PK inhibitors, in parallel with chemotherapeutic agents. We focused then on ATR inhibitors (ATRi) and, to identify putative biomarkers of response and resistance, we analyzed at multiple levels colorectal cancer models highly sensitive or resistant to these drugs. RESULTS: We found that around 30% of colorectal cancers, including those carrying KRAS and BRAF mutations and unresponsive to targeted agents, are sensitive to at least one DDR inhibitor. By investigating potential biomarkers of response to ATRi, we found that ATRi-sensitive cells displayed reduced phospho-RPA32 foci at basal level, while ATRi-resistant cells showed increased RAD51 foci formation in response to replication stress. Lack of ATM and RAD51C expression was associated with ATRi sensitivity. Analysis of mutational signatures and HRDetect score identified a subgroup of ATRi-sensitive models. Organoids derived from patients with metastatic colorectal cancer recapitulated findings obtained in cell lines. CONCLUSIONS: In conclusion, a subset of colorectal cancers refractory to current therapies could benefit from inhibitors of DDR pathways and replication stress. A composite biomarker involving phospho-RPA32 and RAD51 foci, lack of ATM and RAD51C expression, as well as analysis of mutational signatures could be used to identify colorectal cancers likely to respond to ATRi.

Extracellular Vesicles in Human Preterm Colostrum Inhibit Infection by Human Cytomegalovirus In Vitro.

Breast milk is a complex biofluid that nourishes infants, supports their growth and protects them from diseases. However, at the same time, breastfeeding is a transmission route for human cytomegalovirus (HCMV), with preterm infants being at a great risk of congenital disease. The discrepancy between high HCMV transmission rates and the few reported cases of infants with severe clinical illness is likely due to the protective effect of breast milk. The aim of this study was to investigate the anti-HCMV activity of human preterm colostrum and clarify the role of colostrum-derived extracellular vesicles (EVs). Preterm colostrum samples were collected and the EVs were purified and characterized. The in vitro anti-HCMV activity of both colostrum and EVs was tested against HCMV, and the viral replication step inhibited by colostrum-purified EVs was examined. We investigated the putative role EV surface proteins play in impairing HCMV infection using shaving experiments and proteomic analysis. The obtained results confirmed the antiviral action of colostrum against HCMV and demonstrated a remarkable antiviral activity of colostrum-derived EVs. Furthermore, we demonstrated that EVs impair the attachment of HCMV to cells, with EV surface proteins playing a role in mediating this action. These findings contribute to clarifying the mechanisms that underlie the protective role of human colostrum against HCMV infection.

EGFR Blockade Reverts Resistance to KRASG12C Inhibition in Colorectal Cancer.

Most patients with KRAS G12C-mutant non-small cell lung cancer (NSCLC) experience clinical benefit from selective KRASG12C inhibition, whereas patients with colorectal cancer bearing the same mutation rarely respond. To investigate the cause of the limited efficacy of KRASG12C inhibitors in colorectal cancer, we examined the effects of AMG510 in KRAS G12C colorectal cancer cell lines. Unlike NSCLC cell lines, KRAS G12C colorectal cancer models have high basal receptor tyrosine kinase (RTK) activation and are responsive to growth factor stimulation. In colorectal cancer lines, KRASG12C inhibition induces higher phospho-ERK rebound than in NSCLC cells. Although upstream activation of several RTKs interferes with KRASG12C blockade, we identify EGFR signaling as the dominant mechanism of colorectal cancer resistance to KRASG12C inhibitors. The combinatorial targeting of EGFR and KRASG12C is highly effective in colorectal cancer cells and patient-derived organoids and xenografts, suggesting a novel therapeutic strategy to treat patients with KRAS G12C colorectal cancer. SIGNIFICANCE: The efficacy of KRASG12C inhibitors in NSCLC and colorectal cancer is lineage-specific. RTK dependency and signaling rebound kinetics are responsible for sensitivity or resistance to KRASG12C inhibition in colorectal cancer. EGFR and KRASG12C should be concomitantly inhibited to overcome resistance to KRASG12C blockade in colorectal tumors.See related commentary by Koleilat and Kwong, p. 1094.This article is highlighted in the In This Issue feature, p. 1079.

Vitamin C Restricts the Emergence of Acquired Resistance to EGFR-Targeted Therapies in Colorectal Cancer.

The long-term efficacy of the Epidermal Growth Factor Receptor (EGFR)-targeted antibody cetuximab in advanced colorectal cancer (CRC) patients is limited by the emergence of drug-resistant (persister) cells. Recent studies in other cancer types have shown that cells surviving initial treatment with targeted agents are often vulnerable to alterations in cell metabolism including oxidative stress. Vitamin C (VitC) is an antioxidant agent which can paradoxically trigger oxidative stress at pharmacological dose. Here we tested the hypothesis that VitC in combination with cetuximab could restrain the emergence of secondary resistance to EGFR blockade in CRC RAS/BRAF wild-type models. We found that addition of VitC to cetuximab impairs the emergence of drug persisters, limits the growth of CRC organoids, and significantly delays acquired resistance in CRC patient-derived xenografts. Mechanistically, proteomic and metabolic flux analysis shows that cetuximab blunts carbohydrate metabolism by blocking glucose uptake and glycolysis, beyond promoting slow but progressive ROS production. In parallel, VitC disrupts iron homeostasis and further increases ROS levels ultimately leading to ferroptosis. Combination of VitC and cetuximab orchestrates a synthetic lethal metabolic cell death program triggered by ATP depletion and oxidative stress, which effectively limits the emergence of acquired resistance to anti-EGFR antibodies. Considering that high-dose VitC is known to be safe in cancer patients, our findings might have clinical impact on CRC patients treated with anti-EGFR therapies.

High-dose vitamin C enhances cancer immunotherapy.

Vitamin C (VitC) is known to directly impair cancer cell growth in preclinical models, but there is little clinical evidence on its antitumoral efficacy. In addition, whether and how VitC modulates anticancer immune responses is mostly unknown. Here, we show that a fully competent immune system is required to maximize the antiproliferative effect of VitC in breast, colorectal, melanoma, and pancreatic murine tumors. High-dose VitC modulates infiltration of the tumor microenvironment by cells of the immune system and delays cancer growth in a T cell-dependent manner. VitC not only enhances the cytotoxic activity of adoptively transferred CD8 T cells but also cooperates with immune checkpoint therapy (ICT) in several cancer types. Combination of VitC and ICT can be curative in models of mismatch repair-deficient tumors with high mutational burden. This work provides a rationale for clinical trials combining ICT with high doses of VitC.

A Subset of Colorectal Cancers with Cross-Sensitivity to Olaparib and Oxaliplatin.

PURPOSE: Defects in the homologous recombination (HR) repair pathway are of clinical interest due to sensitivity of HR-deficient cells to PARP inhibitors. We were interested in defining PARP vulnerability in patients with metastatic colorectal cancer (mCRC) carrying KRAS and BRAF mutations who display poor prognosis, have limited therapeutic options, and represent an unmet clinical need. EXPERIMENTAL DESIGN: We tested colorectal cancer cell lines, patient-derived organoids (PDO), and patient-derived xenografts (PDX) enriched for KRAS and BRAF mutations for sensitivity to the PARP inhibitor olaparib, and the chemotherapeutic agents oxaliplatin and 5-fluorouracil (5-FU). Genomic profiles and DNA repair proficiency of colorectal cancer models were compared with pharmacologic response. RESULTS: Thirteen of 99 (around 13%) colorectal cancer cell lines were highly sensitive to clinically active concentrations of olaparib and displayed functional deficiency in HR. Response to PARP blockade was positively correlated with sensitivity to oxaliplatin in colorectal cancer cell lines as well as patient-derived organoids. Treatment of PDXs with olaparib impaired tumor growth and maintenance therapy with PARP blockade after initial oxaliplatin response delayed disease progression in mice. CONCLUSIONS: These results indicate that a colorectal cancer subset characterized by poor prognosis and limited therapeutic options is vulnerable to PARP inhibition and suggest that PDO-based drug-screening assays can be used to identify patients with colorectal cancer likely to benefit from olaparib. As patients with mCRC almost invariably receive therapies based on oxaliplatin, "maintenance" treatment with PARP inhibitors warrants further clinical investigation.

Evolving neoantigen profiles in colorectal cancers with DNA repair defects.

BACKGROUND: Neoantigens that arise as a consequence of tumor-specific mutations can be recognized by T lymphocytes leading to effective immune surveillance. In colorectal cancer (CRC) and other tumor types, a high number of neoantigens is associated with patient response to immune therapies. The molecular processes governing the generation of neoantigens and their turnover in cancer cells are poorly understood. We exploited CRC as a model system to understand how alterations in DNA repair pathways modulate neoantigen profiles over time. METHODS: We performed whole exome sequencing (WES) and RNA sequencing (RNAseq) in CRC cell lines, in vitro and in vivo, and in CRC patient-derived xenografts (PDXs) to track longitudinally genomic profiles, clonal evolution, mutational signatures, and predicted neoantigens. RESULTS: The majority of CRC models showed remarkably stable mutational and neoantigen profiles; however, those carrying defects in DNA repair genes continuously diversified. Rapidly evolving and evolutionary stable CRCs displayed characteristic genomic signatures and transcriptional profiles. Downregulation of molecules implicated in antigen presentation occurred selectively in highly mutated and rapidly evolving CRC. CONCLUSIONS: These results indicate that CRCs carrying alterations in DNA repair pathways display dynamic neoantigen patterns that fluctuate over time. We define CRC subsets characterized by slow and fast evolvability and link this phenotype to downregulation of antigen-presenting cellular mechanisms. Longitudinal monitoring of the neoantigen landscape could be relevant in the context of precision medicine.

Radiologic and Genomic Evolution of Individual Metastases during HER2 Blockade in Colorectal Cancer.

Targeting HER2 is effective in 24% of ERBB2 amplified metastatic colorectal cancer; however, secondary resistance occurs in most of the cases. We studied the evolution of individual metastases during treatment to discover spatially resolved determinants of resistance. Circulating tumor DNA (ctDNA) analysis identified alterations associated with resistance in the majority of refractory patients. ctDNA profiles and lesion-specific radiographic reports revealed organ- or metastasis-private evolutionary patterns. When radiologic assessments documented progressive disease in target lesions, response to HER2 blockade was retained in other metastases. Genomic and functional analyses on samples and cell models from eight metastases of a patient co-recruited to a postmortem study unveiled lesion-specific evolutionary trees and pharmacologic vulnerabilities. Lesion size and contribution of distinct metastases to plasma ctDNA were correlated.

Reliance upon ancestral mutations is maintained in colorectal cancers that heterogeneously evolve during targeted therapies.

Attempts at eradicating metastatic cancers with targeted therapies are limited by the emergence of resistant subclones bearing heterogeneous (epi)genetic changes. We used colorectal cancer (CRC) to test the hypothesis that interfering with an ancestral oncogenic event shared by all the malignant cells (such as WNT pathway alterations) could override heterogeneous mechanisms of acquired drug resistance. Here, we report that in CRC-resistant cell populations, phylogenetic analysis uncovers a complex subclonal architecture, indicating parallel evolution of multiple independent cellular lineages. Functional and pharmacological modulation of WNT signalling induces cell death in CRC preclinical models from patients that relapsed during the treatment, regardless of the drug type or resistance mechanisms. Concomitant blockade of WNT and MAPK signalling restrains the emergence of drug-resistant clones. Reliance upon the WNT-APC pathway is preserved throughout the branched genomic drift associated with emergence of treatment relapse, thus offering the possibility of a common therapeutic strategy to overcome secondary drug resistance.

PARP1 expression drives the synergistic antitumor activity of trabectedin and PARP1 inhibitors in sarcoma preclinical models.

BACKGROUND: Enhancing the antitumor activity of the DNA-damaging drugs is an attractive strategy to improve current treatment options. Trabectedin is an isoquinoline alkylating agent with a peculiar mechanism of action. It binds to minor groove of DNA inducing single- and double-strand-breaks. These kinds of damage lead to the activation of PARP1, a first-line enzyme in DNA-damage response pathways. We hypothesized that PARP1 targeting could perpetuate trabectedin-induced DNA damage in tumor cells leading finally to cell death. METHODS: We investigated trabectedin and PARP1 inhibitor synergism in several tumor histotypes both in vitro and in vivo (subcutaneous and orthotopic tumor xenografts in mice). We searched for key determinants of drug synergism by comparative genomic hybridization (aCGH) and gene expression profiling (GEP) and validated their functional role. RESULTS: Trabectedin activated PARP1 enzyme and the combination with PARP1 inhibitors potentiated DNA damage, cell cycle arrest at G2/M checkpoint and apoptosis, if compared to single agents. Olaparib was the most active PARP1 inhibitor to combine with trabectedin and we confirmed the antitumor and antimetastatic activity of trabectedin/olaparib combination in mice models. However, we observed different degree of trabectedin/olaparib synergism among different cell lines. Namely, in DMR leiomyosarcoma models the combination was significantly more active than single agents, while in SJSA-1 osteosarcoma models no further advantage was obtained if compared to trabectedin alone. aCGH and GEP revealed that key components of DNA-repair pathways were involved in trabectedin/olaparib synergism. In particular, PARP1 expression dictated the degree of the synergism. Indeed, trabectedin/olaparib synergism was increased after PARP1 overexpression and reduced after PARP1 silencing. CONCLUSIONS: PARP1 inhibition potentiated trabectedin activity in a PARP1-dependent manner and PARP1 expression in tumor cells might be a useful predictive biomarker that deserves clinical evaluation.

The integrin-linked kinase-associated phosphatase (ILKAP) is a regulatory hub of ovarian cancer cell susceptibility to platinum drugs.

BACKGROUND: Platinum drugs are the most powerful chemotherapeutic agents in the treatment of ovarian cancer. We demonstrated previously that unexpectedly ovarian cancer cells are sensitised to cisplatin (CDDP) by the hepatocyte growth factor (HGF), usually considered an anti-apoptotic factor. METHODS: We used quantitative polymerase chain reaction and Western blot analysis to evaluate gene and protein expression, immunofluorescence to evaluate protein localisation and functional assays to measure cell viability and apoptosis. RESULTS: In ovarian cancer cells, CDDP induced the phosphorylation, i.e. the activation, of the p90RSK. Surprisingly, a 48-h-long cell pre-treatment with HGF reverted this activation. HGF pre-treatment also resulted in the increased expression of the integrin-linked kinase (ILK)-associated phosphatase (ILKAP) that dephosphorylated the p90RSK. Conversely, CDDP down-modulated ILKAP expression. This impaired CDDP efficacy, as ILKAP silencing protected cells from CDDP-induced death. In line, the biochemical inhibition of the p90RSK or the combined silencing of the most expressed RSK isoforms, namely RSK1 and RSK2, increased the efficacy of CDDP. However, p90RSK inhibition was not sufficient to revert cell protection from death after ILKAP suppression, because of the simultaneous increased activity of the anti-apoptotic kinases ILK and ILK substrate AKT, which were both dephosphorylated, i.e. negatively regulated, by ILKAP. Only the combined inhibition of p90RSK and ILK reverted the effect of ILKAP suppression. CONCLUSIONS: As RSKs, ILK and AKT are vital kinases for ovarian cancer onset and progression, data suggest that ILKAP is a regulatory hub of ovarian cancer cell survival by controlling the activation of these kinases.

Peritoneal and hematogenous metastases of ovarian cancer cells are both controlled by the p90RSK through a self-reinforcing cell autonomous mechanism.

The molecular mechanisms orchestrating peritoneal and hematogenous metastases of ovarian cancer cells are assumed to be distinct. We studied the p90RSK family of serine/threonine kinases that lie downstream the RAS-ERK/MAPK pathway and modulate a variety of cellular processes including cell proliferation, survival, motility and invasiveness. We found the RSK1 and RSK2 isoforms expressed in a number of human ovarian cancer cell lines, where they played redundant roles in sustaining in vitro motility and invasiveness. In vivo, silencing of both RSK1 and RSK2 almost abrogated short-term and long-term metastatic engraftment of ovarian cancer cells in the peritoneum. In addition, RSK1/RSK2 silenced cells failed to colonize the lungs after intravenous injection and to form hematogenous metastasis from subcutaneous xenografts. RSK1/RSK2 suppression resulted in lessened ovarian cancer cell spreading on endogenous fibronectin (FN). Mechanistically, RSK1/RSK2 knockdown diminished FN transcription, α5ß1 integrin activation and TGF-ß1 translation. Reduced endogenous FN deposition and TGF-ß1 secretion depended on the lack of activating phosphorylation of the transcription/translation factor YB-1 by p90RSK. Altogether data show how p90RSK activates a self-reinforcing cell autonomous pro-adhesive circuit necessary for metastatic seeding of ovarian cancer cells. Thus, p90RSK inhibitors might hinder both the hematogenous and the peritoneal metastatic spread of human ovarian cancer.

AKT activation drives the nuclear localization of CSE1L and a pro-oncogenic transcriptional activation in ovarian cancer cells.

The human homolog of the yeast cse1 gene (CSE1L) is over-expressed in ovarian cancer. CSE1L forms complex with Ran and importin-α and has roles in nucleocytoplasmic traffic and gene expression. CSE1L accumulated in the nucleus of ovarian cancer cell lines, while it was localized also in the cytoplasm of other cancer cell lines. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells, as the CSE1L protein translocated to the cytoplasm when AKT was inactivated. Moreover, the expression of a constitutively active AKT forced the translocation of CSE1L from the cytoplasm to the nucleus in other cancer cells. Nuclear accrual of CSE1L was associated to the nuclear accumulation of the phosphorylated Ran Binding protein 3 (RanBP3), which depended on AKT as well. Also in samples of human ovarian cancer, AKT activation was associated to nuclear accumulation of CSE1L and phosphorylation of RanBP3. Expression profiling of ovarian cancer cells after CSE1L silencing showed that CSE1L was required for the expression of genes promoting invasion and metastasis. In agreement, CSE1L silencing impaired motility and invasiveness of ovarian cancer cells. Altogether these data show that in ovarian cancer cells activated AKT by affecting RanBP3 phosphorylation determines the nuclear accumulation of CSE1L and likely the nuclear concentration of transcription factors conveying pro-oncogenic signals.

Cells lacking the fumarase tumor suppressor are protected from apoptosis through a hypoxia-inducible factor-independent, AMPK-dependent mechanism.

Loss-of-function mutations of the tumor suppressor gene encoding fumarase (FH) occur in individuals with hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC). We found that loss of FH activity conferred protection from apoptosis in normal human renal cells and fibroblasts. In FH-defective cells, both hypoxia-inducible factor 1α (HIF-1α) and HIF-2α accumulated, but they were not required for apoptosis protection. Conversely, AMP-activated protein kinase (AMPK) was activated and required, as evidenced by the finding that FH inactivation failed to protect AMPK-null mouse embryo fibroblasts (MEFs) and AMPK-depleted human renal cells. Activated AMPK was detected in renal cysts, which occur in mice with kidney-targeted deletion of Fh1 and in kidney cancers of HLRCC patients. In Fh1-null MEFs, AMPK activation was sustained by fumarate accumulation and not by defective energy metabolism. Addition of fumarate and succinate to kidney cells led to extracellular signal-regulated kinase 1/2 (ERK1/2) and AMPK activation, probably through a receptor-mediated mechanism. These findings reveal a new mechanism of tumorigenesis due to FH loss and an unexpected pro-oncogenic role for AMPK that is important in considering AMPK reactivation as a therapeutic strategy against cancer.

The cellular apoptosis susceptibility CAS/CSE1L gene protects ovarian cancer cells from death by suppressing RASSF1C.

The cellular apoptosis susceptibility gene CAS/CSE1L is overexpressed in cancer, although it was originally identified as a gene that renders cells vulnerable to apoptotic stimuli. CAS/CSE1L has roles in the nucleocytoplasmic recycling of importin-α and in the regulation of gene expression, cell migration, and secretion. We identified CAS/CSE1L as a survival factor for ovarian cancer cells in vitro and in vivo. In 3/3 ovarian cancer cell lines, CAS/CSE1L was down-modulated by the unorthodox proapoptotic signaling of the MET receptor. CAS/CSE1L knockdown with RNA interference committed the ovarian cancer cells to death, but not immortalized normal cells and breast and colon cancer cells. In 70 and 95% of these latter cells, respectively, CAS/CSE1L was localized in the cytoplasm, while it accumulated in the nucleus in >90% of ovarian cancer cells. Nuclear localization depended on AKT, which was constitutively active in ovarian cancer cells. In the nucleus, CAS/CSE1L regulated the expression of the proapoptotic Ras-association domain family 1 gene products RASSF1C and RASSF1A, which mediated death signals evoked by depletion of CAS/CSE1L. Our data show that CAS/CSE1L protects ovarian cancer cells from death through transcriptional suppression of a proapoptotic gene and suggest that the localization of CAS/CSE1L dictates its function.

Fumarase tumor suppressor gene and MET oncogene cooperate in upholding transformation and tumorigenesis.

Loss of the fumarate hydratase (FH) tumor suppressor gene results in the development of benign tumors that rarely, but regrettably, progress to very aggressive cancers. Using mouse embryo fibroblasts (MEFs) to model transformation, we found that fh knockdown results in increased expression of the met oncogene-encoded tyrosine kinase receptor through hypoxia-inducible factor (hif) stabilization. MET-increased expression was alone able to stabilize hif, thus establishing a feed forward loop that might enforce tumor progression. The fh-defective MEFs showed increased motility and protection from apoptosis. Motility, but not survival, relied on hif-1alpha and was greatly enhanced by MET ligand hepatocyte growth factor. Met cooperated with a weakly oncogenic ras in making MEFs transformed and tumorigenic, as shown by in vitro and in vivo assays. Loss of fh was not equally effective by itself but enhanced the transformed and tumorigenic phenotype induced by ras and MET. Consistently, the rescue of fumarase expression abrogated the motogenic and transformed phenotype of fh-defective MEFs. In conclusion, the data suggest that the progression of tumors where FH is lost might be boosted by activation of the MET oncogene, which is able to drive cell-autonomous tumor progression and is a strong candidate for targeted therapy.