Patient-matched Normal Mucosa Research Articles

PO-146 Multiple myeloma-derived exosomes carry amphiregulin and are responsible for the uncoupled bone remodelling

IntroductionAnnexins are frequently deregulated in many cancers (‘annexinopathies’). Annexin A1 (ANXA1) overexpression has been reported in gastric, pancreatic and hepatocellular carcinoma; however, it is markedly down-regulated in breast, prostate, esophageal and head and neck cancers. The contrasting patterns of ANXA1 expression in different tumours types is just one of the enigmas in deciphering the underlying regulatory mechanisms and phenotypic specificity of ANXA1. We have previously demonstrated that ANXA1 down-regulation is an early and frequent event in the development of head and neck squamous cell carcinomas (HNSCC).Material and methodsIn an attempt to identify the underlying mechanisms of reduced ANXA1 protein expression, this study investigated ANXA1 protein expression and ANXA1 mRNA levels in HNSCC tissue specimens by immunofluoresce, in situ hybridization and RT-qPCR. Subsequently, the relative levels of miR-196a and miR-196b were quantified in paired HNSCC specimens using Taqman miRNA assays and compared to ANXA1 mRNA levels. Functional studies were performed in HNSCC cells by transfection with specific pre-miR precursors, and also ANXA1 promoter activity using deletion constructs from the 5’ ANXA1 promoter region.Results and discussionResults showed a perfect concordance between the pattern of ANXA1 mRNA and protein detected by immunofluorescence in tumours, precancerous lesions and normal epithelia, reflecting that ANXA1 down-regulation occurs at transcriptional level. We also found that both miR-196a and miR-196b levels inversely correlated with ANXA1 mRNA levels in paired HNSCC tissue samples and patient-matched normal mucosa. In addition, endogenous levels of ANXA1 mRNA and protein were consistently and significantly down-regulated upon miR-196a and miR-196b over-expression in various HNSCC-derived cell lines. The direct interaction of both mature miR-196a and miR-196b was further confirmed by transfection with Anxa1 3’UTR constructs. Combined bioinformatics and functional analysis of ANXA1 promoter activity contributed to identify key regions and potential mediators of ANXA1 transcriptional control.ConclusionThese findings demonstrate that ANXA1 down-regulation in head and neck squamous cell carcinomas occurs via transcriptional control with direct involvement of miR-196a/b.

Annexin A1 down-regulation in head and neck squamous cell carcinoma is mediated via transcriptional control with direct involvement of miR-196a/b

Annexin A1 (ANXA1) down-regulation is an early and frequent event in the development of head and neck squamous cell carcinomas (HNSCC). In an attempt to identify the underlying mechanisms of reduced ANXA1 protein expression, this study investigated ANXA1 mRNA expression in HNSCC specimens by both in situ hybridization and RT-qPCR. Results showed a perfect concordance between the pattern of ANXA1 mRNA and protein detected by immunofluorescence in tumors, precancerous lesions and normal epithelia, reflecting that ANXA1 down-regulation occurs at transcriptional level. We also found that both miR-196a and miR-196b levels inversely correlated with ANXA1 mRNA levels in paired HNSCC tissue samples and patient-matched normal mucosa. In addition, endogenous levels of ANXA1 mRNA and protein were consistently and significantly down-regulated upon miR-196a and miR-196b over-expression in various HNSCC-derived cell lines. The direct interaction of both mature miR-196a and miR-196b was further confirmed by transfection with Anxa1 3′UTR constructs. Combined bioinformatics and functional analysis of ANXA1 promoter activity contributed to identify key regions and potential mediators of ANXA1 transcriptional control. This study unveils that, in addition to miR-196a, miR-196b also directly targets ANXA1 in HNSCC.

OP17 Cystathionine-β-synthase (CBS) is upregulated in colorectal cancer cells and promotes their proliferation

OP17 Cystathionine-β-synthase (CBS) is upregulated in colorectal cancer cells and promotes their proliferation

Extensive telomere erosion in the initiation of colorectal adenomas and its association with chromosomal instability.

Telomere shortening, dysfunction, and fusion may facilitate the acquisition of large-scale genomic rearrangements, driving clonal evolution and tumor progression. The relative contribution that telomere dysfunction and/or APC mutation play in the chromosome instability that occurs during colorectal tumorigenesis is not clear. We used high-resolution telomere length and fusion analysis to analyze 85 adenomatous colorectal polyps obtained from 10 patients with familial adenomatous polyposis and a panel of 50 colorectal carcinomas with patient-matched normal colonic mucosa. Telomerase activity was determined using the telomeric repeat amplification protocol. Array-CGH was used to detect large-scale genomic rearrangements. Pearson correlation and Student t test were used, and all statistical tests were two-sided. Despite the presence of telomerase activity, we observed apparent telomere shortening in colorectal polyps that correlated with large-scale genomic rearrangements (P < .0001) but was independent of polyp size and indistinguishable from that observed in colorectal carcinomas (P = .82). We also observed apparent lengthening of telomeres in both polyps and carcinomas. The extensive differences in mean telomere length of up to 4.6kb between patient-matched normal mucosa and polyps were too large to be accounted for by replicative telomere erosion alone. Telomere fusion events were detected in both polyps and carcinomas; the mutational spectrum accompanying fusion was consistent with alternative nonhomologous end joining. Telomere length distributions observed in colorectal polyps reflect the telomere length composition of the normal originating cells from which clonal growth was initiated. Originating cells containing both short telomeres and APC mutations may give rise to polyps that exhibit short telomeres and are prone to telomere dysfunction, driving genomic instability and progression to malignancy. J Natl Cancer Inst;2013;105:1202-1211.

Quantitative Proteomic Profiling of Colorectal Cancer Stratified by Lymph Node and 8p LOH Status

Purpose: An objective of clinical proteomics, as applied to colorectal cancer (CRC), is to identify patients who are at greater risk of recurrence following initial resection and thus in need of more aggressive management. We applied sensitive proteomic technology to identify biomarkers that correlate with established prognostic indicators including lymph node status and LOH at chromosome 8p. Methods: We compared 48 tissue samples: 12 node-positive CRC, 12 node-negative CRC, and patient-matched normal mucosa. Laser capture microdissection (LCM) was used to isolate pure cell populations from standard frozen sections. About four micrograms of protein was analyzed by two-dimensional difference gel electrophoresis (2D-DIGE). About 200,000 data features were quantified, compared and analyzed using a Significance Analysis of Microarrays (SAM) procedure, which provides a list of candidate biomarkers ranked by priority. Mass spectrometry for molecular identification of candidate biomarkers is in progress. Results: More than 2100 features per gel have been resolved. We project that about 1000 features will be matched as common across 90% or more of the gel images. Interim analysis of 604 well-matched features shows 111 with highly significant changes between cancer and normal (based on SAM analysis with estimated false discovery rate of < 1%). A heat map in Fig. 1 shows an overview of the interim analysis. In addition, as compared between node positive and node negative samples, at least one feature has been identified to date. Characteristics of this and other candidate biomarkers will be discussed.[figure1]FigureConclusion: The combination of LCM and 2D-DIGE provides a comprehensive approach for proteomic analysis of very small amounts of CRC tissue obtained from standard clinical specimens.

Overexpression of the Cytokine Osteopontin Identifies Aggressive Laryngeal Squamous Cell Carcinomas and Enhances Carcinoma Cell Proliferation and Invasiveness

Osteopontin is a secreted cytokine that binds to the cell surface CD44v6 receptor. We studied osteopontin and CD44v6 expression in laryngeal squamous cell carcinomas and correlated osteopontin expression levels with clinicopathologic tumor features. We used immunohistochemistry, immunoblotting, and reverse transcription-PCR to study osteopontin expression in 58 laryngeal squamous cell carcinomas. Cultured squamous carcinoma cells were treated with exogenous osteopontin or with RNA interference to knockdown osteopontin expression. Osteopontin expression was higher in all the invasive carcinomas than in patient-matched normal mucosa. Its expression levels were significantly correlated with tumor stage and grade and with the presence of lymph node and distant metastases. Osteopontin positivity was negatively correlated with overall survival (P = 0.03). Osteopontin expression was paralleled by intense cell surface reactivity for CD44v6. Treatment of squamous carcinoma cells with recombinant osteopontin sharply increased proliferation and Matrigel invasion in comparison with the untreated cells parallel to activation of the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/mitogen-activated protein kinase signaling cascade. Osteopontin knockdown by RNA interference, anti-CD44 antibodies, and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibition prevented these effects. These results identify osteopontin as a marker and a potential therapeutic target in cases of aggressive laryngeal squamous cell carcinomas.

Unfavourable expression of pharmacologic markers in mucinous colorectal cancer

Patients with mucinous colorectal cancer generally have worse prognoses than those with the nonmucinous variety. The reason for this disparity is unclear, but may result from a differential response to adjuvant chemotherapy. We examined known molecular markers for response to common chemotherapy in these two histological subtypes. In all, 21 patients with mucinous and 30 with nonmucinous Dukes C colorectal cancer were reviewed for demographic data and outcome. Total RNA from the tumours and adjacent normal mucosa was isolated and reverse transcribed. Quantitative expression levels of drug pathway genes were determined using TaqMan RT–PCR (5-fluorouracil (5-FU): TYMS, DPYD, ECGF1; oxaliplatin: GSTP1 (glutathione S-transferase pi), ERCC1 and 2; irinotecan: ABCB1, ABCG2, CYP3A4, UGT1A1, CES2, TOP1). Mucinous tumours significantly overexpressed both TYMS and GSTP1 relative to nonmucinous tumours and patient-matched normal mucosa. No significant differences in expression of the remaining markers were found. Mean follow-up was 20 months; 17 patients had recurrent disease. Among patients receiving 5-FU, those with mucinous tumours experienced shorter disease-free survival (DFS) than those with nonmucinous tumours (median DFS 13.8 vs 46.5 months, P=0.053). Mucinous colorectal cancer overexpresses markers of resistance to 5-FU and oxaliplatin. Likewise, DFS may be decreased in patients with mucinous tumours who receive 5-FU. The presence of mucin should be carefully evaluated in developmental trials of new agents for treating colorectal cancer.

Unfavorable expression of pharmacologic markers in mucinous colorectal cancer (CRC)

Abstract Introduction: Patients with mucinous CRC generally have worse prognoses than those with the non-mucinous variety. The reason for this disparity is unclear but may result from a differential response to adjuvant chemotherapy. We examined known molecular markers for response to common chemotherapy in these two histologic subtypes. Methods: Medical records of surgically-treated patients with stage III CRC were reviewed for demographic data and outcome. Twenty-one patients with mucinous and thirty with non-mucinous tumors were matched for age, gender, tumor size, grade and location, and number of lymph node metastases. Total RNA from the tumors and adjacent normal mucosa was isolated and reverse transcribed. Quantitative expression levels of drug pathway genes were determined using TaqMan RT-PCR (5-FU: TYMS, DPYD, ECGF1; oxaliplatin: GSTP1, ERCC1 & 2; irinotecan: ABCB1, ABCG2, CYP3A4, UGT1A1, CES2, TOP1). Results: Mucinous tumors significantly overexpressed both TYMS and GSTP1 relative to non-mucinous tumors and patient-matched normal mucosa. No significant differences in expression of the remaining markers were found. Mean follow-up was 20 months; 17 patients had recurrent disease. Among patients receiving 5-FU, those with mucinous CRC experienced shorter disease-free survival than those with non-mucinous tumors (median DFS 13.8 vs. 46.5 months, p = 0.053). ∗ . Mucinous Non-mucinous Mucosa Tumor Mucosa Tumor TYMS 4.1 ± 0.4 17.7 ± 5.8 ∗† 4.9 ± 0.7 9.6 ± 2.3 ∗ GSTP1 60 ± 9.6 193 ± 40.4 ∗† 51 ± 7.1 139 ± 32.7 ∗ ∗ Denotes p † Denotes p Conclusions: Mucinous tumors overexpressed markers of resistance to 5-FU and oxaliplatin. Additionally, DFS was decreased in patients with mucinous CRC who were given 5-FU. Expression differences in genes influencing chemotherapeutic response may contribute to shorter times to recurrence in patients with mucinous tumors, suggesting that mucin staining should be evaluated in developmental trials for novel CRC treatments.

WISP genes are members of the connective tissue growth factor family that are up-regulated in wnt-1-transformed cells and aberrantly expressed in human colon tumors.

Wnt family members are critical to many developmental processes, and components of the Wnt signaling pathway have been linked to tumorigenesis in familial and sporadic colon carcinomas. Here we report the identification of two genes, WISP-1 and WISP-2, that are up-regulated in the mouse mammary epithelial cell line C57MG transformed by Wnt-1, but not by Wnt-4. Together with a third related gene, WISP-3, these proteins define a subfamily of the connective tissue growth factor family. Two distinct systems demonstrated WISP induction to be associated with the expression of Wnt-1. These included (i) C57MG cells infected with a Wnt-1 retroviral vector or expressing Wnt-1 under the control of a tetracyline repressible promoter, and (ii) Wnt-1 transgenic mice. The WISP-1 gene was localized to human chromosome 8q24.1-8q24.3. WISP-1 genomic DNA was amplified in colon cancer cell lines and in human colon tumors and its RNA overexpressed (2- to >30-fold) in 84% of the tumors examined compared with patient-matched normal mucosa. WISP-3 mapped to chromosome 6q22-6q23 and also was overexpressed (4- to >40-fold) in 63% of the colon tumors analyzed. In contrast, WISP-2 mapped to human chromosome 20q12-20q13 and its DNA was amplified, but RNA expression was reduced (2- to >30-fold) in 79% of the tumors. These results suggest that the WISP genes may be downstream of Wnt-1 signaling and that aberrant levels of WISP expression in colon cancer may play a role in colon tumorigenesis.