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Background: Metabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance.Methods: A survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines.Results: miR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines.Conclusions: We disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties. [ABSTRACT FROM AUTHOR]

Peroxisome proliferator-activated receptor-γ (PPARγ) is a transcription factor of the nuclear hormone receptor superfamily implicated in a wide range of processes, including tumorigenesis. Its role in colorectal cancer (CRC) is still debated; most reports support that PPARγ reduced expression is associated with poor prognosis. We employed 2-Dimensional Differential InGel Electrophoresis (2-D DIGE) followed by Liquid Chromatography (LC)-tandem Mass Spectrometry (MS/MS) to identify differentially expressed proteins and the molecular pathways underlying PPARγ expression in CRC progression. We identified several differentially expressed proteins in HT29 and HCT116 CRC cells and derived clones either silenced or overexpressing PPARγ, respectively. In Ingenuity Pathway Analysis (IPA) they showed reciprocal relation with PPARγ and a strong relationship with networks linked to cell death, growth and survival. Interestingly, five of the identified proteins, ezrin (EZR), isoform C of prelamin-A/C (LMNA), alpha-enolase (ENOA), prohibitin (PHB) and RuvB-like 2 (RUVBL2) were shared by the two cell models with opposite expression levels, suggesting a possible regulation by PPARγ. mRNA and western blot analysis were undertaken to obtain a technical validation and confirm the expression trend observed by 2-D DIGE data. We associated EZR upregulation with increased cell surface localization in PPARγ-overexpressing cells by flow cytometry and immunofluorescence staining. We also correlated EZR and PPARγ expression in our series of CRC specimens and the expression profiling of all five proteins levels in the publicly available colon cancer genomic data from Oncomine and Cancer Genome Atlas (TCGA) colon adenocarcinoma (COAD) datasets. In summary, we identified a panel of proteins correlated with PPARγ expression that could be associated with CRC unveiling new pathways to be investigated for the selection of novel potential prognostic/predictive biomarkers and/or therapeutic targets. [ABSTRACT FROM AUTHOR]

Background: Circadian disruption and deranged molecular clockworks are involved in carcinogenesis. The cryptochrome genes (CRY1 and CRY2) encode circadian proteins important for the functioning of biological oscillators. Their expression in human colorectal cancer (CRC) and in colon cancer cell lines has not been evaluated so far. Methods: We investigated CRY1 and CRY2 expression in fifty CRCs and in the CaCo2, HCT116, HT29, SW480 cell lines. Results: CRY1 (p = 0.01) and CRY2 (p < 0.0001) expression was significantly changed in tumour tissue, as confirmed in a large independent CRC dataset. In addition, lower CRY1 mRNA levels were observed in patients in the age range of 62- 74 years (p = 0.018), in female patients (p = 0.003) and in cancers located at the transverse colon (p = 0.008). Lower CRY2 levels were also associated with cancer location at the transverse colon (p = 0.007). CRC patients displaying CRY1 (p = 0.042) and CRY2 (p = 0.043) expression levels over the median were hallmarked by a poorer survival rate. Survey of selected colon cancer cell lines evidenced variable levels of cryptochrome genes expression and time-dependent changes in their mRNA levels. Moreover, they showed reduced apoptosis, increased proliferation and different response to 5-fluorouracil and oxaliplatin upon CRY1 and CRY2 ectopic expression. The relationship with p53 status came out as an additional layer of regulation: higher CRY1 and CRY2 protein levels coincided with a wild type p53 as in HCT116 cells and this condition only marginally affected the apoptotic and cell proliferation characteristics of the cells upon CRY ectopic expression. Conversely, lower CRY and CRY2 levels as in HT29 and SW480 cells coincided with a mutated p53 and a more robust apoptosis and proliferation upon CRY transfection. Besides, an heterogeneous pattern of ARNTL, WEE and c-MYC expression hallmarked the chosen colon cancer cell lines and likely influenced their phenotypic changes. Conclusion: Cryptochrome gene expression is altered in CRC, particularly in elderly subjects, female patients and cancers located at the transverse colon, affecting overall survival. Altered CRY1 and CRY2 expression patterns and the interplay with the genetic landscape in colon cancer cells may underlie phenotypic divergence that could influence disease behavior as well as CRC patients survival and response to chemotherapy. [ABSTRACT FROM AUTHOR]

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches. [Display omitted] • Serine metabolism is a key driver of 5-FU acquired resistance in CRC • Affecting serine availability impairs DNA damage response in 5-FU-resistant cells • Mitochondrial compartmentalization of serine metabolism sustains 5-FU resistance • Targeting mitochondrial serine metabolism is effective in overcoming 5-FU resistance Pranzini et al. show that 5-FU resistance is supported by increased serine consumption and its mitochondrial compartmentalization to support purine biosynthesis and potentiate DNA damage repair to overcome drug toxicity. Interfering with serine accessibility and targeting mitochondrial serine metabolism overcome 5-FU resistance. [ABSTRACT FROM AUTHOR]

Background INI1 (Integrase interactor 1), also known as SMARCB1, is the most studied subunit of chromatin remodelling complexes. Its role in colorectal tumorigenesis is not known. Methods We examined SMARCB1/INI1 protein expression in 134 cases of colorectal cancer (CRC) and 60 matched normal mucosa by using tissue microarrays and western blot and categorized the results according to mismatch repair status (MMR), CpG island methylator phenotype, biomarkers of tumor differentiation CDX2, CK20, vimentin and p53. We validated results in two independent data sets and in cultured CRC cell lines. Results Herein, we show that negative SMARCB1/INI1 expression (11% of CRCs) associates with loss of CDX2, poor differentiation, liver metastasis and shorter patients' survival regardless of the MMR status or tumor stage. Unexpectedly, even CRCs displaying diffuse nuclear INI1 staining (33%) show an adverse prognosis and vimentin over-expression, in comparison with the low expressing group (56%). The negative association of SMARCB1/INI1-lack of expression with a metastatic behavior is enhanced by the TP53 status. By interrogating global gene expression from two independent cohorts of 226 and 146 patients, we confirm the prognostic results and identify a gene signature characterized by SMARCB1/INI1 deregulation. Notably, the top genes of the signature (BCR, COMT, MIF) map on the long arm of chromosome 22 and are closely associated with SMARCB1/INI1. Conclusion Our findings suggest that SMARCB1/INI1-dysregulation and genetic hot-spots on the long arm of chromosome 22 might play an important role in the CRC metastatic behavior and be clinically relevant as novel biomarkers. [ABSTRACT FROM AUTHOR]

Background: Rhabdoid colorectal tumor (RCT) is a rare, highly aggressive neoplasm recurrent in elderly patients, commonly at the caecum. The molecular mechanisms underlying RCT pathogenesis remain poorly elucidated. The differential diagnosis is with the malignant rhabdoid tumors of infancy characterized by genetic inactivation of SMARCB1 (INI1) or deletions of chromosome 22q12 locus. Materials and methods: To shed light on RCT pathogenesis, we investigated genetic and epigenetic alterations in two cases of pure and composite RCT and compared them with the profiles of matched adenomas and normal mucosa. Immunohistochemical analysis, FISH, methylation specific PCR and DNA sequencing analysis were performed on paraffin-embedded tissues. Results: Loss of epithelial markers, (CK20, CDX2 and E-cadherin) and intense vimentin expression was observed in RCTs but neither in the normal mucosa or adenomas. INI1 expression was detected in normal mucosa, adenomas and retained in pure RCT, while it was undetected in composite RCT. Rearrangement of the 22q12 locus was found only in pure RCT. The APC/ß-catenin pathway was not altered, while MLH1 immunostaining was negative in RCTs and positive in adenomas and normal mucosa. These expression profiles were associated with V600E BRAF mutation, a progressive accumulation of promoter methylation at specific CIMP loci and additional genes from the normal mucosa to tubular adenoma and RCT. Conclusions: Right-sided RCT could be characterized by epigenetic events and molecular features likely similar to those occurring in the serrated pathway and associated with epithelial-mesenchymal transition. These extremely rare tumors may benefit from the use of new biological molecules specific for colorectal carcinoma. [ABSTRACT FROM AUTHOR]

Summary: β-Catenin and p53 play key roles in tumorigenesis. The relationships between these 2 signaling pathways and their contribution to colorectal cancer metastatic progression have not been completely elucidated. We analyzed 141 cases of primary sporadic colorectal cancer, 45 matched metastases, and 80 samples of normal mucosa by immunohistochemistry on paraffin-embedded specimens. The expression profiles were also related to patients'' clinicopathologic features and 5-year survival. In primary tumors, β-catenin immunoreactivity was nuclear (27%), predominantly membrane/cytosolic (46.0%) or negative (27%). This latter subgroup was strongly related to microsatellite instability, in particular to MLH-1 deficiency. Remarkably, β-catenin membrane/cytosolic expression in primary tumors was reduced in the corresponding matched metastases. p53 showed a significant increase in immunoreactivity in (66.7%), whereas it was negative in (33.3%) of tumors. When we considered the expression of both genes, the combination of negative β-catenin and positive p53 nuclear staining (21%) was strongly related to a higher frequency of liver metastases. Such an association was significantly related to a worse prognosis than any other combination. In a multivariate analysis, β-catenin and distant metastases were independent prognostic markers. We suggest that a combination of low β-catenin and high p53 expression in primary colorectal cancers may be a prognostic factor in predicting the progression of the disease, the occurrence of metastasis, and a more severe outcome. [Copyright &y& Elsevier]

Summary: Recent studies have reported cross talk between β-catenin, peroxisome proliferator–activated receptor–γ, and cyclooxygenase 2 signaling pathways. We examined whether molecular changes of these pathways could be related to colorectal cancer metastatic progression. Seventy-two sporadic colorectal cancers and the distant nonneoplastic mucosa were analyzed for β-catenin, peroxisome proliferator–activated receptor–γ, cyclooxygenase 2, and nuclear factor κB levels by immunohistochemistry and Western blot. The expression profiles were correlated with patient outcome and 5-year survival. Nuclear β-catenin staining was detected in only 18.1% of tumors and correlated with poor survival as compared with cases showing cytosolic/membrane accumulation (59.7%, P < .05). This latter group and tumor samples showing cytosolic/nuclear peroxisome proliferator–activated receptor–γ expression (70.8%) were significantly associated with a favorable prognosis (P < .001). Remarkably, reduction or loss of β-catenin (22.2%) and peroxisome proliferator–activated receptor–γ (29.2%) expression was strongly correlated with marked infiltration of tumor-associated macrophages (P < .01), presence of liver metastases, and very short survival (P = .0001). Moreover, β-catenin and peroxisome proliferator–activated receptor–γ levels were inversely correlated with cyclooxygenase 2 (P < .01) and nuclear factor κB expression (P < .05). Our results suggest that reduced expression of β-catenin and peroxisome proliferator–activated receptor–γ could play a key role in aggressive colorectal cancer behavior. This finding may provide a relevant prognostic tool and contribute to early identification of patients at high risk of mortality. [Copyright &y& Elsevier]

Simple Summary: Cellular and mitochondrial metabolism can be dysregulated during tumorigenesis. miR-27a plays a central role in redirecting cell metabolism in colorectal cancer. In this study, we searched for new miR-27a targets that could influence mitochondria and identified FOXJ3 a master regulator of mitochondrial biogenesis. We validated FOXJ3 as an miR-27a target in an in vitro cell model system that was genetically modified for miR-27a expression and showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and regulates other members of the pathway. The miR-27a/FOXJ3 axis also influences mitochondrial dynamics, superoxide production, respiration capacity, and membrane potential. A mouse xenograft model confirmed that miR-27a downregulates FOXJ3 in vivo and a survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and the impact on mitochondrial biogenesis. The miR-27a/FOXJ3 axis is a major actor in regulating mitochondrial homeostasis, and its discovery may contribute to therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities. miR-27a plays a driver role in rewiring tumor cell metabolism. We searched for new miR-27a targets that could affect mitochondria and identified FOXJ3, an apical factor of mitochondrial biogenesis. We analyzed FOXJ3 levels in an in vitro cell model system that was genetically modified for miR-27a expression and validated it as an miR-27a target. We showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and other key members of the pathway, implying multiple levels of control. As assessed by specific markers, the miR-27a/FOXJ3 axis also dysregulates mitochondrial dynamics, resulting in fewer, short, and punctate organelles. Consistently, in high miR-27a-/low FOXJ3-expressing cells, mitochondria are functionally characterized by lower superoxide production, respiration capacity, and membrane potential, as evaluated by OCR assays and confocal microscopy. The analysis of a mouse xenograft model confirmed FOXJ3 as a target and suggested that the miR-27a/FOXJ3 axis affects mitochondrial abundance in vivo. A survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and reinforced cellular component organization or biogenesis as the most affected pathway. The miR-27a/FOXJ3 axis acts as a central hub in regulating mitochondrial homeostasis. Its discovery paves the way for new therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities. [ABSTRACT FROM AUTHOR]

The network of bidirectional homotypic and heterotypic interactions established among parenchymal tumour cells and surrounding mesenchymal stromal cells generates the tumour microenvironment (TME). These intricate crosstalks elicit both beneficial and adverse effects on tumour initiation and progression unbalancing the signals and responses from the neighbouring cells. Here, we highlight the structure, activities and evolution of TME cells considering a novel colorectal cancer (CRC) classification based on differential stromal composition and gene expression profiles. In this scenario, we scrutinise the molecular pathways that either change or become corrupted during CRC development and their relative prognostic value. Finally, we survey the therapeutic molecules directed against TME components currently available in clinical trials as well as those with stronger potential in preclinical studies. Elucidation of dynamic variations in the CRC TME cell composition and their relative contribution could provide novel diagnostic or prognostic biomarkers and allow more personalised therapeutic strategies. [ABSTRACT FROM AUTHOR]

Abstract: MicroRNAs (miRNAs) regulate diverse biological processes by inhibiting translation or inducing degradation of target mRNAs. miR-145 is a candidate tumor suppressor in colorectal carcinoma (CRC). Colorectal carcinogenesis involves deregulation of cellular processes controlled by a number of intertwined chief transcription factors, such as PPARγ and SOX9. Since PPAR family members are able to modulate complex miRNAs networks, we hypothesized a role of miRNA-145 in the interaction between PPARγ and SOX9 in colorectal carcinogenesis. To address this issue, we evaluated gene expression in tissue specimens of CRC patients and we took advantage of invitro models represented by CRC derived cell lines (CaCo2, SW480, HCT116, and HT-29), employing PPARγ activation and/or miRNA-145 ectopic overexpression to analyze how their interplay impact the expression of SOX9 and the development of a malignant phenotype. Results: PPARγ regulates the expression of miR-145 by directly binding to a PPAR response element (PPRE) in its promoter at −1207/−1194bp from the transcription start site. The binding is essential for miR-145 upregulation by PPARγ upon rosiglitazone treatment. Ectopic expression of miR-145, in turn, regulates SOX9 expression through the binding to specific seed motifs. The PPARγ-miR-145-SOX9 axis overarches cell cycle progression, invasiveness and differentiation of CRC derived cell lines. Together, these results suggest that miR-145 is a novel target of PPARγ, acts as a tumor suppressor in CRC cell lines and is a key regulator of intestinal cell differentiation by directly targeting SOX9, a marker of undifferentiated progenitors in the colonic crypts. [Copyright &y& Elsevier]