The p53-inducible
and
genes are frequently silenced in colorectal cancer. To address the
relevance of
function for suppression of intestinal tumor formation, we generated
mice with deletions of the
...and/or
genes separately or in combination. Combined deletion of
increased the number of intestinal stem cells as well as Paneth and Goblet cells, resulting in enlarged intestinal crypts.
-deficient
mice displayed an increased tumor burden and grade and decreased survival.
-deficient adenomas showed elevated proliferation and decreased apoptosis and displayed pronounced bacterial infiltration, which may be due to an observed decrease in infiltrating immune cells and downregulation of barrier proteins. mRNA induction in
-deficient tumors was enriched for miR-34a/b/c seed-matching sites and for mRNAs encoding proteins related to epithelial-mesenchymal transition, stemness, and Wnt signaling. Accordingly, cells explanted from
-deficient adenomas formed tumor organoids at an increased rate. Several upregulated miR-34 targets displayed elevated expression in primary human colorectal cancers that was associated with lymph-node metastases (
and
) and upregulation of
and
in primary colorectal cancer was associated with poor patient survival. In conclusion, our results show that
suppress tumor formation caused by loss of
and control intestinal stem cell and secretory cell homeostasis by downregulation of multiple target mRNAs.
.
Here, we developed and comprehensively characterized a cellular model of colon cancer progression consisting of four defined derivatives of a colon cancer cell line that resulted from consecutive ...epithelial-mesenchymal and mesenchymal-epithelial transitions (EMT/MET) and phenotypically recapitulate the metastatic cascade. Initial EMT was induced by prolonged exposure to IL6, a cytokine also generated by the tumor-stroma. Genome-wide characterization of transcriptional (mRNA, miRNA, and lncRNA) and epigenetic (DNA methylation, H3K4me3, H3K79me3, and H3K27me3 histone modifications) profiles of the cell derivatives, combined with correlative analyses of expression, methylation, and clinical data from the TCGA-COAD database gave insights into the molecular basis of their phenotypic changes. The signatures characterizing invasive, mesenchymal-like cell states as well as the metastases-derived epithelial-like state showed significant association with metastasis, positive nodal status, and poor survival of colon cancer patients. Global hypomethylation of gene-regulatory regions was observed during tumor progression, with the lowest degree of methylation present in cells isolated from metastases. Upregulation of an axon-guidance-related gene signature was the most significant feature of metastatic tumor cells and was also found in primary tumors from colon cancer patients with distant metastases. Furthermore, the microRNAs miR-99a, miR-100, and miR-125b showed elevated expression in mesenchymal-like cells, associated with poor survival, and promoted migration and invasion. Finally, elevated expression of
lncRNA due to promoter demethylation was observed in cells isolated from metastases and was associated with poor survival of colon cancer patients. In the future, our results may be further exploited for the discovery and evaluation of novel metastasis-associated mechanisms and biomarkers.
.
Genotoxic colibactin-producing pks+ Escherichia coli induce DNA double-strand breaks, mutations, and promote tumor development in mouse models of colorectal cancer (CRC). Colibactin's distinct ...mutational signature is reflected in human CRC, suggesting a causal link. Here, we investigate its transformation potential using organoids from primary murine colon epithelial cells. Organoids recovered from short-term infection with pks+ E. coli show characteristics of CRC cells, e.g., enhanced proliferation, Wnt-independence, and impaired differentiation. Sequence analysis of Wnt-independent organoids reveals an enhanced mutational burden, including chromosomal aberrations typical of genomic instability. Although we do not find classic Wnt-signaling mutations, we identify several mutations in genes related to p53-signaling, including miR-34a. Knockout of Trp53 or miR-34 in organoids results in Wnt-independence, corroborating a functional interplay between the p53 and Wnt pathways. We propose larger chromosomal alterations and aneuploidy as the basis of transformation in these organoids, consistent with the early appearance of chromosomal instability in CRC.
Abstract Background & Aims In colorectal tumors, hypoxia causes resistance to therapy and promotes metastasis. Loss of the tumor suppressor p53 (encoded by TP53) provides cancer cells with a ...selective advantage under conditions of hypoxia, but little is known about the mediators of this effect. Methods Isogenic CRC cell lines with different TP53 genotypes were placed under conditions of hypoxia. We examined the effects on levels and activity of microRNA-34 a (MIR34A) in CRC cells. We determined the expression and localization of protein phosphatase 1 regulatory inhibitor subunit 11 (PPP1R11, also called HCGV, IPP3, HCGV, TCTE5, TCTEX5, or CFAP255) in 82 human colon cancers. We analyzed data on human colorectal carcinomas from the Cancer Genome Atlas collection to determine whether expression of PPP1R11 was affected by altered level or activity of p53, markers of EMT, or MIR34A or was associated with metastasis. We determined the effects of disruption miR34a, miR34b, and miR34c in ApcMin/+ mice. DLD-1 cells were transfected with small inhibitor RNAs against PPP1R1, injected into the tail veins of immune-compromised mice, and followed by noninvasive bioluminescence imaging. Results The hypoxia inducible factor 1 alpha subunit (HIF1A) directly repressed the MIR34a gene in p53-defective CRC cells, whereas expression MIR34a was induced in p53-proficient CRC cells exposed to hypoxia. Down-regulation of MIR34a was required for hypoxia-induced epithelial to mesenchymal transition (EMT), invasion and migration, and activation of STAT3 in CRC cells. We identified PPP1R11, whose product inhibits PP1, as a target of MIR34a. PPP1R11 mediates phosphorylation (activation) of STAT3, so expression of MIR34a reduced activation of STAT3 in p53-negative CRC cells. Ectopic expression of PPP1R11 in CRC cells induced EMT, invasion, and migration, which all required STAT3. Increased expression of PPP1R11 in p53-negative CRC cells was required for hypoxia-induced EMT, invasion, migration, and resistance to 5-fluorouracil, as well as metastasis of xenograft tumors to lungs of mice. Adenomas and derived tumoroids of ApcMin/+ mice with disruption miR34a, mir34b, or mir34c had increased levels of PPP1R11. Colorectal tumors from patients had increased levels of PPP1R11 at areas of invasion, compared with other areas of the tumor; increased level PPP1R11 associated with p53 mutations and metastasis to liver. Conclusions HIF1A represses, whereas p53 increases, expression of MIR34a in CRC cells. MIR34a reduces expression of PPP1R11 to prevent activation of STAT3 and inhibit the EMT and metastasis. Strategies to target this pathway might be developed to inhibit CRC metastasis and overcome resistance to therapy associated with hypoxia.
Epithelial-mesenchymal transition (EMT) plays an important role in tumor invasion and metastasis. A comprehensive, bioinformatics analysis of CCLE and TCGA datasets of seven tumor types allowed us to ...identify a novel pan-cancer EMT-associated gene expression signature consisting of 16 epithelial and 4 mesenchymal state-associated mRNAs. Among the identified epithelial cell state-associated factors, down-regulation of the RBM47 (RNA binding motif protein 47) mRNA displayed the most significant association with metastasis and poor survival in multiple cohorts of colorectal cancer (CRC) patients. Moreover, decreased RBM47 protein expression was associated with metastasis in a cohort of primary CRCs. RBM47 was directly suppressed during EMT induced by IL6-activated STAT3 or ectopic SNAIL and SLUG expression via conserved binding motifs of these factors within the RBM47 promoter. Moreover, RNAi-mediated down-regulation of RBM47 in CRC lines resulted in increased cell migration, invasion and metastases formation. As demonstrated by the example of RBM47, the EMT-associated signature characterized here allows to identify biomarkers for predicting clinical outcome of CRC and presumably other cancer entities. In addition, our functional analysis of RBM47 shows that the down-regulation of RBM47 during CRC progression may promote EMT and metastasis.
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IZUM, KILJ, NUK, PILJ, PNG, SAZU, UL, UM, UPUK
In recent years, microRNAs (miRNAs) have been identified as mediators of tumour suppression and stress responses exerted by the p53 tumour suppressor. p53-regulated miRNAs contribute to tumour ...suppression by controlling the expression of central components of multiple processes, including cell cycle progression, epithelial-mesenchymal transition, stemness, metabolism, cell survival and angiogenesis. The expression and activity of p53 itself is also under the control of miRNAs. Finally, genetic and epigenetic alterations identified in the p53-miRNA network indicate that these pathways are important for the initiation and progression of tumours. In the future, knowledge about the p53-miRNA network may be able to be exploited for diagnostic and therapeutic approaches in cancer prevention and treatment.
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DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
About 40% of colorectal cancers have mutations in KRAS accompanied by downstream activation of MAPK signaling, which promotes tumor invasion and progression. Here, we report that MAPK signaling shows ...strong intratumoral heterogeneity and unexpectedly remains regulated in colorectal cancer irrespective of KRAS mutation status. Using primary colorectal cancer tissues, xenograft models, and MAPK reporter constructs, we showed that tumor cells with high MAPK activity resided specifically at the leading tumor edge, ceased to proliferate, underwent epithelial-mesenchymal transition (EMT), and expressed markers related to colon cancer stem cells. In KRAS-mutant colon cancer, regulation of MAPK signaling was preserved through remaining wild-type RAS isoforms. Moreover, using a lineage tracing strategy, we provide evidence that high MAPK activity marked a progenitor cell compartment of growth-fueling colon cancer cells
Our results imply that differential MAPK signaling balances EMT, cancer stem cell potential, and tumor growth in colorectal cancer.
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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 mes- enchymat transition, migration, invasion, and metastasis of various cancer celt 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.
AP4 (TFAP4) encodes a basic helix-loop-helix leucine zipper (bHLH-LZ) transcription factor and is a direct target gene of the oncogenic transcription factor c-MYC. Here, we set out to determine the ...relevance of AP4 in human colorectal cancer (CRC) cells.
A CRISPR/Cas9 approach was employed to generate AP4-deficient CRC cell lines with inducible expression of c-MYC. Colony formation, β-gal staining, immunofluorescence, comet and homologous recombination (HR) assays and RNA-Seq analysis were used to determine the effects of AP4 inactivation. qPCR and qChIP analyses was performed to validate differentially expressed AP4 targets. Expression data from CRC cohorts was subjected to bioinformatics analyses. Immunohistochemistry was used to evaluate AP4 targets in vivo. Ap4-deficient APC
mice were analyzed to determine conservation. Immunofluorescence, chromosome and micronuclei enumeration, MTT and colony formation assays were used to determine the effects of AP4 inactivation and target gene regulation on chromosomal instability (CIN) and drug sensitivity.
Inactivation of AP4 in CRC cell lines resulted in increased spontaneous and c-MYC-induced DNA damage, chromosomal instability (CIN) and cellular senescence. AP4-deficient cells displayed increased expression of the long non-coding RNA MIR22HG, which encodes miR-22-3p and was directly repressed by AP4. Furthermore, Mediator of DNA damage Checkpoint 1 (MDC1), a central component of the DNA damage response and a known target of miR-22-3p, displayed decreased expression in AP4-deficient cells. Accordingly, MDC1 was directly induced by AP4 and indirectly by AP4-mediated repression of miR-22-3p. Adenomas and organoids from Ap4-deficient APC
mice displayed conservation of these regulations. Inhibition of miR-22-3p or ectopic MDC1 expression reversed the increased senescence, DNA damage, CIN and defective HR observed in AP4-deficient CRC cells. AP4-deficiency also sensitized CRC cells to 5-FU treatment, whereas ectopic AP4 conferred resistance to 5-FU in a miR-22-3p and MDC1-dependent manner.
In summary, AP4, miR-22-3p and MDC1 form a conserved and coherent, regulatory feed-forward loop to promote DNA repair, which suppresses DNA damage, senescence and CIN, and contributes to 5-FU resistance. These findings explain how elevated AP4 expression contributes to development and chemo-resistance of colorectal cancer after c-MYC activation.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Recently, the inhibition of epithelial-mesenchymal-transition (EMT) by p53 has been described as a new mode of tumor suppression which presumably prevents metastasis. Here we report that activation ...of p53 down-regulates the EMT-inducing transcription factor SNAIL via induction of the miR-34a/b/c genes. Suppression of miR-34a/b/c caused up-regulation of SNAIL and cells displayed EMT markers and related features, as enhanced migration and invasion. Ectopic miR-34a induced mesenchymal-epithelial-transition (MET) and down-regulation of SNAIL, which was mediated by a conserved miR-34a/b/c seed-matching sequence in the SNAIL 3'-UTR. miR-34a also down-regulated SLUG and ZEB1, as well as the stemness factors BMI1, CD44, CD133, OLFM4 and c-MYC. Conversely, the transcription factors SNAIL and ZEB1 bound to E-boxes in the miR-34a/b/c promoters, thereby repressing miR-34a and miR-34b/c expression. Since ectopic miR-34a prevented TGF-β-induced EMT, the repression of miR-34 genes by SNAIL and related factors is part of the EMT program. In conclusion, the frequent inactivation of p53 and/or miR-34a/b/c found in cancer may shift the equilibrium of these reciprocal regulations towards the mesenchymal state and thereby lock cells in a metastatic state.
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BFBNIB, GIS, IJS, KISLJ, NUK, PNG, UL, UM, UPUK
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