Association between DNA damage response and repair genes and risk of invasive serous ovarian cancer.

BACKGROUND: We analyzed the association between 53 genes related to DNA repair and p53-mediated damage response and serous ovarian cancer risk using case-control data from the North Carolina Ovarian Cancer Study (NCOCS), a population-based, case-control study. METHODS/PRINCIPAL FINDINGS: The analysis was restricted to 364 invasive serous ovarian cancer cases and 761 controls of white, non-Hispanic race. Statistical analysis was two staged: a screen using marginal Bayes factors (BFs) for 484 SNPs and a modeling stage in which we calculated multivariate adjusted posterior probabilities of association for 77 SNPs that passed the screen. These probabilities were conditional on subject age at diagnosis/interview, batch, a DNA quality metric and genotypes of other SNPs and allowed for uncertainty in the genetic parameterizations of the SNPs and number of associated SNPs. Six SNPs had Bayes factors greater than 10 in favor of an association with invasive serous ovarian cancer. These included rs5762746 (median OR(odds ratio)(per allele) = 0.66; 95% credible interval (CI) = 0.44-1.00) and rs6005835 (median OR(per allele) = 0.69; 95% CI = 0.53-0.91) in CHEK2, rs2078486 (median OR(per allele) = 1.65; 95% CI = 1.21-2.25) and rs12951053 (median OR(per allele) = 1.65; 95% CI = 1.20-2.26) in TP53, rs411697 (median OR (rare homozygote) = 0.53; 95% CI = 0.35 - 0.79) in BACH1 and rs10131 (median OR( rare homozygote) = not estimable) in LIG4. The six most highly associated SNPs are either predicted to be functionally significant or are in LD with such a variant. The variants in TP53 were confirmed to be associated in a large follow-up study. CONCLUSIONS/SIGNIFICANCE: Based on our findings, further follow-up of the DNA repair and response pathways in a larger dataset is warranted to confirm these results.

Gelsolin induces colorectal tumor cell invasion via modulation of the urokinase-type plasminogen activator cascade

Gelsolin is a cytoskeletal protein which participates in actin filament dynamics and promotes cell motility and plasticity. Although initially regarded as a tumor suppressor, gelsolin expression in certain tumors correlates with poor prognosis and therapy-resistance. In vitro, gelsolin has anti-apoptotic and pro-migratory functions and is critical for invasion of some types of tumor cells. We found that gelsolin was highly expressed at tumor borders infiltrating into adjacent liver tissues, as examined by immunohistochemistry. Although gelsolin contributes to lamellipodia formation in migrating cells, the mechanisms by which it induces tumor invasion are unclear. Gelsolin's influence on the invasive activity of colorectal cancer cells was investigated using overexpression and small interfering RNA knockdown. We show that gelsolin is required for invasion of colorectal cancer cells through matrigel. Microarray analysis and quantitative PCR indicate that gelsolin overexpression induces the upregulation of invasion-promoting genes in colorectal cancer cells, including the matrix-degrading urokinase-type plasminogen activator (uPA). Conversely, gelsolin knockdown reduces uPA levels, as well as uPA secretion. The enhanced invasiveness of gelsolin-overexpressing cells was attenuated by treatment with function-blocking antibodies to either uPA or its receptor uPAR, indicating that uPA/uPAR activity is crucial for gelsolin-dependent invasion. In summary, our data reveals novel functions of gelsolin in colorectal tumor cell invasion through its modulation of the uPA/uPAR cascade, with potentially important roles in colorectal tumor dissemination to metastatic sites.

A morpho-molecular prognostic model for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the third common cause of cancer-related deaths and its prognostication is still suboptimal. The aim of this study was to establish a new prognostication algorithm for HCC.

Coexpressing shRNA with fluorescence tags for quantification of cell migration studies

Understanding migration of cells has many implications in human physiology; some examples include developmental biology, healing, immune responses and tissue remodeling. On the other hand, invasive migration by tumor cells is pathological and is a major cause of mortality amongst cancer sufferers. Cell migration assays have been widely used to quantify potentially metastatic genes. In recent years, the use of RNAi has significantly increased the tools available in cell migration research due to its specific gene targeting for knockdown. The inability to ensure 100% transfection/transduction efficiency reduces the sensitivity of cell migration assays because cells not successfully transfected/transduced with the RNAi are also included in the calculations. This study introduces a different experimental setup mathematically expressed in our named normalized relative infected cell count (N-RICC) that analyses cell migration assays by co-expressing retrovirally transduced shRNA with fluorescence tags from a single vector. Vectors transduced into cells are visible under fluorescence, thus alleviating the problems involved with transduction efficiency by individually identifying cells with targeted genes. Designed shRNAs were targeted against a list of potentially metastatic genes in a highly migratory breast cancer cell line model, MDA-MB-231. We have successfully applied N-RICC analysis to show greater sensitivity of integrin alpha5 (ITGA5) and Ras homologue A (RhoA) in cell metastasis over conventional methods in scratch-wound assays and migration chambers assays.

Combined genomic and phenotype screening reveals secretory factor SPINK1 as an invasion and survival factor associated with patient prognosis in breast cancer

Secretory factors that drive cancer progression are attractive immunotherapeutic targets. We used a whole-genome data-mining approach on multiple cohorts of breast tumours annotated for clinical outcomes to discover such factors. We identified Serine protease inhibitor Kazal-type 1 (SPINK1) to be associated with poor survival in estrogen receptor-positive (ER+) cases. Immunohistochemistry showed that SPINK1 was absent in normal breast, present in early and advanced tumours, and its expression correlated with poor survival in ER+ tumours. In ER- cases, the prognostic effect did not reach statistical significance. Forced expression and/or exposure to recombinant SPINK1 induced invasiveness without affecting cell proliferation. However, down-regulation of SPINK1 resulted in cell death. Further, SPINK1 overexpressing cells were resistant to drug-induced apoptosis due to reduced caspase-3 levels and high expression of Bcl2 and phospho-Bcl2 proteins. Intriguingly, these anti-apoptotic effects of SPINK1 were abrogated by mutations of its protease inhibition domain. Thus, SPINK1 affects multiple aggressive properties in breast cancer: survival, invasiveness and chemoresistance. Because SPINK1 effects are abrogated by neutralizing antibodies, we suggest that SPINK1 is a viable potential therapeutic target in breast cancer.

DEAD-box helicase DP103 defines metastatic potential of human breast cancers

Despite advancement in breast cancer treatment, 30% of patients with early breast cancers experience relapse with distant metastasis. It is a challenge to identify patients at risk for relapse; therefore, the identification of markers and therapeutic targets for metastatic breast cancers is imperative. Here, we identified DP103 as a biomarker and metastasis-driving oncogene in human breast cancers and determined that DP103 elevates matrix metallopeptidase 9 (MMP9) levels, which are associated with metastasis and invasion through activation of NF-κB. In turn, NF-κB signaling positively activated DP103 expression. Furthermore, DP103 enhanced TGF-β-activated kinase-1 (TAK1) phosphorylation of NF-κB-activating IκB kinase 2 (IKK2), leading to increased NF-κB activity. Reduction of DP103 expression in invasive breast cancer cells reduced phosphorylation of IKK2, abrogated NF-κB-mediated MMP9 expression, and impeded metastasis in a murine xenograft model. In breast cancer patient tissues, elevated levels of DP103 correlated with enhanced MMP9, reduced overall survival, and reduced survival after relapse. Together, these data indicate that a positive DP103/NF-κB feedback loop promotes constitutive NF-κB activation in invasive breast cancers and activation of this pathway is linked to cancer progression and the acquisition of chemotherapy resistance. Furthermore, our results suggest that DP103 has potential as a therapeutic target for breast cancer treatment.

AURKA overexpression accompanies dysregulation of DNA-damage response genes in invasive urothelial cell carcinoma

Invasive urothelial cell carcinoma (UCC) is characterized by increased chromosomal instability and follows an aggressive clinical course in contrast to non-invasive disease. To identify molecular processes that confer and maintain an aggressive malignant phenotype, we used a high-throughput genome-wide approach to interrogate a cohort of high and low clinical risk UCC tumors. Differential expression analyses highlighted cohesive dysregulation of critical genes involved in the G(2)/M checkpoint in aggressive UCC. Hierarchical clustering based on DNA Damage Response (DDR) genes separated tumors according to a pre-defined clinical risk phenotype. Using array-comparative genomic hybridization, we confirmed that the DDR was disrupted in tumors displaying high genomic instability. We identified DNA copy number gains at 20q13.2-q13.3 (AURKA locus) and determined that overexpression of AURKA accompanied dysregulation of DDR genes in high risk tumors. We postulated that DDR-deficient UCC tumors are advantaged by a selective pressure for AURKA associated override of M phase barriers and confirmed this in an independent tissue microarray series. This mechanism that enables cancer cells to maintain an aggressive phenotype forms a rationale for targeting AURKA as a therapeutic strategy in advanced stage UCC.

MT1-MMP regulates urothelial cell invasion via transcriptional regulation of Dickkopf-3

Membrane type-1 matrix metalloproteinase (MT1-MMP) is a zinc-binding endopeptidase, which plays a crucial role in tumour growth, invasion and metastasis. We have shown previously that MT1-MMP has higher expression levels in the human urothelial cell carcinoma (UCC) tissue. We show here that siRNA against MT1-MMP blocks invasion in UCC cell lines. Invasion is also blocked by broad-spectrum protease and MMP inhibitors including tissue inhibitor of metalloproteinase-1 and -2. Membrane type-1-MMP can also regulate transcription. We have used expression arrays to identify genes that are differentially transcribed when siRNA is used to suppress MT1-MMP expression. Upon MT1-MMP knockdown, Dickkopf-3 (DKK3) expression was highly upregulated. The stability of DKK3 mRNA was unaffected under these conditions, suggesting transcriptional regulation of DKK3 by MT1-MMP. Dickkopf-3 has been previously shown to inhibit invasion. We confirm that the overexpression of DKK3 leads to decreased invasive potential as well as delayed wound healing. We show for the first time that the effects of MT1-MMP on cell invasion are mediated in part through changes in DKK3 gene transcription.

Interleukin-8 and its receptor CXCR2 in the tumour microenvironment promote colon cancer growth, progression and metastasis

BACKGROUND: Colorectal cancer (CRC) is a leading cause of death in the United States. Increased level of interleukin-8 (IL-8) and CXCR2 on tumours and in the tumour microenvironment has been associated with CRC growth, progression and recurrence in patients. Here, we aimed to evaluate the effects of tissue microenvironment-encoded IL-8 and CXCR2 on colon cancer progression and metastasis.

METHODS: A novel immunodeficient, skin-specific IL-8-expressing transgenic model was generated to evaluate colon cancer growth and metastasis. Syngeneic mouse colon cancer cells were grafted in CXCR2 knockout (KO) mice to study the contribution of CXCR2 in the microenvironment to cancer growth.

RESULTS: Elevated levels of IL-8 in the serum and tumour microenvironment profoundly enhanced the growth of human and mouse colon cancer cells with increased peri-tumoural angiogenesis, and also promoted the extravasation of the cancer cells into the lung and liver. The tumour growth was inhibited in CXCR2 KO mice with significantly reduced tumour angiogenesis and increased tumour necrosis.

CONCLUSION: Increased expression of IL-8 in the tumour microenvironment enhanced colon cancer growth and metastasis. Moreover, the absence of its receptor CXCR2 in the tumour microenvironment prevented colon cancer cell growth. Together, our study demonstrates the critical roles of the tumour microenvironment-encoded IL-8/CXCR2 in colon cancer pathogenesis, validating the pathway as an important therapeutic target.

POU2F1 activity regulates HOXD10 and HOXD11 promoting a proliferative and invasive phenotype in head and neck cancer

HOX genes are master regulators of organ morphogenesis and cell differentiation during embryonic development, and continue to be expressed throughout post-natal life. To test the hypothesis that HOX genes are dysregulated in head and neck squamous cell carcinoma (HNSCC) we defined their expression profile, and investigated the function, transcriptional regulation and clinical relevance of a subset of highly expressed HOXD genes. Two HOXD genes, D10 and D11, showed strikingly high levels in HNSCC cell lines, patient tumor samples and publicly available datasets. Knockdown of HOXD10 in HNSCC cells caused decreased proliferation and invasion, whereas knockdown of HOXD11 reduced only invasion. POU2F1 consensus sequences were identified in the 5' DNA of HOXD10 and D11. Knockdown of POU2F1 significantly reduced expression of HOXD10 and D11 and inhibited HNSCC proliferation. Luciferase reporter constructs of the HOXD10 and D11 promoters confirmed that POU2F1 consensus binding sites are required for optimal promoter activity. Utilizing patient tumor samples a significant association was found between immunohistochemical staining of HOXD10 and both the overall and the disease-specific survival, adding further support that HOXD10 is dysregulated in head and neck cancer. Additional studies are now warranted to fully evaluate HOXD10 as a prognostic tool in head and neck cancers.

L'ultrasonographie endorectale dans les cancers du rectum [Endorectal ultrasound of rectal cancers].

Endorectal ultrasonography has become the preferred exam to assess the local extent of rectal cancers. From 1990 to 1992, we have examined 28 patients with a rectal cancer. The tumours were classified according to the TNM. The objective of this exam is to identify patients whose tumours have invaded the perirectal fat. These patients are first treated in our clinic by an accelerated hyperfractionated radiotherapy and then operated. The preoperative staging made with the endorectal ultrasound was then compared with the anatomopathologic staging. The depth of the invasion was assessed precisely in 78.5% of cases. The exam's sensitivity to detect the invasion of the perirectal fat was 96% and its specificity 75%. Lymph node involvement was accurately identified in 67.8% of cases with a sensitivity of 81% and a specificity of 50%. This short retrospective study confirms that endorectal ultrasonography is a highly accurate tool for the staging of rectal carcinoma prior to operation and hence to select the patients that can benefit from preoperative irradiation.

Expression of beta-human chorionic gonadotropin (beta-hCG) in non-trophoblastic elements of transitional cell carcinoma of the bladder: possible relationship with the prognosis.

Transitional cell carcinoma (TCC) of the bladder is a neoplasm with variability in its clinical behavior. Although there are several studies correlating stage and ABO isoantigen expression with invasiveness, there is no single predictor factor to assess the potential invasiveness, especially in the low grade, non-invasive TCC. In the present study we evaluated the correlation of histological grade plus stage and the expression of beta human chorionic gonadotropin (beta-hCG), in 100 cases of TCC, with the clinical behavior. These features were correlated with tumor progression in patients with at least two years of follow up. We observed more aggressiveness in G4 group (high grade and invasive) (93% had tumor progression) when compared to G1 group (low grade and superficial) (11% had tumor progression). However in 25.5% of the TCC cases (groups G2: low grade and invasive and G3: high grade and superficial) the clinical behavior was intermediate, showing some limitation in using grading and staging only, as a predictive factor. There was an expression of beta-hCG in 21.4% of the cases in up to 25% of the tumor cells without any trophoblastic morphology. These beta-hCG producing TCC had a strong correlation with aggressiveness: 39.1% and 12.8% of the TCC expressed beta-hCG with and without tumor progression, respectively.

Simple mucin-type carbohydrate antigens (T, sialosyl T, Tn and sialosyl-Tn) in breast carcinogenesis

Immunohistochemical analysis of the expression of simple mucin-type carbohydrate antigens (Tn, sialyl-Tn and T) was performed in a series of 43 cases of intraductal hyperplasia without atypia, 9 cases of intraductal hyperplasia with atypia, 54 cases of ductal carcinoma in situ (DCIS) and 26 cases of invasive breast carcinoma. We also studied 36 cases of isolated breast normal epithelium, 20 cases of 'normal' breast epithelium adjacent to neoplasms and 14 cases of apocrine metaplasia. All antigens were detected in different frequencies in normal, hyperplastic, metaplastic and neoplastic breast epithelium. Tn and sialyl-Tn are expressed more frequently in malignant than in benign breast epithelium; while Tn expression increases from normal to invasive carcinomas, sialyl-Tn increases until DCIS and drops in invasive carcinomas, suggesting that either there is a failure of a proportion of DCIS to progress to invasive carcinoma or loss of expression of sialyl-Tn when some carcinomas become invasive. The high frequency of Tn and sialyl-Tn expression in breast intraductal proliferations probably reflects incomplete glycosylation in these lesions, which is a well-known tumour-associated phenomenon and supports the assumption that such lesions are putative precursors of breast cancer. T antigen was expressed in all groups studied, but its prevalence differed significantly between normal and neoplastic epithelium. The expression of these antigens in epithelium adjacent to carcinomas is similar to that found in isolated normal breast epithelium, whereas apocrine metaplasia has a pattern of simple mucin-type glycosylation that is specific and distinct from that of the normal breast epithelium, with a high frequency of marked expression of Tn and sialyl-Tn. The similarity of the pattern of expression of simple mucin-type antigens in metaplasia and malignant neoplasia reduces the usefulness of these markers from a diagnostic standpoint.

Estudo comparativo dos métodos de ultra-sonografia, tomografia computadorizada e ressonância magnética no estadiamento e invasão das estruturas adjacentes por tumores renais.

BACKGROUND: Ultrasonography (US), Computed Tomography (CT), and Magnetic Resonance imaging (MR) were compared for the staging of renal tumors. The differences between these imaging techniques were also studied for their ability to detect adenopathies, vascular invasion, distant intra-abdominal metastases, and particularly adjacent organ invasion. METHODS: Thirty-one patients with solid or complex renal masses were prospectively studied using US, CT, and MR. Differences between the results obtained were studied using the COCHRAN G test and the McNEMAR test. The sensitivity and specificity of each diagnostic technique were compared against a gold standard of the surgical and histopathological findings. RESULTS: The following sensitivities were obtained: For the detection of adenopathy, US 63.6%, CT and MR 90.9%. For vascular invasion, US 42.8%, CT and MR 85.7%. For the adjacent organ invasion, US 28.5%, CT 85.7%, and MR 71.4%. Some of the criteria that suggest invasion of adjacent structures include: the envelopment of the adjacent structures by the tumor, tumor extension into the adjacent structures with an irregular appearance, and alterations in shape, size, and density of adjacent structures. Loss of fat planes between the tumor and adjacent structures is not a sign of tumor invasion. CONCLUSIONS: Significant differences were found in the detection capacity of US in relation to CT and MR, which were similar. All three techniques were highly sensitive and specific only in the detection of distant abdominal metastases. In addition to the accuracy of these diagnostic modalities for the detection and staging of tumors, invasiveness, risks and cost should be considered in relation to relative costs and benefits.

Melanoma de coróide: Relato de caso insuspeito

The choroidal melanoma is the most frequent intraocular tumor in elderly, occurring mainly in the 60th. This paper shows a misdiagnosis tumor affecting a female aging 36 years-old, misdiagnosed and initially treated as a toxoplasmic uveitis. The tumor had orbit and neck metastasis. The authors call attention to the necessity of early diagnosis to improve the outcome.