Massimo Pancione

Genetic and Epigenetic Events Generate Multiple Pathways in Colorectal Cancer Progression

Colorectal cancer (CRC) is one of the most common causes of death, despite decades of research. Initially considered as a disease due to genetic mutations, it is now viewed as a complex malignancy because of the involvement of epigenetic abnormalities. A functional equivalence between genetic and epigenetic mechanisms has been suggested in CRC initiation and progression. A hallmark of CRC is its pathogenetic heterogeneity attained through at least three distinct pathways: a traditional (adenoma-carcinoma sequence), an alternative, and more recently the so-called serrated pathway. While the alternative pathway is more heterogeneous and less characterized, the traditional and serrated pathways appear to be more homogeneous and clearly distinct. One unsolved question in colon cancer biology concerns the cells of origin and from which crypt compartment the different pathways originate. Based on molecular and pathological evidences, we propose that the traditional and serrated pathways originate from different crypt compartments explaining their genetic/epigenetic and clinicopathological differences. In this paper, we will discuss the current knowledge of CRC pathogenesis and, specifically, summarize the role of genetic/epigenetic changes in the origin and progression of the multiple CRC pathways. Elucidation of the link between the molecular and clinico-pathological aspects of CRC would improve our understanding of its etiology and impact both prevention and treatment.

Reduced β-catenin and peroxisome proliferator–activated receptor–γ expression levels are associated with colorectal cancer metastatic progression: correlation with tumor-associated macrophages, cyclooxygenase 2, and patient outcome

Recent studies have reported cross talk between beta-catenin, peroxisome proliferator-activated receptor-gamma, 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 beta-catenin, peroxisome proliferator-activated receptor-gamma, cyclooxygenase 2, and nuclear factor kappaB levels by immunohistochemistry and Western blot. The expression profiles were correlated with patient outcome and 5-year survival. Nuclear beta-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-gamma expression (70.8%) were significantly associated with a favorable prognosis (P < .001). Remarkably, reduction or loss of beta-catenin (22.2%) and peroxisome proliferator-activated receptor-gamma (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, beta-catenin and peroxisome proliferator-activated receptor-gamma levels were inversely correlated with cyclooxygenase 2 (P < .01) and nuclear factor kappaB expression (P < .05). Our results suggest that reduced expression of beta-catenin and peroxisome proliferator-activated receptor-gamma 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.

UHRF1 coordinates peroxisome proliferator activated receptor gamma (PPARG) epigenetic silencing and mediates colorectal cancer progression

Peroxisome proliferator-activated receptor gamma (PPARG) inactivation has been identified as an important step in colorectal cancer (CRC) progression, although the events involved have been partially clarified. UHRF1 is emerging as a cofactor that coordinates the epigenetic silencing of tumor suppressor genes, but its role in CRC remains elusive. Here, we report that UHRF1 negatively regulates PPARG and is associated with a higher proliferative, clonogenic and migration potential. Consistently, UHRF1 ectopic expression induces PPARG repression through its recruitment on the PPARG promoter fostering DNA methylation and histone repressive modifications. In agreement, UHRF1 knockdown elicits PPARG re-activation, accompanied by positive histone marks and DNA demethylation, corroborating its role in PPARG silencing. UHRF1 overexpression, as well as PPARG-silencing, imparts higher growth rate and phenotypic features resembling those occurring in the epithelial-mesenchymal transition. In our series of 110 sporadic CRCs, high UHRF1-expressing tumors are characterized by an undifferentiated phenotype, higher proliferation rate and poor clinical outcome only in advanced stages III–IV. In addition, the inverse relationship with PPARG found in vitro is detected in vivo and UHRF1 prognostic significance appears closely related to PPARG low expression, as remarkably validated in an independent dataset. The results demonstrate that UHRF1 regulates PPARG silencing and both genes appear to be part of a complex regulatory network. These findings suggest that the relationship between UHRF1 and PPARG may have a relevant role in CRC progression.

Immune escape mechanisms in colorectal cancer pathogenesis and liver metastasis.

Over the past decade, growing evidence indicates that the tumor microenvironment (TME) contributes with genomic/epigenomic aberrations of malignant cells to enhance cancer cells survival, invasion, and dissemination. Many factors, produced or de novo synthesized by immune, stromal, or malignant cells, acting in a paracrine and autocrine fashion, remodel TME and the adaptive immune response culminating in metastasis. Taking into account the recent accomplishments in the field of immune oncology and using metastatic colorectal cancer (mCRC) as a model, we propose that the evasion of the immune surveillance and metastatic spread can be achieved through a number of mechanisms that include (a) intrinsic plasticity and adaptability of immune and malignant cells to paracrine and autocrine stimuli or genotoxic stresses; (b) alteration of positional schemes of myeloid-lineage cells, produced by factors controlling the balance between tumour-suppressing and tumour-promoting activities; (c) acquisition by cancer cells of aberrant immune-phenotypic traits (NT5E/CD73, CD68, and CD163) that enhance the interactions among TME components through the production of immune-suppressive mediators. These properties may represent the driving force of metastatic progression and thus clinically exploitable for cancer prevention and therapy. In this review we summarize results and suggest new hypotheses that favour the growing impact of tumor-infiltrating immune cells on tumour progression, metastasis, and therapy resistance.

Emerging role of the β-catenin-PPARγ axis in the pathogenesis of colorectal cancer.

Multiple lines of evidence indicate that Wnt/β-catenin signaling plays a fundamental role in colorectal cancer (CRC) initiation and progression. Recent genome-wide data have confirmed that in CRC this pathway is one of the most frequently modified by genetic or epigenetic alterations affecting almost 90% of Wnt/β-catenin gene members. A major challenge is thus learning how the corrupted coordination of this pathway is tied to other signalings to enhance cell growth. Peroxisome proliferator activated receptor γ (PPARγ) is emerging as a growth-limiting and differentiation-promoting factor. In tumorigenesis it exerts a tumor suppressor role and is potentially linked with the Wnt/β-catenin pathway. Based on these results, the identification of new selective PPARγ modulators with inhibitory effects on the Wnt/β-catenin pathway is becoming an interesting perspective. Should, in fact, these molecules display such properties, new research avenues would be opened aimed at developing new molecular targeted drugs. Herein, we review the basic principles and present new hypotheses underlying the crosstalk between Wnt/β-catenin and PPARγ signaling. Furthermore, we discuss the advances in our understanding as to how their altered regulation can culminate in colon cancer and the efforts aimed at designing novel PPARγ agonists endowed with Wnt/β-catenin inhibitory effects to be used as therapeutic and/or preventive agents.

CDKN1B V109G polymorphism a new prognostic factor in sporadic medullary thyroid carcinoma

CONTEXT CDKN1B encodes the cyclin-dependent kinase inhibitor p27Kip1 and is mutated in multiple endocrine neoplasia-like syndromes. CDKN1B also harbors single nucleotide polymorphisms; the T/G transversion at nucleotide 326 (the V109G variant) has been reported to be protective in breast, hereditary prostate, and pancreatic tumors. Association of CDNK1B mutations or polymorphisms with sporadic medullary thyroid carcinoma (MTC) has not been investigated yet. OBJECTIVE AND DESIGN We screened germline DNA from 84 patients affected by sporadic MTC and 90 healthy age- and gender-matched controls for CDKN1B mutations or polymorphisms by PCR amplification and sequencing of the amplicons. We also tested all germline and 50 tumor tissue DNA for RET proto-oncogene mutations. Computed tomography, ultrasound scans, and serum calcitonin were carried out before surgery and during the follow-up and associated with CDKN1B polymorphism and disease remission. RESULTS The T/G transversion at nucleotide 326 was the only DNA variation detected. The overall frequency of the T/G and G/G alleles in combination was 46.4%. This variant (V109G) was correlated with post-operative calcitonin levels in the normal range and biochemical remission. Conversely, the wild-type (T/T) allele was associated with post-operative calcitonin levels above normal and a higher risk to develop clinical recurrence and distant metastases. Somatic RET mutations were significantly associated with a more aggressive behavior especially in wild-type allele-bearing patients. CONCLUSIONS Collectively, in sporadic MTC, the CDKN1B V109G polymorphism correlates with a more favorable disease progression than the wild-type allele and might be considered a new promising prognostic marker.

Targeting Angiogenesis and Tumor Microenvironment in Metastatic Colorectal Cancer: Role of Aflibercept

In the last decades, we have progressively observed an improvement in therapeutic options for metastatic colorectal cancer (mCRC) treatment with a progressive prolongation of survival. mCRC prognosis still remains poor with low percentage of 5-year survival. Targeted agents have improved results obtained with standard chemotherapy. Angiogenesis plays a crucial role in colorectal cancer growth, proliferation, and metastasization and it has been investigated as a potential target for mCRC treatment. Accordingly, novel antiangiogenic targeted agents bevacizumab, regorafenib, and aflibercept have been approved for mCRC treatment as the result of several phase III randomized trials. The development of a tumor permissive microenvironment via the aberrant expression by tumor cells of paracrine factors alters the tumor-stroma interactions inducing an expansion of proangiogenic signals. Recently, the VELOUR study showed that addition of aflibercept to FOLFIRI regimen as a second-line therapy for mCRC improved significantly OS, PFS, and RR. This molecule represents a valid second-line therapeutic option and its peculiar ability to interfere with placental growth factor (PlGF)/vascular endothelial growth factor receptor 1 (VEGFR1) axis makes it effective in targeting angiogenesis, inflammatory cells and in overcoming resistances to anti-angiogenic first-line treatment. Here, we discuss about Aflibercept peculiar ability to interfere with tumor microenvironment and angiogenic pathway.

Systems biology analysis reveals NFAT5 as a novel biomarker and master regulator of inflammatory breast cancer.

BackgroundInflammatory breast cancer (IBC) is the most rare and aggressive variant of breast cancer (BC); however, only a limited number of specific gene signatures with low generalization abilities are available and few reliable biomarkers are helpful to improve IBC classification into a molecularly distinct phenotype. We applied a network-based strategy to gain insight into master regulators (MRs) linked to IBC pathogenesis.MethodsIn-silico modeling and Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) on IBC/non-IBC (nIBC) gene expression data (n = 197) was employed to identify novel master regulators connected to the IBC phenotype. Pathway enrichment analysis was used to characterize predicted targets of candidate genes. The expression pattern of the most significant MRs was then evaluated by immunohistochemistry (IHC) in two independent cohorts of IBCs (n = 39) and nIBCs (n = 82) and normal breast tissues (n = 15) spotted on tissue microarrays. The staining pattern of non-neoplastic mammary epithelial cells was used as a normal control.ResultsUsing in-silico modeling of network-based strategy, we identified three top enriched MRs (NFAT5, CTNNB1 or β-catenin, and MGA) strongly linked to the IBC phenotype. By IHC assays, we found that IBC patients displayed a higher number of NFAT5-positive cases than nIBC (69.2% vs. 19.5%; p-value = 2.79 10-7). Accordingly, the majority of NFAT5-positive IBC samples revealed an aberrant nuclear expression in comparison with nIBC samples (70% vs. 12.5%; p-value = 0.000797). NFAT5 nuclear accumulation occurs regardless of WNT/β-catenin activated signaling in a substantial portion of IBCs, suggesting that NFAT5 pathway activation may have a relevant role in IBC pathogenesis. Accordingly, cytoplasmic NFAT5 and membranous β-catenin expression were preferentially linked to nIBC, accounting for the better prognosis of this phenotype.ConclusionsWe provide evidence that NFAT-signaling pathway activation could help to identify aggressive forms of BC and potentially be a guide to assignment of phenotype-specific therapeutic agents. The NFAT5 transcription factor might be developed into routine clinical practice as a putative biomarker of IBC phenotype.

Ensemble of Gene Signatures Identifies Novel Biomarkers in Colorectal Cancer Activated through PPARγ and TNFα Signaling

We describe a novel bioinformatic and translational pathology approach, gene Signature Finder Algorithm (gSFA) to identify biomarkers associated with Colorectal Cancer (CRC) survival. Here a robust set of CRC markers is selected by an ensemble method. By using a dataset of 232 gene expression profiles, gSFA discovers 16 highly significant small gene signatures. Analysis of dichotomies generated by the signatures results in a set of 133 samples stably classified in good prognosis group and 56 samples in poor prognosis group, whereas 43 remain unreliably classified. AKAP12, DCBLD2, NT5E and SPON1 are particularly represented in the signatures and selected for validation in vivo on two independent patients cohorts comprising 140 tumor tissues and 60 matched normal tissues. Their expression and regulatory programs are investigated in vitro. We show that the coupled expression of NT5E and DCBLD2 robustly stratifies our patients in two groups (one of which with 100% survival at five years). We show that NT5E is a target of the TNF-α signaling in vitro; the tumor suppressor PPARγ acts as a novel NT5E antagonist that positively and concomitantly regulates DCBLD2 in a cancer cell context-dependent manner.

Right-sided rhabdoid colorectal tumors might be related to the Serrated Pathway

BackgroundRhabdoid 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 methodsTo 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.ResultsLoss 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/β-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.ConclusionsRight-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.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1641385210804556