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

Activin Signaling in Microsatellite Stable Colon Cancers Is Disrupted by a Combination of Genetic and Epigenetic Mechanisms

Activin receptor 2 (ACVR2) is commonly mutated in microsatellite unstable (MSI) colon cancers, leading to protein loss, signaling disruption, and larger tumors. Here, we examined activin signaling disruption in microsatellite stable (MSS) colon cancers.Fifty-one population-based MSS colon cancers were assessed for ACVR1, ACVR2 and pSMAD2 protein. Consensus mutation-prone portions of ACVR2 were sequenced in primary cancers and all exons in colon cancer cell lines. Loss of heterozygosity (LOH) was evaluated for ACVR2 and ACVR1, and ACVR2 promoter methylation by methylation-specific PCR and bisulfite sequencing and chromosomal instability (CIN) phenotype via fluorescent LOH analysis of 3 duplicate markers. ACVR2 promoter methylation and ACVR2 expression were assessed in colon cancer cell lines via qPCR and IP-Western blots. Re-expression of ACVR2 after demethylation with 5-aza-2'-deoxycytidine (5-Aza) was determined. An additional 26 MSS colon cancers were assessed for ACVR2 loss and its mechanism, and ACVR2 loss in all tested cancers correlated with clinicopathological criteria.Of 51 MSS colon tumors, 7 (14%) lost ACVR2, 2 (4%) ACVR1, and 5 (10%) pSMAD2 expression. No somatic ACVR2 mutations were detected. Loss of ACVR2 expression was associated with LOH at ACVR2 (p<0.001) and ACVR2 promoter hypermethylation (p<0.05). ACVR2 LOH, but not promoter hypermethylation, correlated with CIN status. In colon cancer cell lines with fully methylated ACVR2 promoter, loss of ACVR2 mRNA and protein expression was restored with 5-Aza treatment. Loss of ACVR2 was associated with an increase in primary colon cancer volume (p<0.05).Only a small percentage of MSS colon cancers lose expression of activin signaling members. ACVR2 loss occurs through LOH and ACVR2 promoter hypermethylation, revealing distinct mechanisms for ACVR2 inactivation in both MSI and MSS subtypes of colon cancer

Loss of histone variant macroH2A2 expression associates with progression of anal neoplasm.

AimsThe macroH2A histone variants are epigenetic marks for inactivated chromatin. In this study, we examined the expression of macroH2A2 in anal neoplasm from anal intraepithelial neoplasia (AIN) to anal squamous cell carcinoma (SCC).MethodsAIN and anal SCC samples were analysed for macroH2A2 expression, HIV and human papilloma virus (HPV). The association of macroH2A2 expression with clinical grade, disease recurrence, overall survival and viral involvement was determined.ResultsmacroH2A2 was expressed in normal squamous tissue and lower grade AIN (I and II). Expression was lost in 38% of high-grade AIN (III) and 71% of anal SCC (p=0.002). Patients with AIN with macroH2A2-negative lesions showed earlier recurrence than those with macroH2A2-positive neoplasm (p=0.017). With anal SCC, macroH2A2 loss was more prevalent in the HPV-negative tumours.ConclusionsLoss of histone variant macroH2A2 expression is associated with the progression of anal neoplasm and can be used as a prognostic biomarker for high-grade AIN and SCC

While TGFβ increases p21 expression in the presence of SMAD4, activin decreases nuclear and total p21 independent of SMAD4 status.

<p>A) <i>SMAD4</i>-wild type FET and <i>SMAD4</i>-null SW480 colon cancer cells were treated with vehicle (control), activin, or TGFβ for 24 hours. p21-specific transactivation was determined using a dual luciferase assay with pWWP-luc and pRL-TK (left panel) and mRNA expression levels of p21 were quantified by qPCR and normalized to L19 (right panel). While TGFβ markedly induced both p21-specific transactivation and transcription in the presence of SMAD4, no increase in p21 transactivation and only a modest increase in transcription following activin treatment in the presence of SMAD4 were found (*p<0.05). B) <i>SMAD4</i>-wild type FET and <i>SMAD4</i>-null SW480 cells were treated with control vehicle (C), activin (A), TGFβ(T), or a combination of both ligands (A+T) for 24 hours prior to lysis for total protein, nuclear, and cytoplasmic preparation. Histone H3, α-tubulin, and GAPDH were used as loading controls for the respective fractions. While TGFβ markedly increased p21 levels in all three fractions in the SMAD4 positive cell line only, activin induced a decrease in nuclear and total p21 protein in SMAD4-positive and -negative cells (left panel). Densitometric analysis of all blots revealed statistically significant changes in p21 levels (right panel) (ns  =  non-significant, *p<0.05, **p<0.01, ***p<0.001). C) Initial upregulation of p21 protein is followed by downregulation by 24 h after activin treatment. <i>SMAD4</i>-wild type FET cells were treated with activin or vehicle (control) and harvested at various time points for quantification of p21 protein expression. GAPDH was used as loading control and relative expression was calculated via densitometry. D) While TGFβ-induced upregulation of p21 was SMAD4 dependent, activin-induced downregulation of p21 was still observed in the absence of SMAD4. <i>SMAD4</i>-wild type FET cells were treated with vehicle (CNT), activin or TGFβ in the presence of either scramble siRNA (SC) or SMAD4 siRNA (KD) and total p21 levels were determined. GAPDH was used as loading and C32 cell lysate as p21 positive control.</p

Schematic of proposed differential regulation and effects of activin and TGFβ signaling on p21 in colon cancer cells. * is indicative of total (cytoplasmatic + nuclear) p21.

<p>Schematic of proposed differential regulation and effects of activin and TGFβ signaling on p21 in colon cancer cells. * is indicative of total (cytoplasmatic + nuclear) p21.</p

p21 mediates activin-induced growth suppression and counteracts activin-induced SMAD4-independent migration in the presence of SMAD4.

<p>A) FET cells were treated with either scramble (SC) or p21 specific siRNA (KD). Growth suppression was assessed by MTT-metabolic assay following activin treatment. Activin induced cell growth inhibition in the presence of p21, but the effect was reversed in the absence of p21 (*p<0.05). B) Total viability is decreased in SMAD4 wild type colon cancers following activin treatment in the presence of p21. FET cells were treated with either scramble or p21 specific siRNA. Cell viability was assessed by trypan blue staining following activin treatment. Trypan blue positive cells after activin treatment were decreased in presence of p21, but increased after p21 knockdown (***p<0.001). C) Activin (A) induces cell migration in SMAD4-positive and SMAD4-negative cell lines. Cellular migration is induced in <i>SMAD</i>4-wild type FET cells and <i>SMAD4</i>-null SW480 cells following activin treatment, but more pronounced induction of migration is seen in the absence of SMAD4. Loss of p21 leads to an increase in baseline migration in SMAD4 expressing cells (*p<0.05, **p<0.01, ***p<0.001). D) p21 knockdown increases the overall pro-migratory effect of activin in FET cells. Loss of p21 in the absence of SMAD4 does further increase migratory induction (*p<0.05, ** p<0.01).</p

Nuclear p21 expression in primary colon cancers correlates with ACVR2 and TGFBR2 receptor expression.

<p>A) ACVR2 expression correlates with loss of nuclear p21 in colorectal cancers:</p><p>χ²(1, N = 56) = 15.204, p = 0.0001.</p><p>B) Loss of TGFBR2 expression correlates with loss of nuclear p21 in colorectal cancers:</p><p>χ²(1, N = 56) = 11.755, p = 0.0006.</p

Expression of p21 is lost in a subset of primary colon cancers correlating with the ACVR2/TGFBR2 receptor status.

<p>Fifty-six colon cancers were stained for ACVR2, TGFBR2 and p21. Representative examples for p21 staining are shown: normal colon tissue with nuclear staining (left panel), colon cancer sample with maintained nuclear p21 staining (middle panel), and colon cancer sample with loss of nuclear p21 staining (right panel).</p