Supplementary Tables 1-2 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Supplementary Methods and Materials from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Supplementary Figures 1-3 from PHLDA1 Expression Marks the Putative Epithelial Stem Cells and Contributes to Intestinal Tumorigenesis

Small nucleolar RNA (snoRNA) were one of our earliest recognised classes of non-coding RNA, but were largely ignored by cancer investigators due to an assumption that their activities were confined to the nucleolus. However, as full genome sequences have become available, many new snoRNA genes have been identified, and multiple studies have shown their functions to be diverse. The consensus now is that many snoRNA are dysregulated in cancers, are differentially expressed between cancer types, stages and metastases, and they can actively modify disease progression. In addition, the regulation of the snoRNA class is dominated by the cancer-supporting mTOR signalling pathway, and they may have particular significance to immune cell function and anti-tumour immune responses. Given the recent advent of therapeutics that can target RNA molecules, snoRNA have robust potential as drug targets, either solely or in the context of immunotherapies.

We investigated the influence of selected TP53 SNPs in exon 4 and intron 4 on cancer risk, clinicopathological features and expression of TP53 isoforms. The intron 4 SNPs were significantly over-represented in cohorts of mixed cancers compared to three ethnically matched controls, suggesting they confer increased cancer risk. Further analysis showed that heterozygosity at rs1042522(GC) and either of the two intronic SNPs rs9895829(TC) and rs2909430(AG) confer a 2.34–5.35-fold greater risk of developing cancer. These SNP combinations were found to be associated with shorter patient survival for glioblastoma and prostate cancer. Additionally, these SNPs were associated with tumor-promoting inflammation as evidenced by high levels of infiltrating immune cells and expression of the Δ133TP53 and TP53β transcripts. We propose that these SNP combinations allow increased expression of the Δ133p53 isoforms to promote the recruitment of immune cells that create an immunosuppressive environment leading to cancer progression.

∆122p53 mice (a model of ∆133p53 isoform) are tumour-prone, have extensive inflammation and elevated serum IL-6. To investigate the role of IL-6 we crossed ∆122p53 mice with IL-6 null mice. Here we show that loss of IL-6 reduced JAK-STAT signalling, tumour incidence and metastasis. We also show that ∆122p53 activates RhoA-ROCK signalling leading to tumour cell invasion, which is IL-6-dependent and can be reduced by inhibition of JAK-STAT and RhoA-ROCK pathways. Similarly, we show that Δ133p53 activates these pathways, resulting in invasive and migratory phenotypes in colorectal cancer cells. Gene expression analysis of colorectal tumours showed enrichment of GPCR signalling associated with ∆133TP53 mRNA. Patients with elevated ∆133TP53 mRNA levels had a shorter disease-free survival. Our results suggest that ∆133p53 promotes tumour invasion by activation of the JAK-STAT and RhoA-ROCK pathways, and that patients whose tumours have high ∆133TP53 may benefit from therapies targeting these pathways.

A number of naturally occurring isoforms of the tumour suppressor protein p53 have been discovered, which appear to have differing roles in tumour prevention or promotion. We are investigating the tumour-promoting activities of the Δ133p53 isoform using our mouse model of Δ133p53 (Δ122p53). Here, we report that tumours from Δ122p53 homozygous mice show evidence of invasion and metastasis and that Δ122p53 promotes migration though a 3-dimensional collagen matrix. We also show that Δ122p53 and Δ133p53 promote cell migration in scratch wound and Transwell assays, similar to the 'gain-of-function' phenotypes seen with mutant p53. Using the well-defined B16 mouse melanoma metastatic model, we show that Δ122p53 leads to faster generation of lung metastases. The increased migratory phenotypes are dependent on secreted factors, including the cytokine interleukin-6 and the chemokine CCL2. We propose that Δ122p53 (and Δ133p53) acts in a similar manner to 'gain-of-function' mutant p53 proteins to promote migration, invasion and metastasis, which may contribute to poor survival in patients with Δ133p53-expressing tumours.

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c+ APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo. In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

There is evidence that persistent psychiatric disorders lead to age-related disease and premature mortality. Telomere length has emerged as a promising biomarker in studies that test the hypothesis that internalizing psychiatric disorders are associated with accumulating cellular damage. We tested the association between the persistence of internalizing disorders (depression, generalized anxiety disorder and post-traumatic stress disorder) and leukocyte telomere length (LTL) in the prospective longitudinal Dunedin Study ( n =1037). Analyses showed that the persistence of internalizing disorders across repeated assessments from ages 11 to 38 years predicted shorter LTL at age 38 years in a dose–response manner, specifically in men ( β =−0.137, 95% confidence interval (CI): −0.232, −0.042, P =0.005). This association was not accounted for by alternative explanatory factors, including childhood maltreatment, tobacco smoking, substance dependence, psychiatric medication use, poor physical health or low socioeconomic status. Additional analyses using DNA from blood collected at two time points (ages 26 and 38 years) showed that LTL erosion was accelerated among men who were diagnosed with internalizing disorder in the interim ( β =−0.111, 95% CI: −0.184, −0.037, P =0.003). No significant associations were found among women in any analysis, highlighting potential sex differences in internalizing-related telomere biology. These findings point to a potential mechanism linking internalizing disorders to accelerated biological aging in the first half of the life course, particularly in men. Because internalizing disorders are treatable, the findings suggest the hypothesis that treating psychiatric disorders in the first half of the life course may reduce the population burden of age-related disease and extend health expectancy.

Tumor invasion and metastasis involves complex remodeling of gene expression programs governing epithelial homeostasis. Mutational activation of the RAS-ERK is a frequent occurrence in many cancers and has been shown to drive overexpression of the AP-1 family transcription factor FRA1, a potent regulator of migration and invasion in a variety of tumor cell types. However, the nature of FRA1 transcriptional targets and the molecular pathways through which they promote tumor progression remain poorly understood. We found that FRA1 was strongly expressed in tumor cells at the invasive front of human colorectal cancers (CRCs), and that its depletion suppressed mesenchymal-like features in CRC cells in vitro. Genome-wide analysis of FRA1 chromatin occupancy and transcriptional regulation identified epithelial-mesenchymal transition (EMT)-related genes as a major class of direct FRA1 targets in CRC cells. Expression of the pro-mesenchymal subset of these genes predicted adverse outcomes in CRC patients, and involved FRA-1-dependent regulation and cooperation with TGFβ signaling pathway. Our findings reveal an unexpectedly widespread and direct role for FRA1 in control of epithelial-mesenchymal plasticity in CRC cells, and suggest that FRA1 plays an important role in mediating cross talk between oncogenic RAS-ERK and TGFβ signaling networks during tumor progression.

Activation of the canonical TGF-b signaling pathwayprovides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but towhat extentmutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP)microarray analysis.Mutation spectrawere compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4mutations were associated with mucinous histology. Themutation spectra of SMAD2 and SMAD3were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-XSer motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2–SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented andmutually exclusive to SMAD4mutation, underlining the critical roles of these three proteins within the TGF-b signaling pathway. Cancer Res; 73(2); 725–35. 2012 AACR.

Aim The TGF-b signalling pathway is principally composed of the TGF-b receptors (including TGFBR2) and the SMAD family signalling factors, and is implicated in the control of epithelial mesenchymal transition (EMT). In colorectal cancer, SMAD4 and TGFBR2 are mutated in approximately 15% and 10%, respectively. We hypothesised that cancers harbouring these gene mutations would differ in their biology and perhaps thus clinical progression. Methods Ten cancers with biallelic mutation of TGFBR2, ten with biallelic mutation of SMAD4 and ten wildtype for the two genes were assessed for tumour budding and E-cadherin expression. Tumour sections were stained with H&E, AE1/3 and immunohistochemistry for E-cadherin. Staining intensity and percentage of cell positivity were scored and compared between the three groups. Results Tumour budding was lower in TGFBR2 mutant cancers than the SMAD4 and WT groups when stained by H&E (p<0.05). The percentage of cells staining positive for E-cadherin was significantly higher in the SMAD4 mutant cancers than the TGFBR2 mutant set (p < 0.028), but the difference in intensity was not significant. Conclusions These data suggest that the mutation status of the TGF-b pathway may correlate with colorectal tumour biology and is potentially relevant to clinical progression and metastatic spread. These results warrant further investigation. The TGF-b signalling pathway is principally composed of the TGF-b receptors (including TGFBR2) and the SMAD family signalling factors, and is implicated in the control of epithelial mesenchymal transition (EMT). In colorectal cancer, SMAD4 and TGFBR2 are mutated in approximately 15% and 10%, respectively. We hypothesised that cancers harbouring these gene mutations would differ in their biology and perhaps thus clinical progression. Ten cancers with biallelic mutation of TGFBR2, ten with biallelic mutation of SMAD4 and ten wildtype for the two genes were assessed for tumour budding and E-cadherin expression. Tumour sections were stained with H&E, AE1/3 and immunohistochemistry for E-cadherin. Staining intensity and percentage of cell positivity were scored and compared between the three groups. Tumour budding was lower in TGFBR2 mutant cancers than the SMAD4 and WT groups when stained by H&E (p<0.05). The percentage of cells staining positive for E-cadherin was significantly higher in the SMAD4 mutant cancers than the TGFBR2 mutant set (p < 0.028), but the difference in intensity was not significant. These data suggest that the mutation status of the TGF-b pathway may correlate with colorectal tumour biology and is potentially relevant to clinical progression and metastatic spread. These results warrant further investigation.

Activation of the canonical TGF-β signaling pathway provides growth inhibitory signals in the normal intestinal epithelium. Colorectal cancers (CRCs) frequently harbor somatic mutations in the pathway members TGFBR2 and SMAD4, but to what extent mutations in SMAD2 or SMAD3 contribute to tumorigenesis is unclear. A cohort of 744 primary CRCs and 36 CRC cell lines were sequenced for SMAD4, SMAD2, and SMAD3 and analyzed for allelic loss by single-nucleotide polymorphism (SNP) microarray analysis. Mutation spectra were compared between the genes, the pathogenicity of mutations was assessed, and relationships with clinicopathologic features were examined. The prevalence of SMAD4, SMAD2, and SMAD3 mutations in sporadic CRCs was 8.6% (64 of 744), 3.4% (25 of 744), and 4.3% (32 of 744), respectively. A significant overrepresentation of two genetic hits was detected for SMAD4 and SMAD3, consistent with these genes acting as tumor suppressors. SMAD4 mutations were associated with mucinous histology. The mutation spectra of SMAD2 and SMAD3 were highly similar to that of SMAD4, both in mutation type and location within the encoded proteins. In silico analyses suggested the majority of the mutations were pathogenic, with most missense changes predicted to reduce protein stability or hinder SMAD complex formation. The latter altered interface residues or disrupted the phosphorylation-regulated Ser-Ser-X-Ser motifs within SMAD2 and SMAD3. Functional analyses of selected mutations showed reductions in SMAD3 transcriptional activity and SMAD2-SMAD4 complex formation. Joint biallelic hits in SMAD2 and SMAD3 were overrepresented and mutually exclusive to SMAD4 mutation, underlining the critical roles of these three proteins within the TGF-β signaling pathway.

Abstract Studies employing mouse models have identified crypt base and position +4 cells as strong candidates for intestinal epithelial stem cells. Equivalent cell populations are thought to exist in the human intestine; however robust and specific protein markers are lacking. Here, we show that in the human small and large intestine, PHLDA1 is expressed in discrete crypt base and some position +4 cells. In small adenomas, PHLDA1 was expressed in a subset of undifferentiated and predominantly Ki-67–negative neoplastic cells, suggesting that a basic hierarchy of differentiation is retained in early tumorigenesis. In large adenomas, carcinomas, and metastases PHLDA1 expression became widespread, with increased expression and nuclear localization at invasive margins. siRNA-mediated suppression of PHLDA1 in colon cancer cells inhibited migration and anchorage-independent growth in vitro and tumor growth in vivo. The integrins ITGA2 and ITGA6 were downregulated in response to PHLDA1 suppression, and accordingly cell adhesion to laminin and collagen was significantly reduced. We conclude that PHLDA1 is a putative epithelial stem cell marker in the human small and large intestine and contributes to migration and proliferation in colon cancer cells. Cancer Res; 71(10); 3709–19. ©2011 AACR.