Transforming Growth Factor ß Superfamily Signaling in Development of Colorectal Cancer.

Transforming growth factor (TGF)-ß cytokines signal via a complex network of pathways to regulate proliferation, differentiation, adhesion, migration, and other functions in many cell types. A high percentage of colorectal tumors contain mutations that disrupt TGF-ß family member signaling. We review how TGF-ß family member signaling is altered during development of colorectal cancer, models of study, interaction of pathways, and potential therapeutic strategies.

Hypoxia-induced gene expression results from selective mRNA partitioning to the endoplasmic reticulum.

Protein synthesis is a primary energy-consuming process in the cell. Therefore, under hypoxic conditions, rapid inhibition of global mRNA translation represents a major protective strategy to maintain energy metabolism. How some mRNAs, especially those that encode crucial survival factors, continue to be efficiently translated in hypoxia is not completely understood. By comparing specific transcript levels in ribonucleoprotein complexes, cytoplasmic polysomes and endoplasmic reticulum (ER)-bound ribosomes, we show that the synthesis of proteins encoded by hypoxia marker genes is favoured at the ER in hypoxia. Gene expression profiling revealed that transcripts particularly increased by the HIF-1 transcription factor network show hypoxia-induced enrichment at the ER. We found that mRNAs favourably translated at the ER have higher conservation scores for both the 5'- and 3'-untranslated regions (UTRs) and contain less upstream initiation codons (uAUGs), indicating the significance of these sequence elements for sustained mRNA translation under hypoxic conditions. Furthermore, we found enrichment of specific cis-elements in mRNA 5'- as well as 3'-UTRs that mediate transcript localization to the ER in hypoxia. We conclude that transcriptome partitioning between the cytoplasm and the ER permits selective mRNA translation under conditions of energy shortage.

Shutdown of achaete-scute homolog-1 expression by heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 in hypoxia.

The basic helix-loop-helix transcription factor hASH1, encoded by the ASCL1 gene, plays an important role in neurogenesis and tumor development. Recent findings indicate that local oxygen tension is a critical determinant for the progression of neuroblastomas. Here we investigated the molecular mechanisms underlying the oxygen-dependent expression of hASH1 in neuroblastoma cells. Exposure of human neuroblastoma-derived Kelly cells to 1% O2 significantly decreased ASCL1 mRNA and hASH1 protein levels. Using reporter gene assays, we show that the response of hASH1 to hypoxia is mediated mainly by post-transcriptional inhibition via the ASCL1 mRNA 5'- and 3'-UTRs, whereas additional inhibition of the ASCL1 promoter was observed under prolonged hypoxia. By RNA pulldown experiments followed by MALDI/TOF-MS analysis, we identified heterogeneous nuclear ribonucleoprotein (hnRNP)-A2/B1 and hnRNP-R as interactors binding directly to the ASCL1 mRNA 5'- and 3'-UTRs and influencing its expression. We further demonstrate that hnRNP-A2/B1 is a key positive regulator of ASCL1, findings that were also confirmed by analysis of a large compilation of gene expression data. Our data suggest that a prominent down-regulation of hnRNP-A2/B1 during hypoxia is associated with the post-transcriptional suppression of hASH1 synthesis. This novel post-transcriptional mechanism for regulating hASH1 levels will have important implications in neural cell fate development and disease.

Activin and TGFß use diverging mitogenic signaling in advanced colon cancer.

BACKGROUND: Understanding cell signaling pathways that contribute to metastatic colon cancer is critical to risk stratification in the era of personalized therapeutics. Here, we dissect the unique involvement of mitogenic pathways in a TGFß or activin-induced metastatic phenotype of colon cancer. METHOD: Mitogenic signaling/growth factor receptor status and p21 localization were correlated in primary colon cancers and intestinal tumors from either AOM/DSS treated ACVR2A (activin receptor 2) -/- or wild type mice. Colon cancer cell lines (+/- SMAD4) were interrogated for ligand-induced PI3K and MEK/ERK pathway activation and downstream protein/phospho-isoform expression/association after knockdown and pharmacologic inhibition of pathway members. EMT was assessed using epithelial/mesenchymal markers and migration assays. RESULTS: In primary colon cancers, loss of nuclear p21 correlated with upstream activation of activin/PI3K while nuclear p21 expression was associated with TGFß/MEK/ERK pathway activation. Activin, but not TGFß, led to PI3K activation via interaction of ACVR1B and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFß increased p21 via MEK/ERK pathway through a SMAD4-dependent mechanism. While activin induced EMT via PI3K, TGFß induced EMT via MEK/ERK activation. In vivo, loss of ACVR2A resulted in loss of pAkt, consistent with activin-dependent PI3K signaling. CONCLUSION: Although activin and TGFß share growth suppressive SMAD signaling in colon cancer, they diverge in their SMAD4-independent pro-migratory signaling utilizing distinct mitogenic signaling pathways that affect EMT. p21 localization in colon cancer may determine a dominant activin versus TGFß ligand signaling phenotype warranting further validation as a therapeutic biomarker prior to targeting TGFß family receptors.

Author Correction: Increased stiffness of the tumor microenvironment in colon cancer stimulates cancer associated fibroblast-mediated prometastatic activin A signaling.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Increased stiffness of the tumor microenvironment in colon cancer stimulates cancer associated fibroblast-mediated prometastatic activin A signaling.

Colorectal cancer (CRC) is the second deadliest cancer in the US due to its propensity to metastasize. Stromal cells and especially cancer-associated fibroblasts (CAF) play a critical biophysical role in cancer progression, but the precise pro-metastatic mechanisms are not clear. Activin A, a TGF-ß family member, is a strong pro-metastatic cytokine in the context of CRC. Here, we assessed the link between biophysical forces and pro-metastatic signaling by testing the hypothesis that CAF-generated mechanical forces lead to activin A release and associated downstream effects. Consistent with our hypothesis, we first determined that stromal activin A secretion increased with increasing substrate stiffness. Then we found that stromally-secreted activin A induced ligand-dependent CRC epithelial cell migration and epithelial to mesenchymal transition (EMT). In addition, serum activin A levels are significantly increased in metastatic (stage IV) CRC patients (1.558 ng/ml versus 0.4179 ng/ml, p < 0.05). We propose that increased tumor microenvironment stiffness leads to stromal cell-mediated TGF-ß family signaling relying on the induction and utilization of activin A signaling.

Multiorgan Failure Predicts Mortality in Emphysematous Pancreatitis: A Case Report and Systematic Analysis of the Literature.

Emphysematous pancreatitis (EP) is a subtype of acute necrotizing pancreatitis (ANP) characterized by the presence of gas in and around the pancreas. Although investigators have studied prognostic factors in ANP, less is known about EP. We aimed to determine predictors of mortality and identify changes in management strategies for EP. A PubMed search was performed to identify EP cases. Data were gathered about patient demographics, clinical findings, laboratory results, radiological studies, procedures, outcomes, and mortality. Data were analyzed using univariate and multivariate logistic regression analyses. Including a case from our institution, the study cohort included 64 subjects. The overall mortality rate was 32.8% (21/64). On univariate analysis, age (P = 0.019), hypotension (P = 0.007), gas outside the pancreas on computed tomography imaging (P = 0.003), initial surgical evacuation (P = 0.007), and the development of multiorgan failure (P = 0.008) were associated with mortality. On multivariate analysis, only the development of multiorgan failure was found to be an independent predictor of mortality (P = 0.039). The overall mortality rate of 32.8% for EP is similar to the mortality rates published for ANP. The development of multiorgan failure in EP is strongly associated with increased mortality. Percutaneous and endoscopic approaches have been replacing surgical interventions.

Increased Frequency of KRAS Mutations in African Americans Compared with Caucasians in Sporadic Colorectal Cancer.

OBJECTIVES: The basis for over-representation of colorectal cancer (CRC) in African-American (AA) populations compared with Caucasians are multifactorial and complex. Understanding the mechanisms for this racial disparity is critical for delivery of better care. Several studies have investigated sporadic CRC for differences in somatic mutations between AAs and Caucasians, but owing to small study sizes and conflicting results to date, no definitive conclusions have been reached. METHODS: Here, we present the first systematic literature review and meta-analysis investigating the mutational differences in sporadic CRC between AAs and Caucasians focused on frequent driver mutations (APC,TP53, KRAS,PI3CA, FBXW7,SMAD4, and BRAF). Publication inclusion criteria comprised sporadic CRC, human subjects, English language, information on ethnicity (AA, Caucasian, or both), total subject number >20, and information on mutation frequencies. RESULTS: We identified 6,234 publications. Meta-analysis for APC, TP54, FBXW7, or SMAD4 was not possible owing to paucity of data. KRAS mutations were statistically less frequent in non-Hispanic Whites when compared with AAs (odds ratio, 0.640; 95% confidence interval (CI): 0.5342-0.7666; P=0.0001), while the mutational differences observed in BRAF and PI3CA did not reach statistical significance. CONCLUSIONS: Here, we report the mutational patterns for KRAS, BRAF, and PI3CA in sporadic CRC of AAs and Caucasians in a systematic meta-analysis of previously published data. We identified an increase in KRAS mutations in sporadic CRC in AAs, which may contribute to worse prognosis and increased mortality of CRC in AAs. Future studies investigating health-care disparities in CRC in AAs should control for KRAS mutational frequency.

NFkB is essential for activin-induced colorectal cancer migration via upregulation of PI3K-MDM2 pathway.

Colorectal cancer (CRC) remains a common and deadly cancer due to metastatic disease. Activin and TGFB (TGFß) signaling are growth suppressive pathways that exert non-canonical pro-metastatic effects late in CRC carcinogenesis. We have recently shown that activin downregulates p21 via ubiquitination and degradation associated with enhanced cellular migration independent of SMADs. To investigate the mechanism of metastatic activin signaling, we examined activated NFkB signaling and activin ligand expression in CRC patient samples and found a strong correlation. We hypothesize that activation of the E3 ubiquitin ligase MDM2 by NFkB leads to p21 degradation in response to activin treatment. To dissect the link between activin and pro-carcinogenic NFkB signaling and downstream targets, we found that activin but not TGFB induced activation of NFkB leading to increased MDM2 ubiquitin ligase via PI3K. Further, overexpression of wild type p65 NFkB increased MDM2 expression while the NFkB inhibitors NEMO-binding domain (NBD) and Bay11-7082 blocked the activin-induced increase in MDM2. In conclusion, in colon cancer cell migration, activin utilizes NFkB to induce MDM2 activity leading to the degradation of p21 in a PI3K dependent mechanism. This provides new mechanistic knowledge linking activin and NFkB signaling in advanced colon cancer which is applicable to targeted therapeutic interventions.

Activin signaling is an essential component of the TGF-ß induced pro-metastatic phenotype in colorectal cancer.

Advanced colorectal cancer (CRC) remains a critical health care challenge worldwide. Various TGF-ß superfamily members are important in colorectal cancer metastasis, but their signaling effects and predictive value have only been assessed in isolation. Here, we examine cross-regulation and combined functions of the two most prominent TGF-ß superfamily members activin and TGF-ß in advanced colorectal cancer. In two clinical cohorts we observed by immune-based assay that combined serum and tissue activin and TGF-ß ligand levels predicts outcome in CRC patients and is superior to single ligand assessment. While TGF-ß growth suppression is independent of activin, TGF-ß treatment leads to increased activin secretion in colon cancer cells and TGF-ß induced cellular migration is dependent on activin, indicating pathway cross-regulation and functional interaction in vitro. mRNA expression of activin and TGF-ß pathway members were queried in silico using the TCGA data set. Coordinated ligand and receptor expression is common in solid tumors for activin and TGF-ß pathway members. In conclusion, activin and TGF-ß are strongly connected signaling pathways that are important in advanced CRC. Assessing activin and TGF-ß signaling as a unit yields important insights applicable to future diagnostic and therapeutic interventions.

Activin in acute pancreatitis: Potential risk-stratifying marker and novel therapeutic target.

Acute Pancreatitis is a substantial health care challenge with increasing incidence. Patients who develop severe disease have considerable mortality. Currently, no reliable predictive marker to identify patients at risk for severe disease exists. Treatment is limited to rehydration and supporting care suggesting an urgent need to develop novel approaches to improve standard care. Activin is a critical modulator of inflammatory responses, but has not been assessed in pancreatitis. Here, we demonstrate that serum activin is elevated and strongly correlates with disease severity in two established murine models of acute pancreatitis induced by either cerulein or IL-12 + IL-18. Furthermore, in mice, inhibition of activin conveys survival benefits in pancreatitis. In addition, serum activin levels were measured from a retrospective clinical cohort of pancreatitis patients and high activin levels in patients at admission are predictive of worse outcomes, indicated by longer overall hospital and intensive care unit stays. Taken together, activin is a novel candidate as a clinical marker to identify those acute pancreatitis patients with severe disease who would benefit from aggressive treatment and activin may be a therapeutic target in severe acute pancreatitis.

Expression of an Oncogenic BARD1 Splice Variant Impairs Homologous Recombination and Predicts Response to PARP-1 Inhibitor Therapy in Colon Cancer.

BRCA1-associated RING domain protein 1 (BARD1) stabilizes BRCA1 protein by forming a heterodimeric RING-RING complex, and impacts function of BRCA1, including homologous recombination (HR) repair. Although colon cancer cells usually express wild type BRCA1, presence of an oncogenic BARD1 splice variant (SV) in select cancers may render BRCA1 dysfunctional and allow cells to become sensitive to HR targeting therapies. We previously reported association of loss of full-length (FL) BARD1 with poor prognosis in colon cancer as well as expression of various BARD1 SVs with unknown function. Here we show that loss of BARD1 function through the expression of a BARD1 SV, BARD1ß, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1ß sensitizes colon cancer cells to poly ADP ribose polymerase 1 (PARP-1) inhibition even in a FL BRCA1 background. These results suggest that expression of BARD1ß may serve as a future biomarker to assess suitability of colon cancers for HR targeting with PARP-1 inhibitors in treatment of advanced colon cancer.