Targeting BRCA1 and BRCA2 Deficiencies with G-Quadruplex-Interacting Compounds.

G-quadruplex (G4)-forming genomic sequences, including telomeres, represent natural replication fork barriers. Stalled replication forks can be stabilized and restarted by homologous recombination (HR), which also repairs DNA double-strand breaks (DSBs) arising at collapsed forks. We have previously shown that HR facilitates telomere replication. Here, we demonstrate that the replication efficiency of guanine-rich (G-rich) telomeric repeats is decreased significantly in cells lacking HR. Treatment with the G4-stabilizing compound pyridostatin (PDS) increases telomere fragility in BRCA2-deficient cells, suggesting that G4 formation drives telomere instability. Remarkably, PDS reduces proliferation of HR-defective cells by inducing DSB accumulation, checkpoint activation, and deregulated G2/M progression and by enhancing the replication defect intrinsic to HR deficiency. PDS toxicity extends to HR-defective cells that have acquired olaparib resistance through loss of 53BP1 or REV7. Altogether, these results highlight the therapeutic potential of G4-stabilizing drugs to selectively eliminate HR-compromised cells and tumors, including those resistant to PARP inhibition.

Less correlation between mismatch repair proteins deficiency and decreased expression of HLA class I molecules in endometrial carcinoma: a different propensity from colorectal cancer.

Mismatch repair protein deficiency (dMMR) is a favorable prognostic factor in colorectal cancer. It is also associated with aberrant expression of HLA class I molecules, which are required for cytotoxic T lymphocyte-mediated cancer immunotherapy. Because dMMR is frequently also found in endometrial cancers (ECs), we retrospectively investigated the expression of mismatch repair proteins and HLA class I molecules in 127 EC patients. In this study, EC patients being treated in our hospital were recruited from 2005 to 2009 and observed until December 2017. Lesion specimens were evaluated via immunohistochemistry for MSH6 and PMS2 (mismatch repair proteins) and HLA class I molecules. Expression of these molecules was statistically related to clinical and pathological factors and prognosis. dMMR was detected in 33 patients and did not correlate with the expression level of HLA class I molecules (P = 0.60). On the other hand, unexpectedly, multivariate analysis revealed that intact expression of HLA class I molecules was associated with p53 overexpression (P = 0.004). Neither dMMR nor decreased expression of HLA class I molecules were prognostic factors. These results are inconsistent with previous findings for colorectal cancer. A distinctive local tissue immune microenvironment would underlie the discrepancy in the results between EC and colorectal cancer.

OGDHL silencing promotes hepatocellular carcinoma by reprogramming glutamine metabolism.

BACKGROUND & AIMS: Mitochondrial dysfunction and subsequent metabolic deregulation are commonly observed in cancers, including hepatocellular carcinoma (HCC). When mitochondrial function is impaired, reductive glutamine metabolism is a major cellular carbon source for de novo lipogenesis to support cancer cell growth. The underlying regulators of reductively metabolized glutamine in mitochondrial dysfunction are not completely understood in tumorigenesis. METHODS: We systematically investigated the role of oxoglutarate dehydrogenase-like (OGDHL), one of the rate-limiting components of the key mitochondrial multi-enzyme OGDH complex (OGDHC), in the regulation of lipid metabolism in hepatoma cells and mouse xenograft models. RESULTS: Lower expression of OGDHL was associated with advanced tumor stage, significantly worse survival and more frequent tumor recurrence in 3 independent cohorts totaling 681 postoperative HCC patients. Promoter hypermethylation and DNA copy deletion of OGDHL were independently correlated with reduced OGDHL expression in HCC specimens. Additionally, OGDHL overexpression significantly inhibited the growth of hepatoma cells in mouse xenografts, while knockdown of OGDHL promoted proliferation of hepatoma cells. Mechanistically, OGDHL downregulation upregulated the α-ketoglutarate (αKG):citrate ratio by reducing OGDHC activity, which subsequently drove reductive carboxylation of glutamine-derived αKG via retrograde tricarboxylic acid cycling in hepatoma cells. Notably, silencing of OGDHL activated the mTORC1 signaling pathway in an αKG-dependent manner, inducing transcription of enzymes with key roles in de novo lipogenesis. Meanwhile, metabolic reprogramming in OGDHL-negative hepatoma cells provided an abundant supply of NADPH and glutathione to support the cellular antioxidant system. The reduction of reductive glutamine metabolism through OGDHL overexpression or glutaminase inhibitors sensitized tumor cells to sorafenib, a molecular-targeted therapy for HCC. CONCLUSION: Our findings established that silencing of OGDHL contributed to HCC development and survival by regulating glutamine metabolic pathways. OGDHL is a promising prognostic biomarker and therapeutic target for HCC. LAY SUMMARY: Hepatocellular carcinoma (HCC) is one of the most prevalent tumors worldwide and is correlated with a high mortality rate. In patients with HCC, lower expression of the enzyme OGDHL is significantly associated with worse survival. Herein, we show that silencing of OGDHL induces lipogenesis and influences the chemosensitization effect of sorafenib in liver cancer cells by reprogramming glutamine metabolism. OGDHL is a promising prognostic biomarker and potential therapeutic target in OGDHL-negative liver cancer.

Malignant transformation of liver fatty acid binding protein-deficient hepatocellular adenomas: histopathologic spectrum of a rare phenomenon.

The molecular classification of hepatocellular adenomas highlights a distinctive genotype-phenotype correlation. Malignant transformation is an exceptionally rare complication of hepatocyte nuclear factor 1α (HNF1A)-inactivated hepatocellular adenomas. This subtype is characterized by loss of liver fatty acid binding protein immunoexpression. In this study, we characterized the histopathologic spectrum of 13 liver fatty acid binding protein-deficient hepatocellular adenoma cases showing malignant transformation from multiple centers. Clinicopathologic characteristics of these patients were evaluated. Stains for reticulin, liver fatty acid binding protein, beta-catenin and glutamine synthetase were applied to these lesions. Moreover, the findings were compared to patients with ß-catenin mutated hepatocellular adenoma. Liver fatty acid binding protein-deficient hepatocellular adenomas with borderline features/carcinoma were seen predominantly in females (77%) with an average age of 46 ± 18 years and multiple lesions (77%; five patients with adenomatosis). Meanwhile, ß-catenin mutated hepatocellular adenoma patients with malignant transformation were predominantly male (67%, p = 0.018) with single lesion (86%, p = 0.0009). The largest liver fatty acid binding protein-deficient hepatocellular adenoma nodule in each patient ranged from 4 to 15.5 cm. Loss of liver fatty acid binding protein by immunohistochemistry was noted in all adenoma and borderline/carcinoma components. Features of malignant transformation were pseudoglandular architecture (85%), cytologic atypia (85%), architectural atypia (100%) and lack of steatosis (100%). Other findings included myxoid change (39%), peliosis (46%) and sinusoidal dilatation (46%). Molecular studies confirmed somatic inactivation of HNF1A in 3 cases and absence of TERT promotor and exon 3 CTNNB1 mutations in five cases. To summarize, liver fatty acid binding protein-deficient hepatocellular adenoma with malignant transformation is most frequently seen in female patients with multiple lesions. Most of these lesions demonstrate pseudoglandular architecture, cytologic and architectural atypia, with lack of steatosis. The natural history of these lesions is relatively benign with the exception of disease recurrence in 1 patient.

GADD45G Interacts with E-cadherin to Suppress the Migration and Invasion of Esophageal Squamous Cell Carcinoma.

BACKGROUND/AIMS: Esophageal squamous cell carcinoma (ESCC) is one of the most prevalent cancers with poor prognosis. Metastasis is the leading cause of cancer-related deaths. The growth arrest and DNA damage-inducible 45 gamma (GADD45G) has been reported to correlate with survival, invasion, and metastasis of ESCC. This study was aimed to investigate the role and mechanism of GADD45G in ESCC cell migration and invasion. METHODS: Both the effects of GADD45G and its need for E-cadherin to function on ESCC cell migration and invasion were determined through loss- and gain-of-function approaches via Transwell assays. The interaction between GADD45G and E-cadherin was detected by GST-pull down and IP assays. The expression of E-cadherin upon GADD45G overexpression was evaluated by RT-qPCR and western blot. The level of E-cadherin in cytoplasmic, nuclear, and membrane fractions was examined by western blot following subcellular fractionation. RESULTS: Knockdown of GADD45G increased the migration and invasion abilities of KYSE150 cells, while overexpression of GADD45G showed the opposite effects on YES2 and KYSE30 cells. GADD45G could interact with E-cadherin and enhanced its membrane level. Knockdown of E-cadherin abolished the inhibitory effects of GADD45G on ESCC cell migration and invasion. Intriguingly, dimer-dissociating mutant of GADD45G could not interact with E-cadherin and almost lost its ability to suppress the ESCC cell migration and invasion. CONCLUSIONS: This study reveals a novel role for GADD45G in inhibiting the ESCC cell migration and invasion, which will provide a new insight in understanding the ESCC metastatic mechanism.

MTNR1B loss promotes chordoma recurrence by abrogating melatonin-mediated ß-catenin signaling repression.

Chordoma is an extremely rare malignant bone tumor with a high rate of relapse. While cancer stem cells (CSCs) are closely associated with tumor recurrence, which depend on its capacity to self-renew and induce chemo-/radioresistance, whether and how CSCs participate in chordoma recurrence remains unclear. The current study found that tumor cells in recurrent chordoma displayed more dedifferentiated CSC-like properties than those in corresponding primary tumor tissues. Meanwhile, MTNR1B deletion along with melatonin receptor 1B (MTNR1B) down-regulation was observed in recurrent chordoma. Further investigation revealed that activation of Gαi2 by MTNR1B upon melatonin stimulation could inhibit SRC kinase activity via recruiting CSK and SRC, increasing SRC Y530 phosphorylation, and decreasing SRC Y419 phosphorylation. This subsequently suppressed ß-catenin signaling and stemness via decreasing ß-catenin p-Y86/Y333/Y654. However, MTNR1B loss in chordoma mediated increased CSC properties, chemoresistance, and tumor progression by releasing melatonin's repression of ß-catenin signaling. Clinically, MTNR1B deletion was found to correlate with patients' survival. Together, our study establishes a novel convergence between melatonin and ß-catenin signaling pathways and reveals the significance of this cross talk in chordoma recurrence. Besides, we propose that MTNR1B is a potential biomarker for prediction of chordoma prognosis and selection of treatment options, and chordoma patients might benefit from targeting MTNR1B/Gαi2/SRC/ß-catenin axis.

CDKN2A loss and PIK3CA mutation in myoepithelial-like metaplastic breast cancer.

Metaplastic breast carcinoma comprises a heterogeneous group of tumours with poorly understood pathogenesis. A subset of metaplastic breast cancers show myoepithelial differentiation and constitute a morphological spectrum with ill-defined borders from fibromatosis-like spindle cell carcinoma to myoepithelial carcinoma. In a series of 34 metaplastic breast cancers with spindle cell and myoepithelial differentiation, we found recurrent genetic aberrations, which set them apart from other metaplastic breast cancers and suggest a unique pathogenesis. The majority of cases (28 of 34 patients; 82.4%) showed distinct chromosomal loss in the 9p21.3 region, including CDKN2A and CDKN2B. Biallelic loss of the CDKN2A/B region was found in 50% of deleted cases. Expression of the cyclin-dependent kinase inhibitor CDKN2A (p16) was missing in all samples affected by 9p21.3 loss. Other genomic alterations frequently occurring in triple-negative and metaplastic breast cancer were absent or found in only a minority of cases. Gains of whole chromosome 5 and chromosomal region 5p were observed in nine cases, and were associated with recurrences (p < 0.001). In 64.3% of cases, 9p21.3 loss was accompanied by concurrent PIK3CA mutation. Both genomic abnormalities were also detectable in adenomyoepitheliomas (4/12), which are considered to represent the precursor lesion of myoepithelial metaplastic breast cancer. In adenomyoepithelioma, PIK3CA mutation was present in both luminal epithelial and myoepithelial cells, whereas p16 loss was found only in the latter. We conclude that 9p21.3 (CDKN2A) loss and PIK3CA mutation characterize a subgroup of metaplastic breast cancers with myoepithelial and spindle cell differentiation. Myoepithelial cells in adenomyoepithelioma may show identical aberrations. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Impact of loss-of-function mutations at the RNF43 locus on colorectal cancer development and progression.

RNF43 mutations are frequently detected in colorectal cancer cells and lead to a loss of function of the ubiquitin E3 ligase. Here, we investigated the clinical significance of RNF43 mutations in a large Japanese cohort and the role of RNF43 at various stages of colorectal cancer development and progression. Mutation analysis of the RNF43 gene locus with pyrosequencing technology detected RNF43 hotspot mutations in one (0.88%) of 113 colorectal polyp cases and in 30 (6.45%) of 465 colorectal cancer cases. Moreover, patients with colorectal cancer harbouring mutated RNF43 experienced a higher recurrence rate than those harbouring non-mutated RNF43. In addition, the growth of RNF43 wild-type colorectal cancer cell lines was significantly increased by RNF43 silencing. We generated Rnf43 knockout mice in a C57BL/6 N background by using the CRISPR-Cas9 system. Although intestinal organoids from Rnf43 knockout mice did not show continuous growth in the absence of R-spondin, an azoxymethane/dextran sodium sulphate mouse model demonstrated that tumours were markedly larger in Rnf43 knockout mice than in wild-type mice. These findings provide evidence that Wnt signalling activation by RNF43 mutations during the tumourigenic stage enhances tumour growth and promotes a high recurrence rate in colorectal cancer patients. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

A rare castration-resistant progenitor cell population is highly enriched in Pten-null prostate tumours.

Castration-resistant prostate cancer is a lethal disease. The cell type(s) that survive androgen deprivation remain poorly described, despite global efforts to understand the various mechanisms of therapy resistance. We recently identified in wild-type (WT) mouse prostates a rare population of luminal progenitor cells that we called LSCmed according to their FACS profile (Lin- /Sca-1+ /CD49fmed ). Here, we investigated the prevalence and castration resistance of LSCmed in various mouse models of prostate tumourigenesis (Pb-PRL, Ptenpc-/- , and Hi-Myc mice). LSCmed prevalence is low (∼8%, similar to WT) in Hi-Myc mice, where prostatic androgen receptor signalling is unaltered, but is significantly higher in the two other models, where androgen receptor signalling is decreased, rising up to more than 80% in Ptenpc-/- prostates. LSCmed tolerate androgen deprivation and persist or are enriched 2-3 weeks after castration. The tumour-initiating properties of LSCmed from Ptenpc-/- mice were demonstrated by regeneration of tumours in vivo. Transcriptomic analysis revealed that LSCmed represent a unique cell entity as their gene expression profile is different from luminal and basal/stem cells, but shares markers of each. Their intrinsic androgen signalling is markedly decreased, explaining why LSCmed tolerate androgen deprivation. This also illuminates why Ptenpc-/- tumours are castration-resistant since LSCmed represent the most prevalent cell type in this model. We validated CK4 as a specific marker for LSCmed on sorted cells and prostate tissues by immunostaining, allowing for the detection of LSCmed in various mouse prostate specimens. In castrated Ptenpc-/- prostates, there was significant proliferation of CK4+ cells, further demonstrating their key role in castration-resistant prostate cancer progression. Taken together, this study identifies LSCmed as a probable source of prostate cancer relapse after androgen deprivation and as a new therapeutic target for the prevention of castrate-resistant prostate cancer. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Passenger deletions generate therapeutic vulnerabilities in cancer.

Inactivation of tumour-suppressor genes by homozygous deletion is a prototypic event in the cancer genome, yet such deletions often encompass neighbouring genes. We propose that homozygous deletions in such passenger genes can expose cancer-specific therapeutic vulnerabilities when the collaterally deleted gene is a member of a functionally redundant family of genes carrying out an essential function. The glycolytic gene enolase 1 (ENO1) in the 1p36 locus is deleted in glioblastoma (GBM), which is tolerated by the expression of ENO2. Here we show that short-hairpin-RNA-mediated silencing of ENO2 selectively inhibits growth, survival and the tumorigenic potential of ENO1-deleted GBM cells, and that the enolase inhibitor phosphonoacetohydroxamate is selectively toxic to ENO1-deleted GBM cells relative to ENO1-intact GBM cells or normal astrocytes. The principle of collateral vulnerability should be applicable to other passenger-deleted genes encoding functionally redundant essential activities and provide an effective treatment strategy for cancers containing such genomic events.

Clinicopathological and molecular characterization of SMARCA4-deficient thoracic sarcomas with comparison to potentially related entities.

A growing number of studies suggest critical tumor suppressor roles of the SWI/SNF chromatin remodeling complex in a variety of human cancers. The recent discovery of SMARCA4-deficient thoracic sarcomas has added to the list of tumor groups with the SMARCA4 inactivating mutation. To better characterize these tumors and establish their nosological status, we undertook a clinicopathological and molecular analysis of 12 SMARCA4-deficient thoracic sarcomas and compared them with three potentially related disease entities. Eleven men and one woman with SMARCA4-deficient thoracic sarcomas (aged 27-82 years, median 39 years) were included in the study. Most of the patients had heavy smoking exposure and pulmonary emphysema/bullae. The primary tumors were large and involved the thoracic region in all cases and simultaneously affected the abdominal cavity in 3 cases. The patients followed a rapid course, with a median survival of 7 months. Histologically, all tumors showed diffuse sheets of mildly dyscohesive, relatively monotonous, and undifferentiated epithelioid cells with prominent nucleoli. Immunohistochemically, all tumors demonstrated a complete absence (8 cases) or diffuse severe reduction (4 cases) of SMARCA4 expression. Cytokeratin, CD34, SOX2, SALL4, and p53 were expressed in 6/12, 10/12, 10/12, 10/12, and 7/10 cases, respectively. SMARCA2 expression was deficient in 11/12 cases, and none (0/8) expressed claudin-4. Targeted sequencing was performed in 5 cases and demonstrated the inactivating SMARCA4 mutation in each case and uncovered alterations in TP53 (5/5), NF1 (2/5), CDKN2A (2/5), KRAS (1/5), and KEAP1 (1/5), among others. Comparative analysis supported the distinctiveness of SMARCA4-deficient thoracic sarcomas as they were distinguishable from 13 malignant rhabdoid tumors, 15 epithelioid sarcomas, and 12 SMARCA4-deficient lung carcinomas based on clinicopathological and immunohistochemical grounds. SMARCA4-deficient thoracic sarcomas constitute a unique, highly lethal entity that requires full recognition and differentiation from other epithelioid malignancies involving the thoracic region.

Silencing of epidermal growth factor, latrophilin and seven transmembrane domain-containing protein 1 (ELTD1) via siRNA-induced cell death in glioblastoma.

The failure of therapies targeting tumor angiogenesis may be caused by anti-angiogenic resistance mechanisms induced by VEGF and non-VEGF pathways alterations. Anti-angiogenic therapy failure is also attributed to immune system, acting by tumor-associated macrophages that release pro-angiogenic factors and a consequent increase of blood vessels. Recently, in a study by Rheal et al., a new angiogenic receptor, epidermal growth factor, latrophilin, and 7 trans-membrane domain-containing protein 1 on chromosome 1(ELTD1) has been identified as a promising glioma biomarker. In this study we aim to analyse whether this receptor may be used as a target molecule in glioblastoma therapy. Our results showed that small interfering RNA silencing ELTD1 caused cytotoxicity in glioblastoma cells. We also found that PDGFR, VEGFR, and their common PI3K/mTOR intracellular pathway inactivation-induced cytotoxicity in glioblastoma cells. Further, we found high percent of cytotoxicity in a low passage glioblastoma cell line after BEZ235 (a dual inhibitor of PI3K/mTOR pathway) treatment at nanomolar concentrations, compared to AG1433 (a PDGFR inhibitor) and SU1498 (a VEGFR inhibitor) that were only cytotoxic at micromolar ranges. In the future, these could prove as attractive therapeutic targets in single therapy or coupled with classic therapeutic approaches such as chemotherapy of radiotherapy.

Decreased circadian component Bmal1 predicts tumor progression and poor prognosis in human pancreatic ductal adenocarcinoma.

The circadian clock has been demonstrated playing important roles in human tumorigenic process; however, the detailed clinical implications of circadian disruption on tumors have not been well understood. In this study, we investigated the expression pattern of Bmal1, the core component of the circadian system, in human pancreatic ductal adenocarcinoma (PDA). Our immunohistochemistry analysis showed that the protein level of Bmal1 was significantly decreased in tumor tissues from 87 patients with PDA compared with adjacent non-cancerous tissues. Low Bmal1 expression was associated with the TNM/clinical stage, histological differentiation, and vascular invasion of PDA; but no significant relevance to patient age, gender, the tumor location, or the size. Furthermore, Kaplan-Meier survival analysis revealed that PDA patients with low Bmal1 expression had shorter overall survival (OS) times as well as disease-free times (DFS) compared to the patients with high Bmal1 expression. Lastly, univariate and multivariate analyses identified low Bmal1 expression as an independent prognostic factor for poor survival outcome for patients with PDA. Collectively, our present study demonstrated that the decreased expression of Bmal1 is correlated with the tumor progression and poor prognosis in human PDA, which implicated its potential to be used as a biomarker for diagnosis and prognosis of PDA.

Host sphingosine kinase 1 worsens pancreatic cancer peritoneal carcinomatosis.

BACKGROUND: There are no effective treatments for pancreatic cancer peritoneal carcinomatosis (PC) or cancer dissemination in abdominal cavity. Sphingosine-1-phosphate (S1P), a bioactive lipid mediator produced by sphingosine kinases (SphK1 and SphK2), plays critical roles in cancer progression. We reported that SphK1, but not SphK2, is responsible for S1P export from breast cancer cells and recently discovered that S1P is linked to inflammation and cancer in colitis-associated cancer progression. Given the fact that inflammation is known to be essential for the establishment and progression of PC, we hypothesized that SphK1 in the host animals is involved in progression of pancreatic cancer PC. METHODS: Murine pancreatic adenocarcinoma panc02-luc cells were intraperitoneally injected into wildtype or SphK1 knockout (KO) mice to generate a syngeneic PC model. Cell proliferation and apoptosis were determined by Ki67 and TUNEL staining, respectively. RESULTS: All the animals developed panc02-luc PC. SphK1 KO mice developed significantly less tumor burden, less total tumor weight, and fewer number of PC nodules at 14 d after implantation. Histologically, less inflammatory cell infiltration and less cancer cell proliferation were observed in the tumors. There was no difference in apoptosis. CONCLUSIONS: Our results raise an intriguing possibility that S1P generated by SphK1 in the host promotes pancreatic cancer PC progression by stimulation of proliferation of cancer cells.

Loss of DAXX and ATRX are associated with chromosome instability and reduced survival of patients with pancreatic neuroendocrine tumors.

BACKGROUND & AIMS: Sporadic pancreatic neuroendocrine tumors (pNETs) are rare and genetically heterogeneous. Chromosome instability (CIN) has been detected in pNETs from patients with poor outcomes, but no specific genetic factors have been associated with CIN. Mutations in death domain-associated protein gene (DAXX) or ATR-X gene (ATRX) (which both encode proteins involved in chromatin remodeling) have been detected in 40% of pNETs, in association with activation of alternative lengthening of telomeres. We investigated whether loss of DAXX or ATRX, and consequent alternative lengthening of telomeres, are related to CIN in pNETs. We also assessed whether loss of DAXX or ATRX is associated with specific phenotypes of pNETs. METHODS: We collected well-differentiated primary pNET samples from 142 patients at the University Hospital Zurich and from 101 patients at the University Hospital Bern (both located in Switzerland). Clinical follow-up data were obtained for 149 patients from general practitioners and tumor registries. The tumors were reclassified into 3 groups according to the 2010 World Health Organization classification. Samples were analyzed by immunohistochemistry and telomeric fluorescence in situ hybridization. We correlated loss of DAXX, or ATRX, expression, and activation of alternative lengthening of telomeres with data from comparative genomic hybridization array studies, as well as with clinical and pathological features of the tumors and relapse and survival data. RESULTS: Loss of DAXX or ATRX protein and alternative lengthening of telomeres were associated with CIN in pNETs. Furthermore, loss of DAXX or ATRX correlated with tumor stage and metastasis, reduced time of relapse-free survival, and decreased time of tumor-associated survival. CONCLUSIONS: Loss of DAXX or ATRX is associated with CIN in pNETs and shorter survival times of patients. These results support the hypothesis that DAXX- and ATRX-negative tumors are a more aggressive subtype of pNET, and could lead to identification of strategies to target CIN in pancreatic tumors.

Reduced LIMK2 expression in colorectal cancer reflects its role in limiting stem cell proliferation.

OBJECTIVE: Colorectal cancer (CRC) is a major contributor to cancer mortality and morbidity. LIM kinase 2 (LIMK2) promotes tumour cell invasion and metastasis. The objectives of this study were to determine how LIMK2 expression is associated with CRC progression and patient outcome, and to use genetically modified Drosophila and mice to determine how LIMK2 deletion affects gastrointestinal stem cell regulation and tumour development. DESIGN: LIMK2 expression and activity were measured by immunostaining tumours from CRC-prone mice, human CRC cell lines and 650 human tumours. LIMK knockdown in Drosophila or Limk2 deletion in mice allowed for assessment of their contributions to gastrointestinal stem cell homeostasis and tumour development. RESULTS: LIMK2 expression was reduced in intestinal tumours of cancer-prone mice, as well as in human CRC cell lines and tumours. Reduced LIMK2 expression and substrate phosphorylation were associated with shorter patient survival. Genetic analysis in Drosophila midgut and intestinal epithelial cells isolated from genetically modified mice revealed a conserved role for LIMK2 in constraining gastrointestinal stem cell proliferation. Limk2 deletion increased colon tumour size in a colitis-associated colorectal mouse cancer model. CONCLUSIONS: This study revealed that LIMK2 expression and activity progressively decrease with advancing stage, and supports the hypothesis that there is selective pressure for reduced LIMK2 expression in CRC to relieve negative constraints imposed upon gastrointestinal stem cells.

Frequent mono-allelic loss associated with deficient PTEN expression in imatinib-resistant gastrointestinal stromal tumors.

Insufficiency of phosphatase and tensin homolog (PTEN) occurs in numerous tumor types and has been implicated as a resistance mechanism to receptor tyrosine kinase-targeted therapies in human cancer. In this study, we have performed a comprehensive molecular and immunohistochemical characterization of PTEN in 58 imatinib-naïve and 54 imatinib-treated gastrointestinal stromal tumors (GISTs). The findings were correlated with clinicopathological data. At the genomic level, PTEN was affected mainly by mono-allelic loss, which was significantly less frequent in imatinib-naïve vs imatinib-resistant tumors (9% vs 39%, P<0.001). Neither PTEN mutations nor PTEN promoter hyper-methylation were found. By immunohistochemistry, PTEN depletion was clearly related to GIST progression. Low PTEN protein expression was common (50%) and often paralleled with total immunonegativity in imatinib-resistant tumors. The abnormal PTEN protein expression correlated with PTEN loss at the genomic level (P=0.001). In addition, the effect of small interfering RNA (siRNA) PTEN knockdown on KIT signaling was examined in GIST-T1 and GIST430 cell lines, in the absence or presence of a dual PI3K/mTOR inhibitor NVP-BEZ235, alone or in combination with imatinib. In both cell lines, siRNA silencing of PTEN resulted in the substantial upregulation of PI3K-AKT and MAPK pathways. The MAPK hyperactivation was further potentiated by NVP-BEZ235 in the imatinib-sensitive GIST-T1 cells; yet, this effect was counteracted efficiently by combined treatment. In the imatinib-resistant GIST430 cells, neither NVP-BEZ235 alone or in combination with imatinib yielded sufficient inhibition of hyper-phosphorylated MAPK and downstream intermediate S6 protein. In conclusion, depleted PTEN expression associated with mono-allelic PTEN loss occurs frequently in imatinib-resistant GIST and might serve as a biomarker for stratifying patients for optimal treatment. In vitro, the PTEN insufficiency leads to hyperactivation of AKT and MAPK pathways in tumor cells. Novel therapies targeting multiple components of the integrated KIT receptor signaling pathways in imatinib-resistant GIST warrant further studies.

FAM190A deficiency creates a cell division defect.

Like the p16, SMAD4, and RB1 genes, FAM190A (alias CCSER1) lies at a consensus site of homogeneous genomic deletions in human cancer. FAM190A transcripts in 40% of cancers also contain in-frame deletions of evolutionarily conserved exons. Its gene function was unknown. We found an internal deletion of the FAM190A gene in a pancreatic cancer having prominent focal multinuclearity. The experimental knockdown of FAM190A expression by shRNA caused focal cytokinesis defects, multipolar mitosis, and multinuclearity as observed in time-lapse microscopy. FAM190A was localized to the γ-tubulin ring complex of early mitosis and to the midbody in late cytokinesis by immunofluorescence assay and was present in the nuclear fraction of unsynchronized cells by immunoblot. FAM190A interacted with EXOC1 and Ndel1, which function in cytoskeletal organization and the cell division cycle. Levels of FAM190A protein peaked 12 hours after release from thymidine block, corresponding to M-phase. Slower-migrating phosphorylated forms accumulated toward M-phase and disappeared after release from a mitotic block and before cytokinesis. Studies of FAM190A alterations may provide mechanistic insights into mitotic dysregulation and multinuclearity in cancer. We propose that FAM190A is a regulator or structural component required for normal mitosis and that both the rare truncating mutations and common in-frame deletion alteration of FAM190A may contribute to the chromosomal instability of cancer.

Can we accurately report PTEN status in advanced colorectal cancer?

BACKGROUND: Loss of phosphatase and tensin homologue (PTEN) function evaluated by loss of PTEN protein expression on immunohistochemistry (IHC) has been reported as both prognostic in metastatic colorectal cancer and predictive of response to anti-EGFR monoclonal antibodies although results remain uncertain. Difficulties in the methodological assessment of PTEN are likely to be a major contributor to recent conflicting results. METHODS: We assessed loss of PTEN function in 51 colorectal cancer specimens using Taqman® copy number variation (CNV) and IHC. Two blinded pathologists performed independent IHC assessment on each specimen and inter-observer variability of IHC assessment and concordance of IHC versus Taqman® CNV was assessed. RESULTS: Concordance between pathologists (PTEN loss vs no loss) on IHC assessment was 37/51 (73%). In specimens with concordant IHC assessment, concordance between IHC and Taqman® copy number in PTEN loss assessment was 25/37 (68%). CONCLUSION: Assessment PTEN loss in colorectal cancer is limited by the inter-observer variability of IHC, and discordance of CNV with loss of protein expression. An understanding of the genetic mechanisms of PTEN loss and implementation of improved and standardized methodologies of PTEN assessment are required to clarify the role of PTEN as a biomarker in colorectal cancer.

The expanding family of SMARCB1(INI1)-deficient neoplasia: implications of phenotypic, biological, and molecular heterogeneity.

Since the description of atypical teratoid/rhabdoid tumors of the central nervous system and renal/extrarenal malignant rhabdoid tumors in children, the clinicopathologic spectrum of neoplasms having in common a highly variable rhabdoid cell component (0% to 100%) and consistent loss of nuclear SMARCB1 (INI1) expression has been steadily expanding to include cribriform neuroepithelial tumor of the ventricle, renal medullary carcinoma and a subset of collecting duct carcinoma, epithelioid sarcoma, subsets of miscellaneous benign and malignant soft tissue tumors, and rare rhabdoid carcinoma variants of gastroenteropancreatic, sinonasal, and genitourinary tract origin. Although a majority of SMARCB1-deficient neoplasms arise de novo, the origin of SMARCB1-deficient neoplasia in the background of a phenotypically or genetically definable differentiated SMARCB1-intact "parent neoplasm" has been convincingly demonstrated, highlighting the rare occurrence of rhabdoid tumors as "double-hit neoplasia." As a group, SMARCB1-deficient neoplasms occur over a wide age range (0 to 80 y), may be devoid of rhabdoid cells or display uniform rhabdoid morphology, and follow a clinical course that varies from benign to highly aggressive causing death within a few months irrespective of aggressive multimodality therapy. Generally applicable criteria that would permit easy recognition of these uncommon neoplasms do not exist. Diagnosis is based on site-specific and entity-specific sets of clinicopathologic, immunophenotypic, and/or molecular criteria. SMARCB1 immunohistochemistry has emerged as a valuable tool in confirming or screening for SMARCB1-deficient neoplasms. This review summarizes the different phenotypic and topographic subgroups of SMARCB1-deficient neoplasms including sporadic and familial, benign and malignant, and rhabdoid and nonrhabdoid variants, highlighting their phenotypic heterogeneity and molecular complexity.