Human Colorectal Cancer Tissue Samples Research Articles

A Necessary Role for Cyclin D2 Induction During Colon Cancer Progression Mediated by L1

The cell adhesion molecule L1CAM (L1), mainly known for its function in brain cells, is a Wnt/β-catenin signaling target gene in colorectal cancer (CRC) cells, where it promotes invasion and liver metastasis. We interrogated which genes are expressed at increased levels in human CRC tissue and induced in CRC cell lines overexpressing L1. We found increased cyclin D2 levels in CRC tissue and LS 174T and HCT 116 human CRC cells overexpressing L1. Increased cyclin D2 in CRC cells was associated with higher proliferation rates, faster motility, tumorigenesis, and liver metastasis. The suppression of cyclin D2 expression by shRNA to cyclin D2 blocked the increase in these cellular properties of L1-expressing cells. The overexpression of cyclin D2 in the absence of L1 also conferred tumorigenic properties similar to L1 expression. The pathways involved in the elevation of cyclin D2 by L1 include NF-κB, Akt, and β-catenin signaling but not the Erk pathway. We found that in a significant percentage of human CRC tissue samples, cyclin D2 is expressed at high levels in the nuclei of cancer cells. At the same time, the adjacent normal mucosa was negative for cyclin D2 staining. The results suggest that the increased cyclin D2 expression by L1 is required to induce proliferative, motile tumor development in CRC tissue and can serve as a diagnostic marker and a target for CRC therapy.

Circ_0060967 contributes to colorectal cancer progression by sponging miR-1184 to up-regulate SRC proto-oncogene

Background and study aimsCircular RNAs (circRNAs) are closely associated with cancer pathogenesis. The purpose of our current study was to explore the role and mechanism of circ_0060967 in colorectal cancer (CRC) development. Patients and methodsHuman CRC specimens and paired healthy tissues were used to examine variable expression. The expression of circ_0060967 and microRNA (miR)-1184 was examined by quantitative reverse transcription-PCR. The protein levels of proliferating cell nuclear antigen, BCL2-associated X, apoptosis regulator (Bax), proto-oncogene nonreceptor tyrosine kinase Src (SRC), nuclear factor-κB inhibitor alpha (IκBα), phosphorylated-IκBα (p-IκBα), RELA proto-oncogene, nuclear factor-κB subunit (p65), and phosphorylated-p65 (p-p65) were determined by western blot. Proliferation and motility of HCT-116 and SW480 CRC cells were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) and transwell assays, respectively. Dual-luciferase reporter assay and RNA immunoprecipitation assay were used to determine the binding relation between miR-1184 and circ_0060967 or SRC. Animal studies were used to detect the role of circ_0060967 in CRC cell tumorigenicity. ResultsCirc_0060967 abundance was enhanced in human CRC tissue samples versus paired normal colorectal tissues and in HCT-116 and SW480 CRC cells versus normal HCO cells. Decreased expression of circ_0060967 could suppress cell growth, motility, and invasiveness of CRC cells in vitro and tumor growth in vivo. Circ_0060967 sponged miR-1184, and miR-1184 targeted SRC. Furthermore, we also found circ_0060967 affected cell growth by modulating miR-1184/SRC axis in CRC. ConclusionThis study demonstrates a novel circ_0060967/miR-1184/SRC regulatory cascade in affecting CRC cell malignant behaviors, which can have a broad effect on the field of molecularly targeted therapeutics.

Downregulation of the Tumor Suppressor TFF1 Is Required during Induction of Colon Cancer Progression by L1.

Simple SummaryAberrant activation of Wnt/β-catenin signaling and the subsequent induction of downstream target genes is a hallmark of colorectal cancer (CRC) development. Previously, we found that overexpression of the immunoglobulin-like cell adhesion receptor L1CAM (L1), a target of the Wnt/β-catenin pathway, confers enhanced proliferation, motility, tumorigenesis, and liver metastasis in CRC cells. Transcriptomic and proteomic analyses revealed changes in both pro-tumorigenic and potential tumor-suppressor genes in L1-overexpressing CRC cells. We wished to identify such tumor suppressor/s, and found that trefoil family factor 1 (TFF1) was involved in L1-mediated CRC progression. TFF1 overexpression suppressed the growth, motility and tumorigenesis of L1-expressing CRC cells by inhibiting the NF-κB pathway. In human CRC tissue, TFF1-positive staining was evident in goblet cells of the normal mucosa, while in CRC tissue, TFF1 expression was lost in >50% of the tumor samples. Our results support a tumor-suppressor role of TFF1 in human CRC, and we suggest that TFF1 could be used for CRC detection and as a novel therapeutic target in L1-mediated CRC.The immunoglobulin family cell adhesion receptor L1 is induced in CRC cells at the invasive front of the tumor tissue, and confers enhanced proliferation, motility, tumorigenesis, and liver metastasis. To identify putative tumor suppressors whose expression is downregulated in L1-expressing CRC cells, we blocked the L1–ezrin–NF-κB signaling pathway and searched for genes induced under these conditions. We found that TFF1, a protein involved in protecting the mucus epithelial layer of the colon, is downregulated in L1-expressing cells and displays characteristics of a tumor suppressor. Overexpression of TFF1 in L1-transfected human CRC cells blocks the pro-tumorigenic and metastatic properties conferred by L1 by suppressing NF-κB signaling. Immunohistochemical analyses revealed that human CRC tissue samples often lose the expression of TFF1, while the normal mucosa displays TFF1 in goblet cells. Identifying TFF1 as a tumor suppressor in CRC cells could provide a novel marker for L1-mediated CRC development and a potential target for therapy.

Involvement of TMEM16A/ANO1 upregulation in the oncogenesis of colorectal cancer

The Ca2+-activated Cl− channel ANO1 is widely expressed in epithelial cells, and ANO1 upregulation is implicated in the oncogenesis of many epithelium-originated cancers. However, whether ANO1 plays a causal role in the tumorigenesis of colorectal cancer remains largely unknown. Here, we show that ANO1 channel protein is upregulated in human colorectal cancer tissue samples and its upregulation is correlated with the TNM staging, histological type, pathological differentiation and poor prognosis. Knockdown or pharmacological inhibition of ANO1 suppresses colorectal cancer cell proliferation and induces cell apoptosis. Furthermore, ANO1 knockdown inhibits the growth of subcutaneous xenograft tumors implanted with colorectal cancer HT-29 cells in nude mice. Mechanically, knockdown of endogenous ANO1 inactivates the Wnt/β-catenin signaling through downregulating critical components, such as Frizzled protein 1, β-catenin and upregulating GSK3β. Taken together, our results demonstrate that ANO1 upregulation is involved in the tumorigenesis of colorectal cancer, and inhibition of ANO1 upregulation or inactivating downstream Wnt/β-catenin signaling may have therapeutic potential for colorectal cancer.

ZRANB1 enhances stem-cell-like features and accelerates tumor progression by regulating Sox9-mediated USP22/Wnt/β-catenin pathway in colorectal cancer

The pathogenesis of colorectal cancer (CRC) is a multistep process characterized by the accumulation of gene mutations and epigenetic alterations. Tumor necrosis factor receptor-associated factor-binding protein domain (ZRANB1) is a deubiquitinase that mediates tumor growth and metastasis by deubiquitinating target proteins. In this study, we examined the regulatory effects of ZRANB1 on the maintenance of cancer stem cell (CSC) properties and tumor growth in CRC. Human CRC tissue samples and matched normal tissues were collected for the analysis of ZRANB1 expression. ZRANB1 was upregulated in CRC human tissues and cell lines, and its expression was positively correlated with advanced tumor stage and poor survival of CRC patients. The overexpression of ZRANB1 also induced the expression of CSC markers in CRC cells. Then, a xenograft model was established by inoculating BALB/c mice with CRC cells. The upregulation of ZRANB1 promoted tumorigenesis in vivo. Sox9 is a transcription factor that acts as an oncogene in human cancers. ZRANB1 increased the stability of Sox9 in CRC cells by decelerating its ubiquitination. Further analysis revealed that Sox9 regulated the transcription activity of USP22 by binding to its promoter. Moreover, ZRANB1 enhances stem-cell-like features of CRC cells and activated the Wnt/β-catenin pathway through USP22. Our results highlighted the role of ZRANB1 as a molecular target for CRC treatment, which may contribute to the development of novel therapies with better efficacy.

MiR-26a enhances colorectal cancer cell growth by targeting RREB1 deacetylation to activate AKT-mediated glycolysis.

We previously reported the inhibitory effects of microRNA-26a (miR-26a) on the conversion of pyruvate to acetyl coenzyme A in glucose metabolism by directly targeting pyruvate dehydrogenase protein X component in colorectal cancer (CRC) cells (Chen B et al., BMC Cancer 2014). Here, using microRNA in situ hybridization, we confirmed that miR-26a levels were elevated in 77 human CRC tissue samples and further investigated the key miR-26a-mediated metabolic regulation elements and signaling pathways in CRC cells through quantitative proteomic dissection combined with cancer cell biology and biochemical loss-of-function analysis. We found that AKT transcription signaling was a target pathway via miR-26a-mediated deacetylation modification of Ras-responsive element-binding protein 1 (RREB1) at the Lys-60 residue. miR-26a improved the deacetylation level of RREB1, thus contributing to RREB1 binding to the AKT1 promoter to activate AKT transcription and its related signaling pathway in glycolysis. Moreover, miR-26a promoted CRC tumorigenesis in CRC cells and subcutaneous xenograft mice. Thus, miR-26a is a key regulator of CRC tumorigenesis that mediates the deacetylation modification of RREB1 to enhance AKT1 transcription and downstream target gene expression in glycolysis for CRC growth.

Nuclear translocation of ATG5 induces DNA mismatch repair deficiency (MMR‐D)/microsatellite instability (MSI) via interacting with Mis18α in colorectal cancer

It is well known that microsatellite instability-high (MSI-H) is associated with 5-fluorouracil (5-FU) resistance in colorectal cancer. MSI-H is the phenotype of DNA mismatch repair deficiency (MMR-D), mainly occurring due to hypermethylation of MLH1 promoter CpG island. However, the mechanisms of MMR-D/MSI-H are unclear. We aim to investigate the pathway of MMR-D/MSI-H involved in 5-FU resistance. Human colorectal cancer specimens were diagnosed for MSI-H by immunohistochemistry and western blotting. Proteome microarray interactome assay was performed to screen nuclear proteins interacting with ATG5. Nuclear ATG5 and ATG5-Mis18α overexpression were analysed in ATG5high colorectal cancer bearing mice. The methylation assay determined the hypermethylation of hMLH1 promoter CpG island in freshly isolated human colorectal cancer tissue samples and HT29atg5 and SW480atg5 cancer cells. In ATG5high colorectal cancer patients, 5-FU-based therapy resulted in nuclear translocation of ATG5, leading to MSI-H. Colorectal cancer in Atg5 Tg mice demonstrated 5-FU resistance, compared to Atg5+/- and WT mice. Proteome microarray assay identified Mis18α, a protein localized on the centromere and a source for methylation of the underlying chromatin, which responded to the translocated nuclear ATG5 leading to ATG5-Mis18α conjugate overexpression. This resulted in MLH1 deficiency due to hypermethylation of hMLH1 promoter CpG island, while the deletion of nuclear Mis18α failed to induce ATG5-Mis18α complex and MMR-D/MSI-H. Nuclear ATG5 resulted in MMR-D/MSI-H through its interaction with Mis18α in ATG5high colorectal cancer cells. We suggest that ATG5-Mis18α or Mis18α may be a therapeutic target for treating colorectal cancer.

YAP promotes the proliferation and migration of colorectal cancer cells through the Glut3/AMPK signaling pathway.

Yes-associated protein (YAP), as a major downstream effector in the Hippo signaling pathway, is considered as an oncogene in cancer. The present study aimed to investigate the potential role of YAP in the development and progression of colorectal cancer (CRC). The mRNA and protein expression levels of YAP in human CRC tissue samples and adjacent normal tissue were analyzed using public databases, as well as clinical samples. The potential roles of YAP and the underlying mechanism regulating the proliferation and migration of CRC cells were examined using genetic manipulation in vitro. The correlation between the expression of the YAP gene and epithelial-to-mesenchymal transition (EMT) markers was investigated in order to determine the mechanism underlying the observed effects of YAP. YAP mRNA expression levels were significantly upregulated in CRC tissue compared with in normal tissue, as determined using datasets obtained from Oncomine. Similarly, in clinical samples, the protein expression levels of YAP were significantly upregulated in CRC tissue samples compared with in normal tissue samples. YAP knockdown inhibited the proliferation and migration of CRC cells in vitro, whereas its overexpression resulted in the opposite effect. The expression levels of the YAP gene were positively correlated with those of EMT markers (such as vimentin and N-cadherin) and EMT-inducing transcription factors (such as Snail1, Slug and zinc finger E-box binding homeobox 1 and 2) in CRC samples from Gene Expression Profiling Interactive Analysis. Furthermore, YAP silencing increased the protein expression of E-cadherin and decreased that of vimentin in CRC cells. By contrast, the overexpression of YAP had the opposite effect. YAP promoted the glucose transporter 3 (Glut3)/AMP-activated protein kinase (AMPK) signaling pathway in CRC cells. In conclusion, YAP promoted the proliferation and migration of CRC cells, as well as the expression of EMT markers, possibly by regulating the Glut3/AMPK signaling pathway.

Stromal reaction inhibitor and immune-checkpoint inhibitor combination therapy attenuates excluded-type colorectal cancer in a mouse model.

Despite recent advances in cancer immunotherapy, the efficacy of colorectal cancer (CRC) immunotherapy regimens is limited. This study evaluated the combined effect of an anti-PD-1 antibody and a platelet-derived growth factor receptor inhibitor (imatinib) on CRC progression using an orthotopic transplanted mouse model that reproduced the three histological phenotypes of CRC (inflamed-, excluded-, and desert-type). The frequency of each of these phenotypes in 196 human CRC tissue samples was also evaluated. Excluded-type CRC had the highest frequency in human tissue samples. In the mouse model, imatinib suppressed stromal reaction and increased sensitivity to anti-PD-1 treatment in excluded-type CRC. Antitumor effect was observed in mice with excluded-type tumors only after concomitant administration of anti-PD-1 antibody and imatinib. Immunohistological analysis revealed a reduction in stromal volume and an increase in the number of CD8-positive T cells in the tumor nest following combination therapy. RNA sequencing revealed significant activation of immune-related pathways and suppression of stromal-related pathways in transplanted tumors treated with combination therapy compared with tumors treated with anti-PD-1 antibody monotherapy. This combination therapy may prove effective for CRC cases that are unresponsive to anti-PD-1 antibody monotherapy.

Integrin-Linked-Kinase Overexpression Is Implicated in Mechanisms of Genomic Instability in Human Colorectal Cancer.

Genomic instability is a hallmark of cancer cells contributing to tumor development and progression. Integrin-linked kinase (ILK) is a focal adhesion protein with well-established role in carcinogenesis. We have previously shown that ILK overexpression is critically implicated in human colorectal cancer (CRC) progression. In light of the recent findings that ILK regulates centrosomes and mitotic spindle formation, we aimed to determine its implication in mechanisms of genomic instability in human CRC. Association of ILK expression with markers of genomic instability (micronuclei formation, nucleus size, and intensity) was investigated in diploid human colon cancer cells HCT116 upon ectopic ILK overexpression, by immunofluorescence and in human CRC samples by Feulgen staining. We also evaluated the role of ILK in mitotic spindle formation, by immunofluorescence, in HCT116 cells upon inhibition and overexpression of ILK. Finally, we evaluated association of ILK overexpression with markers of DNA damage (p-H2AX, p-ATM/ATR) in human CRC tissue samples by immunohistochemistry and in ILK-overexpressing cells by immunofluorescence. We showed that ILK overexpression is associated with genomic instability markers in human colon cancer cells and tissues samples. Aberrant mitotic spindles were observed in cells treated with specific ILK inhibitor (QLT0267), while ILK-overexpressing cells failed to undergo nocodazole-induced mitotic arrest. ILK overexpression was also associated with markers of DNA damage in HCT116 cells and human CRC tissue samples. The above findings indicate that overexpression of ILK is implicated in mechanisms of genomic instability in CRC suggesting a novel role of this protein in cancer.

MiR-29a-5p/STAT3 Positive Feedback Loop Regulates TETs in Colitis-Associated Colorectal Cancer.

Interleukin (IL)-6/signal transducers and activators of transcription 3 (STAT3) signaling plays an important role in the development of colitis-associated colorectal cancer (CAC). The mechanism of CAC formation remains unclear, and the relationship between miRNAs and the IL-6/STAT3 signaling pathway in the development of CAC is not well understood. In this study, we investigated the relationship between miR-29a-5p and the IL-6/STAT3 signaling pathway in the development of CAC and alterations in 10-11 translocations (TETs) regulated by this network. miR-29a-5p was screened in a CAC mouse model by high-throughput microarray analysis and investigated in human colorectal cancer tissue samples and colon cell lines by quantitative reverse transcription polymerase chain reaction (Q-RTPCR). The expression of miR-29a and TETs was detected by Q-RTPCR, and the expression of STAT3/P-STAT3 and TET3 was detected via Western blot assay. The expression of TET1 and 5-hydroxymethylcytosine (5hmC) was detected through immunofluorescence. Our results showed that miR-29a-5p was significantly upregulated and was accompanied by STAT3 activation in the colon tissues of CAC mouse and human colorectal cancer tissues, as compared with normal colon tissues. In contrast, the levels of TETs and 5hmC were decreased. In vitro, overexpression of miR-29a-5p in colonic cell lines (HCT-116 and IEC-6) and RAW264.7 cells increased STAT3 expression, but decreased that of TET3, TET1, and 5hmC. miR-29a-5p downregulation in HCT-116 and IEC-6 cell lines could rescue the expression of STAT3 and TET3. Notably, STAT3 activation induced by IL-6 upregulated miR-29a-5p expression and reduced TET expression in vitro, although STAT3 inhibitor treatment downregulated miR-29a-5p expression, which was induced by IL-6. Our studies showed that tumor development occurred with inflammation. The miR-29a-5p/STAT3 signaling axis could play an important role in the development of CAC, and the miR-29a-5p/STAT3 positive feedback loop may amplify the effects of inflammation, lead to decreased levels of TET and 5hmC, and eventually lead to the development of CAC.

Comparative analysis of the bioenergetics of human adenocarcinoma Caco-2 cell line and postoperative tissue samples from colorectal cancer patients.

The aim of this work was to explore the key bioenergetic properties for mitochondrial respiration in the widely-used Caco-2 cell line and in human colorectal cancer (HCC) postoperational tissue samples. Oxygraphy and metabolic control analysis (MCA) were applied to estimate the function of oxidative phosphorylation in cultured Caco-2 cells and HCC tissue samples. The mitochondria of Caco-2 cells and HCC tissues displayed larger functional activity of respiratory complex (C)II compared with CI, whereas in normal colon tissue an inverse pattern in the ratio of CI to CII activity was observed. MCA showed that the respiration in Caco-2 and HCC tissue cells is regulated by different parts of electron transport chain. In HCC tissues, this control is performed essentially at the level of respiratory chain complexes I-IV, whereas in Caco-2 cells at the level of CIV (cytochrome c oxidase) and the ATP synthasome. The differences we found in the regulation of respiratory chain activity and glycose index could represent an adaptive response to distinct growth conditions; this highlights the importance of proper validation of results obtained from in-vitro models before their extrapolation to the more complex in-vivo systems.

Long non‑coding RNA 00152 functions as a competing endogenous RNA to regulate NRP1 expression by sponging with miRNA‑206 in colorectal cancer.

Colorectal cancer (CRC) is the third most common type of cancer; however, the molecular mechanisms underlying colorectal tumor metastasis and growth remain elusive. Recently, accumulating evidence has indicated that long non‑coding RNAs (lncRNAs) play a critical role in CRC progression and metastasis; however, the biological role and clinical significance of lncRNA 00152 (lnc00152) in CRC remains largely unknown. Thus, in this study, lnc00152 expression was measured in 80human CRC tissue samples, 40non‑cancerous tissue samples, and 3CRC cell lines (SW480, SW620 and LoVo) using RT‑qPCR. We examined the effects of lnc00152 on CRC cells following transfection with lnc00152 overexpression plasmid or respective siRNA invitro and invivo. Luciferase assays revealed the mechanism driving competitive endogenous RNA (ceRNA). We identified that lnc00152 was aberrantly overexpressed in colorectal tumors and cancer cells and that lnc00152 was modulated by miRNA‑206. lnc00152 overexpression enhanced the proliferative and invasive ability of CRC cells invitro, promoted tumor growth invivo, and was associated with the shorter overall survival of patients with CRC. In addition, lnc00152 overexpression promoted epithelial-mesenchymal transition(EMT) and increased neuropilin‑1 (NRP1) expression in the CRC cells. By contrast, lnc00152 silencing exerted a counteractive effect. Collectively, these findings demonstrate the critical role of lnc00152 in tumor growth and progression in CRC, and identify a novel therapeutic target associated with CRC development and progression.

In Silico Modeling of Immunotherapy and Stroma-Targeting Therapies in Human Colorectal Cancer.

Despite the fact that the local immunological microenvironment shapes the prognosis of colorectal cancer, immunotherapy has shown no benefit for the vast majority of colorectal cancer patients. A better understanding of the complex immunological interplay within the microenvironment is required. In this study, we utilized wet lab migration experiments and quantitative histological data of human colorectal cancer tissue samples (n = 20) including tumor cells, lymphocytes, stroma, and necrosis to generate a multiagent spatial model. The resulting data accurately reflected a wide range of situations of successful and failed immune surveillance. Validation of simulated tissue outcomes on an independent set of human colorectal cancer specimens (n = 37) revealed the model recapitulated the spatial layout typically found in human tumors. Stroma slowed down tumor growth in a lymphocyte-deprived environment but promoted immune escape in a lymphocyte-enriched environment. A subgroup of tumors with less stroma and high numbers of immune cells showed high rates of tumor control. These findings were validated using data from colorectal cancer patients (n = 261). Low-density stroma and high lymphocyte levels showed increased overall survival (hazard ratio 0.322, P = 0.0219) as compared with high stroma and high lymphocyte levels. To guide immunotherapy in colorectal cancer, simulation of immunotherapy in preestablished tumors showed that a complex landscape with optimal stroma permeabilization and immune cell activation is able to markedly increase therapy response in silico These results can help guide the rational design of complex therapeutic interventions, which target the colorectal cancer microenvironment. Cancer Res; 77(22); 6442-52. ©2017 AACR.

AF1q Mediates Tumor Progression in Colorectal Cancer by Regulating AKT Signaling.

The up-regulation of ALL1-fused gene from chromosome 1q (AF1q) is commonly seen in aggressive hematologic malignancies as well as in several solid tumor tissues. However, its expression and intrinsic function in human colorectal cancer (CRC) remains largely undefined. To explore the role of AF1q in human CRC progression, AF1q expression was analyzed in human CRC tissue samples and CRC cell lines. Clinical specimens revealed that AF1q was up-regulated in human CRC tissues, and that this up-regulation was associated with tumor metastasis and late tumor, lymph node, metastasis (TNM) stage. AF1q knockdown by shRNA inhibited tumor cell proliferation, migration, invasion, and epithelial-mesenchymal transition in vitro, as well as tumorigenesis and liver metastasis in vivo, whereas these effects were reversed following AF1q overexpression. These AF1q-mediated effects were modulated by the protein kinase B (AKT) signaling pathway, and inhibition of AKT signaling attenuated AF1q-induced tumor promotion. Thus, AF1q contributes to CRC tumorigenesis and progression through the activation of the AKT signaling pathway. AF1q might therefore serve as a promising new target in the treatment of CRC.

Analysis of the expression of SDF-1 splicing variants in human colorectal cancer and normal mucosa tissues.

C-X-C motif chemokine ligand 12 (CXCL12), also termed stromal cell-derived factor-1 (SDF-1) is a small protein 8-14 kDa in length that is expressed as six isoforms, consisting of SDF-1α, SDF-1β, SDF-1γ, SDF-1δ, SDF-1ε and SDF-1θ. All six isoforms are encoded by the single CXCL12 gene on chromosome 10. This gene regulates leukocyte trafficking and is variably expressed in a number of normal and cancer tissues. The potential role of the novel CXCL12 splice variants as components of the CXCR4 axis in cancer development is not fully understood. The present study aimed to analyze the expression profile of the various SDF-1 isoforms and SDF-1 polymorphisms, and the association with the clinicopathological features and overall survival of patients with colorectal cancer (CRC). SDF-1 polymorphism analysis was performed using restriction fragment length polymorphism (RFLP) analysis in 73 histologically confirmed human CRC tissue samples at various stages of disease. The expression pattern of the SDF-1 isoforms was analyzed by reverse transcription-polymerase chain reaction in 40 histologically confirmed human CRC tissue samples obtained at various stages of disease, as well as in matched adjacent normal mucosa samples. The presence of the CXCL12 gene polymorphism rs1801157 demonstrated an association with local progression of the primary tumor, as indicated by the T stage. The frequency of the GG genotype was slightly increased in patients with stage 3 and 4 tumors (78.0%) compared with the incidence of the GA/AA genotype (69.5%; P=0.067). The expression of SDF-1β was associated with the presence of metastases (P=0.0656) and the expression of SDF-1γ was significantly associated with tumor size (P=0.0423). The present study is the first to analyze the association between the expression profile of the chemokine CXCL12 splice variants in human CRC tissues and their clinical relevance. The present results reveal that the CXCL12 G801A polymorphism is a low-penetrance risk factor for the development of CRC, and was associated with the T stage. All six isoforms of SDF-1 were expressed in CRC tissues. The expression of SDF-1β was found to be associated with metastases and SDF-1γ appears to be a possible tumor marker for local tumor progression.

DNA hypermethylation and decreased mRNA expression of MAL, PRIMA1, PTGDR and SFRP1 in colorectal adenoma and cancer.

BackgroundColorectal cancer (CRC) development is accompanied by changes in expression for several genes; but the details of the underlying regulatory procesess remain unknown. Our aims were to assess the role of epigenetic processes in tumour formation and to identify characteristic DNA methylation and miRNA alterations in the colorectal adenoma-carcinoma sequence.MethodsWhole genome expression profiling was performed on colonic biopsy samples (49 healthy normal, 49 colorectal adenoma (AD), 49 CRC); on laser capture microdissected (LCM) epithelial and stromal cells from 6 CRC-normal adjacent tissue (NAT) samples pairs, and on demethylated human CRC cell lines using HGU133 Plus 2.0 microarrays (Affymetrix). Methylation status of genes with gradually altering expression along the AD-CRC sequence was further analysed on 10–10 macrodissected and 5–5 LCM samples from healthy colon, from adenoma and from CRC biopsy samples using bisulfite-sequencing PCR (BS-PCR) followed by pyrosequencing. In silico miRNA prediction for the selected genes was performed with miRWALK algorithm, miRNA expression was analysed on 3 CRC-NAT sample pairs and 3 adenoma tissue samples using the Human Panel I + II (Exiqon). SFRP1 immunohistochemistry experiments were performed.ResultsA set of transcripts (18 genes including MAL, SFRP1, SULT1A1, PRIMA1, PTGDR) showed decreasing expression (p < 0.01) in the biopsy samples along the adenoma-carcinoma sequence. Three of those (COL1A2, SFRP2, SOCS3) showed hypermethylation and THBS2 showed hypomethylation both in AD and in CRC samples compared to NAT, while BCL2, PRIMA1 and PTGDR showed hypermethylation only in the CRC group. miR-21 was found to be significantly (p < 0.01) upregulated in adenoma and tumour samples compared to the healthy colonic tissue controls and could explain the altered expression of genes for which DNA methylation changes do not appear to play role (e.g. BCL2, MAL, PTGS2). Demethylation treatment could upregulate gene expression of genes that were found to be hypermethylated in human CRC tissue samples. Decreasing protein levels of SFRP1 was also observed along the adenoma-carcinoma sequence.ConclusionHypermethylation of the selected markers (MAL, PRIMA1, PTGDR and SFRP1) can result in reduced gene expression and may contribute to the formation of colorectal cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1687-x) contains supplementary material, which is available to authorized users.

Abstract 3442: Ganetespib sensitizes colorectal cancer in vitro and in vivo to a clinically relevant regimen of radiation and chemotherapy

Abstract Purpose: We describe the efficacy of the second generation HSP90 inhibitor ganetespib in combination with radiation and chemotherapy against colorectal adenocarcinoma in vitro and in vivo. Methods and Materials: Ganetespib's effects on colorectal adenocarcinoma proliferation and ability to sensitize to radiation and/or chemotherapy were analyzed in the colorectal cell lines HCT-116 and HT-29. Ganetespib's ability to modulate apoptosis when combined with radiation and chemotherapy was characterized, and its effects on the AKT, ERK and PI3K pathways were investigated. Tumor growth delay of colorectal adenocarcinoma with ganetespib alone and in combination with radiation and chemotherapy was analyzed in xenografts. Lastly, immunohistochemical analyses of HSP90 and p-AKT levels where performed in paired normal and tumor colorectal tissue samples from 10 patients. Results: At the clinically achievable dose of 50 nM, ganetespib inhibited colorectal cancer cells proliferation (p&amp;lt;0.014), potentiated 5-FU chemotherapy and 2Gy of ionizing radiation (p&amp;lt;0.0001), increased pro-apoptotic signaling proteins, and decreased levels of p-AKT, PI3K, and p-ERK in vitro. Combinations of ganetespib, 5-FU, and radiation therapy exhibited synergistic efficacy in human xenograft models in vivo (p&amp;lt;0.0001). Finally, HSP90 and p-AKT were both co-overexpressed in human colorectal cancer tissue samples relative to normal colon tissue. Conclusion: Altogether, these data show the clinically relevant combination of ganetespib with chemotherapy and radiation is effective in colorectal adenocarcinoma in vitro and in vivo. We are currently evaluating ganetespib's safety with chemotherapy and radiation therapy in a phase I study of patients with localized colorectal adenocarcinoma. Citation Format: Ganji Purnachandra Nagaraju, Kirtesh Patel, Jing Wen, Roberto Diaz, El-Rayes F. Bassel. Ganetespib sensitizes colorectal cancer in vitro and in vivo to a clinically relevant regimen of radiation and chemotherapy. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3442. doi:10.1158/1538-7445.AM2015-3442

Mutations in MicroRNA Genes and Their Binding Sites are Infrequently Associated with Human Colorectal Cancer in the Kashmiri Population.

MicroRNAs are small non-coding RNAs, 19-24 nucleotides in length that bind to the 3'UTR of target mRNAs and thus regulate gene expression post transcriptionally. MiRNAs have been implicated in various biological and pathological processes. The binding of miRNAs to 3'UTR is crucial for regulating the mRNA level and hence protein expression. The complementarity between the miRNA and its target mRNA is critical for the outcome of the miRNA mediated translational regulation. Changes in the nucleotide sequence of either the miRNA or its target binding site can deregulate gene expression and hence lead to the development of various pathological conditions, including tumorigenesis. To determine whether sequence alterations in miRNA genes and their target sites in mRNAs are associated with the colorectal cancers, we screened two miRNA genes-Let-7c, mir-206 and selected miRNA binding regions on KRAS, TP53 and GJA1 3'UTR. This study was carried out on 60 human colorectal cancer tissue samples. Our sequencing results did not reveal any mutation/single-nucleotide polymorphism in either the miRNAs or the miRNA binding sites in any of the tumor samples. This data suggests that mutations/SNPs targeting miRNA genes or their binding sites in 3'-untranslated regions are infrequent events in the development of colorectal cancer in Kashmiri population.

Dipeptidase 1 (DPEP1) is a marker for the transition from low-grade to high-grade intraepithelial neoplasia and an adverse prognostic factor in colorectal cancer

Background:Colorectal cancer (CRC) is the second leading cause of cancer-related deaths worldwide. Improvements in the understanding of its molecular mechanism and the characterisation of CRC-specific biomarkers facilitating early detection are considered to increase overall survival.Methods:A meta-analysis of microarray and Serial Analysis of Gene Expression (SAGE) has been performed to identify differentially regulated genes in CRC. Dipeptidase 1 (DPEP1/MDP/RDP) and Syntenin-2 (SDCBP2/SITAC18) were found to be differentially expressed in tumour tissue compared with normal mucosa. Expression of DPEP1 was assessed in a validation set of 87 normal mucosa samples, 20 hyperplastic polyps, 46 CR adenomas with low- and high-grade intraepithelial neoplasia (IEN) and 217 well-documented CRCs by immunohistochemistry and partially by immunoblotting and real-time PCR.Results:Expression of DPEP1 was specifically increased in human CRC tissue samples compared with normal mucosa (P<0.0001, Mann–Whitney U-test), showing a striking upregulation in high-grade compared with low-grade IEN. Furthermore, high DPEP1 expression was found to strongly correlate with histological stage (P<0.0001, chi-square test) as well as localisation (P<0.0001, chi-square test) and has been recognised as an independent adverse prognostic factor, showing significant prognostic values with an ROC (receiver operating characteristic)-AUC of 0.9230.Conclusion:Dipeptidase 1 has been identified as an excellent marker of high-grade IEN and CRC, and may thus be applied for screening of early neoplastic lesions and for prognostic stratification.