Heiko Hermeking

MicroRNA-34a regulates cardiac ageing and function

Ageing is the predominant risk factor for cardiovascular diseases and contributes to a significantly worse outcome in patients with acute myocardial infarction. MicroRNAs (miRNAs) have emerged as crucial regulators of cardiovascular function and some miRNAs have key roles in ageing. We propose that altered expression of miRNAs in the heart during ageing contributes to the age-dependent decline in cardiac function. Here we show that miR-34a is induced in the ageing heart and that in vivo silencing or genetic deletion of miR-34a reduces age-associated cardiomyocyte cell death. Moreover, miR-34a inhibition reduces cell death and fibrosis following acute myocardial infarction and improves recovery of myocardial function. Mechanistically, we identified PNUTS (also known as PPP1R10) as a novel direct miR-34a target, which reduces telomere shortening, DNA damage responses and cardiomyocyte apoptosis, and improves functional recovery after acute myocardial infarction. Together, these results identify age-induced expression of miR-34a and inhibition of its target PNUTS as a key mechanism that regulates cardiac contractile function during ageing and after acute myocardial infarction, by inducing DNA damage responses and telomere attrition.

Frequent concomitant inactivation of miR-34a and miR-34b/c by CpG methylation in colorectal, pancreatic, mammary, ovarian, urothelial, and renal cell carcinomas and soft tissue sarcomas

The microRNA encoding genes miR-34a and miR-34b/c represent direct p53 target genes and possess tumor suppressive properties as they mediate apoptosis, cell cycle arrest, and senescence. We previously reported that the miR-34a gene is subject to epigenetic inactivation by CpG methylation of its promoter region in primary prostate cancer and melanomas, and in 110 different cancer cell lines of diverse origin. Here we analyzed the methylation status of miR-34a and miR-34b/c in additional primary tumors of divergent sites. We found methylation of miR-34a or miR-34b/c in formalin-fixed, paraffin-embedded (FFPE) tumor samples from 178 patients with the following frequencies: colorectal cancer (74% miR-34a, 99% miR-34b/c; n = 114), pancreatic cancer (64%, 100%; n = 11), mammary cancer (60%, 90%; n = 10), ovarian cancer (62%, 69%; n = 13), urothelial cancer (71%, 57%; n = 7), and renal cell cancer (58%, 100%; n = 12). Furthermore, soft tissue sarcomas showed methylation of miR-34 gene promoters in FFPE samples (64%, 45%; n = 11), in explanted, cultured cells (53%, 40%; n = 40), and in frozen tissue samples (75%, 75%, n = 8). In the colorectal cancer samples a statistically significant correlation of miR-34a methylation and the absence of p53 mutation was detected. With the exception of sarcoma cell lines, the inactivation of miR-34a and miR-34b/c was concomitant in most cases. These results show that miR-34 inactivation is a common event in tumor formation, and suggest that CpG methylation of miR-34a and miR-34-b/c may have diagnostic value. The mutual exclusiveness of miR-34a methylation and p53 mutation indicates that miR-34a inactivation may substitute for loss of p53 function in cancer.

Frequent Downregulation of miR-34 Family in Human Ovarian Cancers

Purpose: The miR-34 family is directly transactivated by tumor suppressor p53, which is frequently mutated in human epithelial ovarian cancer (EOC). We hypothesized that miR-34 expression would be decreased in EOC and that reconstituted miR-34 expression might reduce cell proliferation and invasion of EOC cells. Experimental Designs: miR-34 expression was determined by quantitative reverse transcription-PCR and in situ hybridization in a panel of 83 human EOC samples. Functional characterization of miR-34 was accomplished by reconstitution of miR-34 expression in EOC cells with synthetic pre-miR molecules followed by determining changes in proliferation, apoptosis, and invasion. Results: miR-34a expression is decreased in 100%, and miR-34b*/c in 72%, of EOC with p53 mutation, whereas miR-34a is also downregulated in 93% of tumors with wild-type p53. Furthermore, expression of miR-34b*/c is significantly reduced in stage IV tumors compared with stage III (P = 0.0171 and P = 0.0029, respectively). Additionally, we observed promoter methylation and copy number variations at mir-34. In situ hybridization showed that miR-34a expression is inversely correlated with MET immunohistochemical staining, consistent with translational inhibition by miR-34a. Finally, miR-34 reconstitution experiments in p53 mutant EOC cells resulted in reduced proliferation, motility, and invasion, the latter of which was dependent on MET expression. Conclusions: Our work suggests that miR-34 family plays an important role in EOC pathogenesis and reduced expression of miR-34b*/c may be particularly important for progression to the most advanced stages. Part of miR-34 effects on motility and invasion may be explained by regulation of MET, which is frequently overexpressed in EOC. Clin Cancer Res; 16(4); 1119–28

Genome-wide Characterization of miR-34a Induced Changes in Protein and mRNA Expression by a Combined Pulsed SILAC and Microarray Analysis

The gene encoding the miR-34a microRNA is a transcriptional target of the p53 tumor suppressor protein and subject to epigenetic inactivation in colorectal cancer and numerous other tumor types. Here, we combined pulsed SILAC (pSILAC) and microarray analyses to identify miR-34a-induced changes in protein and mRNA expression. pSILAC allowed to quantify the de novo protein synthesis of 1206 proteins after activation of a conditional miR-34a allele in a colorectal cancer cell line. ∼19% of the detected proteins were differentially regulated, with 113 proteins being down- and 115 up-regulated. The proteins with a miR-34a seed-matching-sequence in the 3′-untranslated region (UTR) of the corresponding mRNA showed a clear bias toward translational repression. Proteins involved in DNA replication, e.g. the MCM proteins, and cell proliferation, were over-represented among indirectly down-regulated proteins lacking a miR-34a seed-match. The decrease in de novo protein synthesis of direct miR-34a targets correlated with reduced levels of the corresponding mRNA in most cases, indicating an interdependence of both types of regulation. In addition, 43 mRNAs encoding proteins not detected by pSILAC were down-regulated after miR-34a expression and contained miR-34a seed-matches. The direct regulation of selected miR-34a target-mRNAs was confirmed using reporter assays. Via down-regulation of the proteins encoded by these mRNAs miR-34a presumably inhibits glycolysis (LDHA), WNT-signaling (LEF1), invasion/migration (AXL) and lipid metabolism (ACSL1, ACSL4). Furthermore, miR-34a may activate p53 by inhibiting its acetylation (MTA2, HDAC1) and degradation (YY1). In summary, miR-34a presumably participates in multiple tumor suppressive pathways by directly and indirectly suppressing the expression of numerous, critical proteins.

The p53/miR-34 axis in development and disease

The tumor suppressor p53 is one of the most frequently mutated genes in human cancers. MicroRNAs (miRNAs) are small non-protein coding RNAs that regulate gene expression on the post-transcriptional level. Recently, it was shown that p53 regulates the expression of several miRNAs, thereby representing an important mechanism of p53 signaling. Several independent studies identified the members of the miR-34 family as the most prevalent p53-induced miRNAs. miR-34s are frequently silenced in variety of tumor entities, suggesting that they are important tumor suppressors. Indeed, ectopic expression of miR-34s inhibits proliferation, epithelial to mesenchymal transition, migration, invasion, and metastasis of various cancer cell entities. Moreover, delivery or re-expression of miR-34 leads to notable repression of tumor growth and metastasis in cancer mouse models, and may therefore represent an efficient strategy for future cancer therapeutics. Besides their crucial functions in cancer, members of the miR-34 family also play important roles in spermatogenesis, stem cell differentiation, neuronal development, aging, and cardiovascular functions. Consequently, miR-34 has also been implicated in various non-cancerous diseases, such as brain disorders, osteoporosis, and cardiovascular complications.

Wild-type p53 controls cell motility and invasion by dual regulation of MET expression

Recent observations suggest that p53 mutations are responsible not only for growth of primary tumors but also for their dissemination. However, mechanisms involved in p53-mediated control of cell motility and invasion remain poorly understood. By using the primary ovarian surface epithelium cell culture, we show that conditional inactivation of p53 or expression of its mutant forms results in overexpression of MET receptor tyrosine kinase, a crucial regulator of invasive growth. At the same time, cells acquire increased MET-dependent motility and invasion. Wild-type p53 negatively regulates MET expression by two mechanisms: (i) transactivation of MET-targeting miR-34, and (ii) inhibition of SP1 binding to MET promoter. Both mechanisms are not functional in p53 absence, but mutant p53 proteins retain partial MET promoter suppression. Accordingly, MET overexpression, cell motility, and invasion are particularly high in p53-null cells. These results identify MET as a critical effector of p53 and suggest that inhibition of MET may be an effective antimetastatic approach to treat cancers with p53 mutations. These results also show that the extent of advanced cancer traits, such as invasion, may be determined by alterations in individual components of p53/MET regulatory network.

Detection of miR-34a Promoter Methylation in Combination with Elevated Expression of c-Met and β-Catenin Predicts Distant Metastasis of Colon Cancer

Purpose: Here, we determined whether epigenetic inactivation of miR-34a and miR-34b/c genes may serve as a prognostic marker for distant metastases in colon cancer. Experimental Design: Using a case–control study design of 94 primary colon cancer samples with and without liver metastases, we determined CpG methylation frequencies of miR-34a and miR-34b/c promoters, expression of miR-34a, and its targets c-Met, Snail, and β-catenin and their prognostic value. Results: miR-34a methylation was detected in 45.1% (n = 42 of 93) of the samples and strongly associated with metastases to the liver (P = 0.003) and lymph nodes (P = 0.006). miR-34b/c methylation was detected in 91.9% of the samples (n = 79/86). A significant inverse correlation between miR-34a methylation and expression of mature miR-34a (P = 0.018) was detected. Decreased miR-34a expression was associated with upregulation of c-Met, Snail, and β-catenin protein levels (P = 0.031, 0.132, and 0.004), which were associated with distant metastases (P = 0.001, 0.017, and 0.005). In a confounder-adjusted multivariate regression model miR-34a methylation, high c-Met and β-catenin levels provided the most significant prognostic information about metastases to the liver (P = 0.014, 0.031, and 0.058) and matched pairs showed a higher prevalence of these risk factors in the samples with distant spread (P = 0.029). Finally, we obtained statistical evidence indicating that the simultaneous detection of these three markers has the highest prognostic value. Conclusions: Silencing of miR-34a and upregulation of c-Met, Snail, and β-catenin expression is associated with liver metastases of colon cancer. Detection of miR-34a silencing in resected primary colon cancer may be of prognostic value, especially in combination with detection of c-Met and β-catenin expression. Clin Cancer Res; 19(3); 710–20. ©2012 AACR.

AP4 is a mediator of epithelial–mesenchymal transition and metastasis in colorectal cancer

The transcription factor AP4 is a critical regulator of epithelial–mesenchymal transition, migration, invasion, and metastasis in colorectal cancer cells.

SNAIL and miR-34a feed-forward regulation of ZNF281/ZBP99 promotes epithelial-mesenchymal transition

Here, we show that expression of ZNF281/ZBP‐99 is controlled by SNAIL and miR‐34a/b/c in a coherent feed‐forward loop: the epithelial–mesenchymal transition (EMT) inducing factor SNAIL directly induces ZNF281 transcription and represses miR‐34a/b/c, thereby alleviating ZNF281 mRNA from direct down‐regulation by miR‐34. Furthermore, p53 activation resulted in a miR‐34a‐dependent repression of ZNF281. Ectopic ZNF281 expression in colorectal cancer (CRC) cells induced EMT by directly activating SNAIL, and was associated with increased migration/invasion and enhanced β‐catenin activity. Furthermore, ZNF281 induced the stemness markers LGR5 and CD133, and increased sphere formation. Conversely, experimental down‐regulation of ZNF281 resulted in mesenchymal–epithelial transition (MET) and inhibition of migration/invasion, sphere formation and lung metastases in mice. Ectopic c‐MYC induced ZNF281 protein expression in a SNAIL‐dependent manner. Experimental inactivation of ZNF281 prevented EMT induced by c‐MYC or SNAIL. In primary CRC samples, expression of ZNF281 increased during tumour progression and correlated with recurrence. Taken together, these results identify ZNF281 as a component of EMT‐regulating networks, which contribute to metastasis formation in CRC.

Tissue-specific opposing functions of the inflammasome adaptor ASC in the regulation of epithelial skin carcinogenesis

A chronic inflammatory microenvironment favors tumor progression through molecular mechanisms that are still incompletely defined. In inflammation-induced skin cancers, IL-1 receptor- or caspase-1–deficient mice, or mice specifically deficient for the inflammasome adaptor protein ASC (apoptosis-associated speck-like protein containing a CARD) in myeloid cells, had reduced tumor incidence, pointing to a role for IL-1 signaling and inflammasome activation in tumor development. However, mice fully deficient for ASC were not protected, and mice specifically deficient for ASC in keratinocytes developed more tumors than controls, suggesting that, in contrast to its proinflammatory role in myeloid cells, ASC acts as a tumor-suppressor in keratinocytes. Accordingly, ASC protein expression was lost in human cutaneous squamous cell carcinoma, but not in psoriatic skin lesions. Stimulation of primary mouse keratinocytes or the human keratinocyte cell line HaCaT with UVB induced an ASC-dependent phosphorylation of p53 and expression of p53 target genes. In HaCaT cells, ASC interacted with p53 at the endogenous level upon UVB irradiation. Thus, ASC in different tissues may influence tumor growth in opposite directions: it has a proinflammatory role in infiltrating cells that favors tumor development, but it also limits keratinocyte proliferation in response to noxious stimuli, possibly through p53 activation, which helps suppressing tumors.