CD66 and CD49f expressing cells are associated with distinct neoplastic phenotypes and progression in human cervical cancer.

BACKGROUND: In this study, building on our recent work identifying a subset of CD66+ve cells with distinctive tumourigenic properties in human cervical cancers, we examine patterns of expression and function of these cells; to generate insights into the process of metastasis. METHODS: Our broad approach in this study has been to compare the expression and function of two subsets marked by CD66 and CD49f. We use a combination of histopathology, immunostaining and flow cytometry, functional analysis of an established cervical cancer cell line and a retrospective analysis of a cohort of cervical cancer. RESULTS: We noted CD66 expression associated with clusters of cells which are spindle shaped, SMA+ve, podoplanin+ve, phalloidin high, fibronectin high, plakoglobin low, ki67-ve and CK10+ve at the migratory phase along with features of partial EMT. Further, TGFß1 a well known regulator of EMT, positively correlated with CD66 expression. The additional CD49f+ve subset at the leading invading front of migration was SMA-ve, phalloidin low, fibronectin low, plakoglobin high, Ki67+ve and CK14+ve. These data are consistent with a role for CD66 cells in metastatic invasion with a collective cell migration process co-opting the CD49f subset. Our retrospective analysis of a cohort is consistent with a role for CD66 in metastasis. However, the broad analysis of CD66, CD49f and TGFß1 expression with patterns of overall survival points to a possible protective effect particularly for local recurrences. Hence, future studies focussing on potential heterogeneity within the CD66 subset along with the possible role of isoforms and intra-cellular roles would be essential.

Assessment of the radiation sensitivity of cervical cancer cell lines.

The aim of radiotherapy is to kill tumor cells in a primary tumor, in draining lymph nodes, and/or in small metastatic lesions. The response of tumor cells to radiation depends on the dose, an individual's radiosensitivity, the duration of radiation exposure (i.e., the timing), the fraction size, and the presence of other variables (e.g., chemotherapy). Sensitivity of the cells to radiation can be determined by cell proliferation and clonogenicity assays, which assess the ability of the cells to survive at low cell densities and to successfully initiate and sustain cell proliferation over time yielding viable colonies or clones after irradiation with a range of doses (0-10Gy). Apart from assessing the sensitivity of the cells to radiation, these assays are now being increasingly used to test for the effects of drugs/genes on the growth and proliferative characteristics of cells in vitro. Additionally, they are being used to determine the combinatorial effect of novel agents or inhibitors, which can modify the response to radiation for a favorable therapeutic outcome. The rates of cell survival and proliferation obtained from these assays help in identifying the most sensitive and resistant cell lines among particular cancer types. Because of their wide range of application, from identifying the most sensitive and resistant cell lines, to evaluating novel therapeutic agents, we describe here the basic steps involved in assessing the radiosensitivity of cervical cancer cell lines.

Dominant negative Ubiquitin-conjugating enzyme E2C sensitizes cervical cancer cells to radiation.

PURPOSE: To find the radiation sensitivity of human cervical carcinoma cell lines and to investigate the effect of the dominant negative-Ubiquitin-conjugating enzyme E2C (DN-UBE2C) on cell proliferation and radiation response. MATERIALS AND METHODS: Radiation sensitivities of human cervical cell lines (SiHa, HeLa, BU25TK, ME 180, and C33A) were analyzed by assessing their cell survival after irradiation by MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Soft agar cloning assay, growth curve and radiation response of DN-UBE2C stably transfected SiHa and HeLa cell lines were assessed by MTS assay and Clonogenic assay. RESULTS: Difference in sensitivity to radiation was observed among the cervical cancer cell lines studied. SiHa was found to be the most resistant cell line whereas C33A cells were the most sensitive. The growth rate of SiHa and HeLa transfected with DN-UBE2C was significantly reduced compared to vector control. Furthermore, DN-UBE2C-mediated radiosensitivity was correlated with a significant decrease in resistance to radiation by SiHa and HeLa cells after transfection with the DN-UBE2C when compared to control cultures. CONCLUSION: These results suggested that the Ubiquitin-conjugating enzyme E2C (UBE2C) gene is a potential therapeutic target for cervical cancer treatment.

Identification and validation of genes involved in cervical tumourigenesis.

BACKGROUND: Cervical cancer is the most common cancer among Indian women. This cancer has well defined pre-cancerous stages and evolves over 10-15 years or more. This study was undertaken to identify differentially expressed genes between normal, dysplastic and invasive cervical cancer. MATERIALS AND METHODS: A total of 28 invasive cervical cancers, 4 CIN3/CIS, 4 CIN1/CIN2 and 5 Normal cervix samples were studied. We have used microarray technique followed by validation of the significant genes by relative quantitation using Taqman Low Density Array Real Time PCR. Immunohistochemistry was used to study the protein expression of MMP3, UBE2C and p16 in normal, dysplasia and cancers of the cervix. The effect of a dominant negative UBE2C on the growth of the SiHa cells was assessed using a MTT assay. RESULTS: Our study, for the first time, has identified 20 genes to be up-regulated and 14 down-regulated in cervical cancers and 5 up-regulated in CIN3. In addition, 26 genes identified by other studies, as to playing a role in cervical cancer, were also confirmed in our study. UBE2C, CCNB1, CCNB2, PLOD2, NUP210, MELK, CDC20 genes were overexpressed in tumours and in CIN3/CIS relative to both Normal and CIN1/CIN2, suggesting that they could have a role to play in the early phase of tumorigenesis. IL8, INDO, ISG15, ISG20, AGRN, DTXL, MMP1, MMP3, CCL18, TOP2A AND STAT1 were found to be upregulated in tumours. Using Immunohistochemistry, we showed over-expression of MMP3, UBE2C and p16 in cancers compared to normal cervical epithelium and varying grades of dysplasia. A dominant negative UBE2C was found to produce growth inhibition in SiHa cells, which over-expresses UBE2C 4 fold more than HEK293 cells. CONCLUSIONS: Several novel genes were found to be differentially expressed in cervical cancer. MMP3, UBE2C and p16 protein overexpression in cervical cancers was confirmed by immunohistochemistry. These will need to be validated further in a larger series of samples. UBE2C could be evaluated further to assess its potential as a therapeutic target in cervical cancer.

A 7 gene expression score predicts for radiation response in cancer cervix.

BACKGROUND: Cervical cancer is the most common cancer among Indian women. The current recommendations are to treat the stage IIB, IIIA, IIIB and IVA with radical radiotherapy and weekly cisplatin based chemotherapy. However, Radiotherapy alone can help cure more than 60% of stage IIB and up to 40% of stage IIIB patients. METHODS: Archival RNA samples from 15 patients who had achieved complete remission and stayed disease free for more than 36 months (No Evidence of Disease or NED group) and 10 patients who had failed radical radiotherapy (Failed group) were included in the study. The RNA were amplified, labelled and hybridized to Stanford microarray chips and analyzed using BRB Array Tools software and Significance Analysis of Microarray (SAM) analysis. 20 genes were selected for further validation using Relative Quantitation (RQ) Taqman assay in a Taqman Low-Density Array (TLDA) format. The RQ value was calculated, using each of the NED sample once as a calibrator. A scoring system was developed based on the RQ value for the genes. RESULTS: Using a seven gene based scoring system, it was possible to distinguish between the tumours which were likely to respond to the radiotherapy and those likely to fail. The mean score +/- 2 SE (standard error of mean) was used and at a cut-off score of greater than 5.60, the sensitivity, specificity, Positive predictive value (PPV) and Negative predictive value (NPV) were 0.64, 1.0, 1.0, 0.67, respectively, for the low risk group. CONCLUSION: We have identified a 7 gene signature which could help identify patients with cervical cancer who can be treated with radiotherapy alone. However, this needs to be validated in a larger patient population.