Heterogeneity and plasticity of epidermal stem cells.

The epidermis is an integral part of our largest organ, the skin, and protects us against the hostile environment. It is a highly dynamic tissue that, during normal steady-state conditions, undergoes constant turnover. Multiple stem cell populations residing in autonomously maintained compartments facilitate this task. In this Review, we discuss stem cell behaviour during normal tissue homeostasis, regeneration and disease within the pilosebaceous unit, an integral structure of the epidermis that is responsible for hair growth and lubrication of the epithelium. We provide an up-to-date view of the pilosebaceous unit, encompassing the heterogeneity and plasticity of multiple discrete stem cell populations that are strongly influenced by external cues to maintain their identity and function.

High expression of GEM and EDNRA is associated with metastasis and poor outcome in patients with advanced bladder cancer.

BACKGROUND: The standard treatment for non-metastatic muscle-invasive bladder cancer (stages T2-T4a) is radical cystectomy with lymphadenectomy. However, patients undergoing cystectomy show metastatic spread in 25% of cases and these patients will have limited benefit from surgery. Identification of patients with high risk of lymph node metastasis will help select patients that may benefit from neoadjuvant and/or adjuvant chemotherapy. METHODS: RNA was procured by laser micro dissection of primary bladder tumors and corresponding lymph node metastases for Affymetrix U133 Plus 2.0 Gene Chip expression profiling. A publically available dataset was used for identification of the best candidate markers, and these were validated using immunohistochemistry in an independent patient cohort of 368 patients. RESULTS: Gene Set Enrichment Analysis showed significant enrichment for e.g. metastatic signatures in the metastasizing tumors, and a set of 12 genes significantly associated with lymph node metastasis was identified. Tumors did not cluster according to their metastatic ability when analyzing gene expression profiles using hierarchical cluster analysis. However, half (6/12) of the primary tumor clustered together with matching lymph node metastases, indicating a large degree of intra-patient similarity in these patients. Immunohistochemical analysis of 368 tumors from cystectomized patients showed high expression of GEM (P = 0.033; HR = 1.46) and EDNRA (P = 0.046; HR = 1.60) was significantly associated with decreased cancer-specific survival. CONCLUSIONS: GEM and EDNRA were identified as promising prognostic markers for patients with advanced bladder cancer. The clinical relevance of GEM and EDNRA should be evaluated in independent prospective studies.

MiRNA-362-3p induces cell cycle arrest through targeting of E2F1, USF2 and PTPN1 and is associated with recurrence of colorectal cancer.

Colorectal cancer (CRC) is one of the leading causes of cancer deaths in Western countries. A significant number of CRC patients undergoing curatively intended surgery subsequently develop recurrence and die from the disease. MicroRNAs (miRNAs) are aberrantly expressed in cancers and appear to have both diagnostic and prognostic significance. In this study, we identified novel miRNAs associated with recurrence of CRC, and their possible mechanism of action. TaqMan(®) Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosas and 46 microsatellite stable CRC tumors. Four miRNAs (miR-362-3p, miR-570, miR-148 a* and miR-944) were expressed at a higher level in tumors from patients with no recurrence (p<0.015), compared with tumors from patients with recurrence. A significant association with increased disease free survival was confirmed for miR-362-3p in a second independent cohort of 43 CRC patients, using single TaqMan(®) microRNA assays. In vitro functional analysis showed that over-expression of miR-362-3p in colon cancer cell lines reduced cell viability, and proliferation mainly due to cell cycle arrest. E2F1, USF2 and PTPN1 were identified as potential miR-362-3p targets by mRNA profiling of HCT116 cells over-expressing miR-362-3p. Subsequently, these genes were confirmed as direct targets by Luciferase reporter assays and their knockdown in vitro phenocopied the effects of miR-362-3p over-expression. We conclude that miR-362-3p may be a novel prognostic marker in CRC, and hypothesize that the positive effects of augmented miR-362-3p expression may in part be mediated through the targets E2F1, USF2 and PTPN1.

Alternative splicing of SLC39A14 in colorectal cancer is regulated by the Wnt pathway.

Alternative splicing is a crucial step in the generation of protein diversity and its misregulation is observed in many human cancer types. By analyzing 143 colorectal samples using exon arrays, SLC39A14, a divalent cation transporter, was identified as being aberrantly spliced in tumor samples. SLC39A14 contains two mutually exclusive exons 4A and 4B and the exon 4A/4B ratio was significantly altered in adenomas (p = 3.6 × 10(-10)) and cancers (p = 9.4 × 10(-11)), independent of microsatellite stability status. The findings were validated in independent exon array data sets and by quantitative real-time reverse-transcription PCR (qRT-PCR). Aberrant Wnt signaling is a hallmark of colorectal tumorigenesis and is characterized by nuclear ß-catenin. Experimental inactivation of Wnt signaling in DLD1 and Ls174T cells by knockdown of ß-catenin or overexpression of dominant negative TCFs (TCF1 and TCF4) altered the 4A/4B ratio, indicating that SLC39A14 splicing is regulated by the Wnt pathway. An altered 4A/4B ratio was also observed in gastric and lung cancer where Wnt signaling is also known to be aberrantly activated. The splicing factor SRSF1 and its regulator, the kinase SRPK1, were found to be deregulated upon Wnt inactivation in colorectal carcinoma cells. SRPK1 was also found up-regulated in both adenoma samples (p = 1.5 × 10(-5)) and cancer samples (p = 5 × 10(-4)). In silico splicing factor binding analysis predicted SRSF1 to bind predominantly to the cancer associated exon 4B, hence, it was hypothesized that SRPK1 activates SRSF1 through phosphorylation, followed by SRSF1 binding to exon 4B and regulation of SLC39A14 splicing. Indeed, siRNA-mediated knockdown of SRPK1 and SRSF1 in DLD1 and SW480 colorectal cancer cells led to a change in the 4A/4B isoform ratio, supporting a role of these factors in the regulation of SLC39A14 splicing. In conclusion, alternative splicing of SLC39A14 was identified in colorectal tumors and found to be regulated by the Wnt pathway, most likely through regulation of SRPK1 and SRSF1.

Expression of TIP60 (tat-interactive protein) and MRE11 (meiotic recombination 11 homolog) predict treatment-specific outcome of localised invasive bladder cancer.

UNLABELLED: What's known on the subject? and What does the study add? Several studies have shown that defects in DNA-damage response are associated with good survival after chemotherapy and radiotherapy. Furthermore, loss of cell cycle regulators may be prognostic indicators of poor survival after cystectomy. However, the potential clinical impact of previous findings is hampered by insufficient validation of significant results in suitable cystectomy and radiotherapy cohorts. Here we use a large cohort of patients receiving radiotherapy to successfully validate the importance of MRE11 as a predictive marker of disease-specific survival (DSS). Furthermore, using two independent patient cohorts we show for the first time that TIP60 is a predictive marker of DSS after cystectomy. We show that combined use of TIP60 and MRE11 may hold the potential to guide treatment decisions. OBJECTIVE: • To determine the association between the proteins: tat-interactive protein 60 kDa (TIP60), p16, meiotic recombination 11 homolog (MRE11), phosphorylated ataxia telangiectasia mutated (ATM), retinoblastoma protein (Rb), Ki67, and p53 and clinical outcome in invasive lymph node-negative bladder cancer. PATIENTS AND METHODS: • Protein expression was measured by immunohistochemistry in cancer specimens from two independent cohorts of patients with bladder cancer treated with cystectomy (162 patients and 273) and one cohort of patients receiving radiotherapy (148). • Disease-specific survival (DSS) was used as the outcome measure, and patients with no disease-specific death were followed for a minimum of 36 months. RESULTS: • TIP60 was significantly correlated with DSS in both cystectomy cohorts (hazard ratio [HR] 0.42, 95% confidence interval [CI] 0.26-0.68, P < 0.001 and HR 0.45, 95% CI 0.28-0.72, P = 0.001). • MRE11 was significantly correlated with DSS in the cohort receiving radiotherapy (HR 0.64, 95% CI 0.47-0.86, P = 0.005). • P16 was significantly correlated with DSS in all three cohorts (HR 0.46, 95% CI 0.30-0.75, P = 0.032; HR 0.60, 95% CI 0.37-0.97, P = 0.032; HR 0.52, 95% CI 0.28-0.96, P = 0.001). • Rb was significantly correlated with DSS in one cystectomy cohort (HR 1.71, 95% CI 1.13-2.75, P = 0.017). • Ki67, p53, and pATM were not significantly correlated with DSS in any of the cohorts. CONCLUSIONS: • TIP60 protein expression was a predictive marker for DSS after cystectomy in two independent cohorts. This novel marker was the strongest predictive factor in multivariate analysis in patients receiving cystectomy. • MRE11 was shown to be a predictive marker for DSS after radiotherapy. • We have shown that TIP60 and MRE11 hold the potential to guide patients with invasive bladder cancer to either cystectomy or radiotherapy. This study was based on retrospective material and consequently we suggest that these markers should be validated in a prospective study.

Tumor-specific usage of alternative transcription start sites in colorectal cancer identified by genome-wide exon array analysis.

BACKGROUND: Approximately half of all human genes use alternative transcription start sites (TSSs) to control mRNA levels and broaden the transcriptional output in healthy tissues. Aberrant expression patterns promoting carcinogenesis, however, may arise from alternative promoter usage. RESULTS: By profiling 108 colorectal samples using exon arrays, we identified nine genes (TCF12, OSBPL1A, TRAK1, ANK3, CHEK1, UGP2, LMO7, ACSL5, and SCIN) showing tumor-specific alternative TSS usage in both adenoma and cancer samples relative to normal mucosa. Analysis of independent exon array data sets corroborated these findings. Additionally, we confirmed the observed patterns for selected mRNAs using quantitative real-time reverse-transcription PCR. Interestingly, for some of the genes, the tumor-specific TSS usage was not restricted to colorectal cancer. A comprehensive survey of the nine genes in lung, bladder, liver, prostate, gastric, and brain cancer revealed significantly altered mRNA isoform ratios for CHEK1, OSBPL1A, and TCF12 in a subset of these cancer types.To identify the mechanism responsible for the shift in alternative TSS usage, we antagonized the Wnt-signaling pathway in DLD1 and Ls174T colorectal cancer cell lines, which remarkably led to a shift in the preferred TSS for both OSBPL1A and TRAK1. This indicated a regulatory role of the Wnt pathway in selecting TSS, possibly also involving TP53 and SOX9, as their transcription binding sites were enriched in the promoters of the tumor preferred isoforms together with their mRNA levels being increased in tumor samples. Finally, to evaluate the prognostic impact of the altered TSS usage, immunohistochemistry was used to show deregulation of the total protein levels of both TCF12 and OSBPL1A, corresponding to the mRNA levels observed. Furthermore, the level of nuclear TCF12 had a significant correlation to progression free survival in a cohort of 248 stage II colorectal cancer samples. CONCLUSIONS: Alternative TSS usage in colorectal adenoma and cancer samples has been shown for nine genes, and OSBPL1A and TRAK1 were found to be regulated in vitro by Wnt signaling. TCF12 protein expression was upregulated in cancer samples and correlated with progression free survival.

Lrig1 marks a population of gastric epithelial cells capable of long-term tissue maintenance and growth in vitro.

The processes involved in renewal of the epithelium that lines the mouse stomach remain unclear. Apart from the cells in the isthmus, several other populations located deeper in the gastric glands have been suggested to contribute to the maintenance of the gastric epithelium. Here, we reveal that Lrig1 is expressed in the basal layer of the forestomach and the lower part of glands in the corpus and pylorus. In the glandular epithelium of the stomach, Lrig1 marks a heterogeneous population comprising mainly non-proliferative cells. Yet, fate-mapping experiments using a knock-in mouse line expressing Cre specifically in Lrig1+ cells demonstrate that these cells are able to contribute to the long-term maintenance of the gastric epithelium. Moreover, when cultured in vitro, cells expressing high level of Lrig1 have much higher organoid forming potential than the corresponding cellular populations expressing lower levels of Lrig1. Taken together, these observations show that Lrig1 is expressed primarily by differentiated cells, but that these cells can be recruited to contribute to the maintenance of the gastric epithelium. This confirms previous observations that cells located in the lower segments of gastric glands can participate in tissue replenishment.

Functional screening identifies miRNAs influencing apoptosis and proliferation in colorectal cancer.

MicroRNAs (miRNAs) play a critical role in many biological processes and are aberrantly expressed in human cancers. Particular miRNAs function either as tumor suppressors or oncogenes and appear to have diagnostic and prognostic significance. Although numerous miRNAs are dys-regulated in colorectal cancer (CRC) only a small fraction has been characterized functionally. Using high-throughput functional screening and miRNA profiling of clinical samples the present study aims at identifying miRNAs important for the control of cellular growth and/or apoptosis in CRC. The high-throughput functional screening was carried out in six CRC cell lines transfected with a pre-miR library including 319 synthetic human pre-miRs. Phenotypic alterations were evaluated by immunostaining of cleaved cPARP (apoptosis) or MKI67 (proliferation). Additionally, TaqMan Human MicroRNA Array Set v2.0 was used to profile the expression of 667 miRNAs in 14 normal colon mucosa and 46 microsatellite stable stage II CRC patients. Among the miRNAs that induced growth arrest and apoptosis in the CRC cell lines, and at same time were dys-regulated in the clinical samples, miR-375 was selected for further analysis. Independent in vitro analysis of transient and stable transfected CRC cell lines confirmed that miR-375 reduces cell viability through the induction of apoptotic death. We identified YAP1 as a direct miR-375 target in CRC and show that HELLS and NOLC1 are down-stream targets. Knock-down of YAP1 mimicked the phenotype induced by miR-375 over-expression indicating that miR-375 most likely exerts its pro-apoptotic role through YAP1 and its anti-apoptotic down-stream targets BIRC5 and BCL2L1. Finally, in vivo analysis of mouse xenograft tumors showed that miR-375 expression significantly reduced tumor growth. We conclude that the high-throughput screening successfully identified miRNAs that induce apoptosis and/or inhibit proliferation in CRC cells. Finally, combining the functional screening with profiling of CRC tissue samples we identified clinically relevant miRNAs and miRNA targets in CRC.

Emerging roles of microRNAs in the Wnt signaling network.

The Wnt signaling network is known to regulate many cellular processes and is of crucial importance during development and in pathological conditions, including cancer. Small noncoding RNAs from the microRNA family (miRNAs) are important elements in the post-transcriptional control of gene expression. In this work, I review the cross talk between miRNAs and the canonical Wnt signaling pathway in various biological processes with particular emphasis on carcinogenesis. Because alterations of miRNA activity and aberrant Wnt signaling are each intimately linked to tumor biology, deciphering the complex interplay between these two regulatory modules is essential to advance our understanding of the integrated functions of miRNAs in signal transduction cascades and develop rational treatment regimens against cancer.

p53-Responsive micrornas 192 and 215 are capable of inducing cell cycle arrest.

microRNAs provide a novel layer of regulation for gene expression by interfering with the stability and/or translation of specific target mRNAs. Overall levels of microRNAs are frequently down-regulated in cancer cells, and reducing general microRNA processing increases cancerogenesis in transgenic models, suggesting that at least some microRNAs might act as effectors in tumor suppression. Accordingly, the tumor suppressor p53 up-regulates miR-34a, a microRNA that contributes to apoptosis and acute senescence. Here, we used array hybridization to find that p53 induces two additional, mutually related clusters of microRNAs, leading to the up-regulation of miR-192, miR-194, and miR-215. The same microRNAs were detected at high levels in normal colon tissue but were severely reduced in many colon cancer samples. On the other hand, miR-192 and its cousin miR-215 can each contribute to enhanced CDKN1A/p21 levels, colony suppression, cell cycle arrest, and cell detachment from a solid support. These effects were partially dependent on the presence of wild-type p53. Antagonizing endogenous miR-192 attenuated 5-fluorouracil-induced accumulation of p21. Hence, miR-192 and miR-215 can act as effectors as well as regulators of p53; they seem to suppress cancerogenesis through p21 accumulation and cell cycle arrest.

Diagnostic and prognostic microRNAs in stage II colon cancer.

MicroRNAs (miRNA) are a class of small noncoding RNAs with important posttranscriptional regulatory functions. Recent data suggest that miRNAs are aberrantly expressed in many human cancers and that they may play significant roles in carcinogenesis. Here, we used microarrays to profile the expression of 315 human miRNAs in 10 normal mucosa samples and 49 stage II colon cancers differing with regard to microsatellite status and recurrence of disease. Several miRNAs were differentially expressed between normal tissue and tumor microsatellite subtypes, with miR-145 showing the lowest expression in cancer relative to normal tissue. Microsatellite status for the majority of cancers could be correctly predicted based on miRNA expression profiles. Furthermore, a biomarker based on miRNA expression profiles could predict recurrence of disease with an overall performance accuracy of 81%, indicating a potential role of miRNAs in determining tumor aggressiveness. The expression levels of miR-320 and miR-498, both included in the predictive biomarker, correlated with the probability of recurrence-free survival by multivariate analysis. We successfully verified the expression of selected miRNAs using real-time reverse transcription-PCR assays for mature miRNAs, whereas in situ hybridization was used to detect the accumulation of miR-145 and miR-320 in normal epithelial cells and adenocarcinoma cells. Functional studies showed that miR-145 potently suppressed growth of three different colon carcinoma cell lines. In conclusion, our results suggest that perturbed expression of numerous miRNAs in colon cancer may have a functional effect on tumor cell behavior, and, furthermore, that some miRNAs with prognostic potential could be of clinical importance.

Clusterin expression can be modulated by changes in TCF1-mediated Wnt signaling.

BACKGROUND: Clusterin (CLU) is an enigmatic molecule associated with various physiological processes and disease states. Different modes of cellular stress lead to increased CLU levels, and additionally numerous growth factors and cytokines affect the expression of the CLU gene. APC and c-MYC, both intimately linked to the Wnt signaling pathway have previously been shown to influence CLU levels, and we therefore investigated if changes in Wnt signaling activity in vitro could regulate the expression of one, or more, of several CLU mRNA and protein variants. RESULTS: Over-expression of the cytoplasmic domain of E-cadherin tagged with GFP was used to abrogate Wnt signaling activity in LS174T and HCT116 colon carcinoma cells. This fusion construct sequestered signaling competent beta-catenin whereby Wnt signaling was abrogated, and consequently cytoplasmic CLU protein levels increased as demonstrated by immunofluorescence. To determine which branch of the Wnt pathway was mediating the CLU response, we over-expressed dominant negative (dn) TCF1 and TCF4 transcription factors in stably transfected LS174T cells. We observed both intra- and extracellular levels of CLU protein to be induced by dnTCF1 but not dnTCF4. Subsequent analysis of the expression levels of three CLU mRNA variants by real time RT-PCR revealed only one CLU mRNA variant to be responsive to dnTCF1 over-expression. 5'-end RACE indicated that this CLU mRNA variant was shorter at the 5'-end than previously reported, and accordingly the translated protein was predicted to be shorter at the N-terminus and destined to the secretory pathway which fit our observations. Examination of the immediate expression kinetics of CLU after dnTCF1 over-expression using real time RT-PCR indicated that CLU might be a secondary Wnt target. CONCLUSION: In conclusion, we have demonstrated that the Wnt signaling pathway specifically regulates one out of three CLU mRNA variants via TCF1. This CLU transcript is shorter at the 5' end than reported by the RefSeq database, and produces the intracellular 60 kDa CLU protein isoform which is secreted as a ~80 kDa protein after post-translational processing.

Clusterin expression in normal mucosa and colorectal cancer.

The gene Clusterin is a target for cancer therapy in clinical trials. The indication for intervention is up-regulated Clusterin expression. Clusterin has been reported to be deregulated in multiple cancer types, including colorectal cancer (CRC). However, for CRC the studies have disagreed on whether Clusterin is up- or down-regulated by neoplastic cells. In the present study we sought to clarify the expression and distribution of Clusterin mRNAs and proteins in normal and neoplastic colorectal tissue through laser microdissection, variant-specific real time RT-PCR, immunohistochemistry, immunofluorescence, Western blotting, and array-based transcriptional profiling. At the transcript level we demonstrated the expression of two novel Clusterin transcripts in addition to the known transcript, and at the protein level we demonstrated two Clusterin isoforms. Our analysis of normal epithelial cells revealed that among these, Clusterin was only expressed by rare neuroendocrine subtype. Furthermore our analysis showed that in the normal mucosa the majority of the observed Clusterin protein originated from the stromal compartment. In tumors we found that Clusterin was de novo synthesized by non-neuroendocrine cancer cells in approximately 25% of cases. Moreover we found that the overall Clusterin level in tumors often appeared to be lower than in normal mucosa due to the stromal compartment often being suppressed in tumors. Although Clusterin in normal neuroendocrine cells showed a basal localization, the localization in cancer cells was often apical and in some cases associated with apical secretion. Collectively our results indicate that Clusterin expression is very complex. We conclude that Clusterin expression is associated with neuroendocrine differentiation in normal epithelia and that the Clusterin observed in neoplastic cells is de novo synthesized. The cases with de novo synthesized Clusterin define a distinct subgroup of CRC that may be of clinical importance as anti-Clusterin therapeutics are now in clinical trials.