Jessica Bauer

TGF-β: Duality of Function Between Tumor Prevention and Carcinogenesis

Several mechanisms underlying tumor progression have remained elusive, particularly in relation to transforming growth factor beta (TGF-β). Although TGF-β initially inhibits epithelial growth, it appears to promote the progression of advanced tumors. Defects in normal TGF-β pathways partially explain this paradox, which can lead to a cascade of downstream events that drive multiple oncogenic pathways, manifesting as several key features of tumorigenesis (uncontrolled proliferation, loss of apoptosis, epithelial-to-mesenchymal transition, sustained angiogenesis, evasion of immune surveillance, and metastasis). Understanding the mechanisms of TGF-β dysregulation will likely reveal novel points of convergence between TGF-β and other pathways that can be specifically targeted for therapy.

Lipid Alterations in Experimental Murine Colitis: Role of Ceramide and Imipramine for Matrix Metalloproteinase-1 Expression

Background Dietary lipids or pharmacologic modulation of lipid metabolism are potential therapeutic strategies in inflammatory bowel disease (IBD). Therefore, we analysed alterations of bioactive lipids in experimental models of colitis and examined the functional consequence of the second messenger ceramide in inflammatory pathways leading to tissue destruction. Methodology/Principal Findings Chronic colitis was induced by dextran-sulphate-sodium (DSS) or transfer of CD4+CD62L+ cells into RAG1−/−-mice. Lipid content of isolated murine intestinal epithelial cells (IEC) was analysed by tandem mass spectrometry. Concentrations of MMP-1 in supernatants of Caco-2-IEC and human intestinal fibroblasts from patients with ulcerative colitis were determined by ELISA. Imipramine was used for pharmacologic inhibition of acid sphingomyelinase (ASM). Ceramide increased by 71% in chronic DSS–induced colitis and by 159% in the transfer model of colitis. Lysophosphatidylcholine (LPC) decreased by 22% in both models. No changes were detected for phosphatidylcholine. Generation of ceramide by exogenous SMase increased MMP-1-protein production of Caco-2-IEC up to 7-fold. Inhibition of ASM completely abolished the induction of MMP-1 by TNF or IL-1β in Caco-2-IEC and human intestinal fibroblasts. Conclusions/Significance Mucosal inflammation leads to accumulation of ceramide and decrease of LPC in the intestinal epithelium. One aspect of ceramide generation is an increase of MMP-1. Induction of MMP-1 by TNF or IL-1β is completely blocked by inhibition of ASM with imipramine. Therefore, inhibition of ASM may offer a treatment strategy to reduce MMP-1 expression and tissue destruction in inflammatory conditions.

Effects of Activin and TGFβ on p21 in Colon Cancer

Activin and TGFβ share SMAD signaling and colon cancers can inactivate either pathway alone or simultaneously. The differential effects of activin and TGFβ signaling in colon cancer have not been previously dissected. A key downstream target of TGFβ signaling is the cdk2 inhibitor p21 (p21cip1/waf1). Here, we evaluate activin-specific effects on p21 regulation and resulting functions. We find that TGFβ is a more potent inducer of growth suppression, while activin is a more potent inducer of apoptosis. Further, growth suppression and apoptosis by both ligands are dependent on SMAD4. However, activin downregulates p21 protein in a SMAD4-independent fashion in conjunction with increased ubiquitination and proteasomal degradation to enhance migration, while TGFβ upregulates p21 in a SMAD4-dependent fashion to affect growth arrest. Activin-induced growth suppression and cell death are dependent on p21, while activin-induced migration is counteracted by p21. Further, primary colon cancers show differential p21 expression consistent with their ACVR2/TGFBR2 receptor status. In summary, we report p21 as a differentially affected activin/TGFβ target and mediator of ligand-specific functions in colon cancer, which may be exploited for future risk stratification and therapeutic intervention.

Loss of TGFβ signaling promotes colon cancer progression and tumor-associated inflammation.

TGFβ has both tumor suppressive and tumor promoting effects in colon cancer. Also, TGFβ can affect the extent and composition of inflammatory cells present in tumors, contextually promoting and inhibiting inflammation. While colon tumors display intratumoral inflammation, the contributions of TGFβ to this process are poorly understood. In human patients, we found that epithelial loss of TGFβ signaling was associated with increased inflammatory burden; yet overexpression of TGFβ was also associated with increased inflammation. These findings were recapitulated in mutant APC models of murine tumorigenesis, where epithelial truncation of TGFBR2 led to lethal inflammatory disease and invasive colon cancer, mediated by IL8 and TGFβ1. Interestingly, mutant APC mice with global suppression of TGFβ signals displayed an intermediate phenotype, presenting with an overall increase in IL8-mediated inflammation and accelerated tumor formation, yet with a longer latency to the onset of disease observed in mice with epithelial TGFBR-deficiency. These results suggest that the loss of TGFβ signaling, particularly in colon epithelial cells, elicits a strong inflammatory response and promotes tumor progression. This implies that treating colon cancer patients with TGFβ inhibitors may result in a worse outcome by enhancing inflammatory responses.

Matrix metalloproteinase-1 expression induced by IL-1β requires acid sphingomyelinase

Matrix metalloproteinase‐1 (MMP‐1) is increased in inflammatory conditions leading to destruction of extracellular matrix. Many inflammatory stimuli activate sphingomyelinases (SMases), which generate ceramide. We aimed to define the relevance and type of SMase responsible for the regulation of MMP‐1. Acid sphingomyelinase (ASM)‐deficient human fibroblasts failed to phosphorylate extracellular signal‐regulated kinase (ERK), or upregulate MMP‐1 mRNA and protein expression upon stimulation with interleukin‐1 beta (IL‐1β), whereas phosphorylation of p38 mitogen‐activated protein kinase and IL‐8 production remained unaffected. Transfection of ASM restored MMP‐1 production. Addition of exogenous SMase was sufficient to restore activation of ERK and increase MMP‐1 mRNA. Inhibition of ASM with imipramine completely abrogated MMP‐1 induction. The results suggest that IL‐1β‐induced expression of MMP‐1 is dependent on ASM.

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

BackgroundUnderstanding 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 TGFβ or activin-induced metastatic phenotype of colon cancer.MethodMitogenic 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.ResultsIn primary colon cancers, loss of nuclear p21 correlated with upstream activation of activin/PI3K while nuclear p21 expression was associated with TGFβ/MEK/ERK pathway activation. Activin, but not TGFβ, led to PI3K activation via interaction of ACVR1B and p85 independent of SMAD4, resulting in p21 downregulation. In contrast, TGFβ increased p21 via MEK/ERK pathway through a SMAD4-dependent mechanism. While activin induced EMT via PI3K, TGFβ induced EMT via MEK/ERK activation. In vivo, loss of ACVR2A resulted in loss of pAkt, consistent with activin-dependent PI3K signaling.ConclusionAlthough activin and TGFβ 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 TGFβ ligand signaling phenotype warranting further validation as a therapeutic biomarker prior to targeting TGFβ family receptors.

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, BARD1β, results in a more malignant phenotype with decreased RAD51 foci formation, reduced BRCA1 E3 ubiquitin ligase activity, and decreased nuclear BRCA1 protein localization. BARD1β 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 BARD1β 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.

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

Advanced colorectal cancer (CRC) remains a critical health care challenge worldwide. Various TGF-β 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-β superfamily members activin and TGF-β in advanced colorectal cancer. In two clinical cohorts we observed by immune-based assay that combined serum and tissue activin and TGF-β ligand levels predicts outcome in CRC patients and is superior to single ligand assessment. While TGF-β growth suppression is independent of activin, TGF-β treatment leads to increased activin secretion in colon cancer cells and TGF-β induced cellular migration is dependent on activin, indicating pathway cross-regulation and functional interaction in vitro. mRNA expression of activin and TGF-β 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-β pathway members. In conclusion, activin and TGF-β are strongly connected signaling pathways that are important in advanced CRC. Assessing activin and TGF-β signaling as a unit yields important insights applicable to future diagnostic and therapeutic interventions.

Anti-p21 autoantibodies detected in colorectal cancer patients: A proof of concept study

Whereas the presence of autoantibodies in cancer patients has been acknowledged, their diagnostic or therapeutic significance has yet to be established. This is due, at least in part, to the lack of robust screening techniques to detect and characterize such antibodies for further assessment. In this study, we screened colorectal cancer (CRC) patient sera for antibodies specifically targeting the key cell cycle inhibitory factor p21 encoded by the cyclin-dependent kinase inhibitor 1A (CDKN1A). Anti-p21 antibody titers were higher in CRC patient samples versus controls, correlating with a more advanced disease stage and lymph node involvement. Further, we isolated for the first time a specific human antibody fragment against p21, which could potentially be useful as a tool to study tumorigenicity in CRC patients.

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 (TGFβ) 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.