Direct repression of MYB by ZEB1 suppresses proliferation and epithelial gene expression during epithelial-to-mesenchymal transition of breast cancer cells

Introduction Epithelial-to-mesenchymal transition (EMT) promotes cell migration and is important in metastasis. Cellular proliferation is often downregulated during EMT, and the reverse transition (MET) in metastases appears to be required for restoration of proliferation in secondary tumors. We studied the interplay between EMT and proliferation control by MYB in breast cancer cells. Methods MYB, ZEB1, and CDH1 expression levels were manipulated by lentiviral small-hairpin RNA (shRNA)-mediated knockdown/overexpression, and verified with Western blotting, immunocytochemistry, and qRT-PCR. Proliferation was assessed with bromodeoxyuridine pulse labeling and flow cytometry, and sulforhodamine B assays. EMT was induced with epidermal growth factor for 9 days or by exposure to hypoxia (1% oxygen) for up to 5 days, and assessed with qRT-PCR, cell morphology, and colony morphology. Protein expression in human breast cancers was assessed with immunohistochemistry. ZEB1-MYB promoter binding and repression were determined with Chromatin Immunoprecipitation Assay and a luciferase reporter assay, respectively. Student paired t tests, Mann–Whitney, and repeated measures two-way ANOVA tests determined statistical significance (P < 0.05). Results Parental PMC42-ET cells displayed higher expression of ZEB1 and lower expression of MYB than did the PMC42-LA epithelial variant. Knockdown of ZEB1 in PMC42-ET and MDA-MB-231 cells caused increased expression of MYB and a transition to a more epithelial phenotype, which in PMC42-ET cells was coupled with increased proliferation. Indeed, we observed an inverse relation between MYB and ZEB1 expression in two in vitro EMT cell models, in matched human breast tumors and lymph node metastases, and in human breast cancer cell lines. Knockdown of MYB in PMC42-LA cells (MYBsh-LA) led to morphologic changes and protein expression consistent with an EMT. ZEB1 expression was raised in MYBsh-LA cells and significantly repressed in MYB-overexpressing MDA-MB-231 cells, which also showed reduced random migration and a shift from mesenchymal to epithelial colony morphology in two dimensional monolayer cultures. Finally, we detected binding of ZEB1 to MYB promoter in PMC42-ET cells, and ZEB1 overexpression repressed MYB promoter activity. Conclusions This work identifies ZEB1 as a transcriptional repressor of MYB and suggests a reciprocal MYB-ZEB1 repressive relation, providing a mechanism through which proliferation and the epithelial phenotype may be coordinately modulated in breast cancer cells.

LCC15-MB : a vimentin-positive human breast cancer cell line from a femoral bone metastasis

The LCC15-MB cell line was established from a femoral bone metastasis that arose in a 29-year-old woman initially diagnosed with an infiltrating ductal mammary adenocarcinoma. The tumor had a relatively high (8%) S-phase fraction and 1/23 positive lymph nodes (LN). Both the primary tumor and LN metastasis were positive for estrogen receptor (ER) and progesterone receptor (PgR), but lacked erbB2 expression. Approximately one year later, the patient presented with a 0.8 cm comedo-type intraductal mammary adenocarcinoma in the left breast that was negative for ER and PgR, but positive for erbB2. Thirty-five months after the initial diagnosis she was treated for acute skeletal metastasis, and stabilized with a hip replacement. At this time, tumor cells were removed from surplus involved bone, inoculated into cell culture, and developed into the LCC15-MB cell line. The bone metastasis was a poorly differentiated adenocarcinoma lacking ER, PgR, and erbB2, characteristics shared by the LCC15-MB cells, although ER can be re-expressed by treatment of the LCC15-MB cells for 5 days with 75 μM 5-aza-2'-deoxycytidine. The LCC15-MB cell line is tumorigenic when implanted subcutaneously in NCr nu/nu mice and produces long-bone metastases after intracardiac injection. Although the bone metastasis from which the LCC15-MB cell line was derived lacked vimentin (VIM) expression, the original primary tumor and lymph node metastasis were strongly VIM positive, as are LCC15-MB cells in vitro and in nude mice. The karyotype and isozyme profiles of LCC15-MB cells are consistent with its origin from a human female, with most chromosome counts in the hypertriploid range. Thirty-two marker chromosomes are present. These cells provide an in vitro/in vivo model in which to study the inter-relationships between ER, VIM, and bone metastasis in human breast cancer.

Vimentin expression in cervical carcinomas : association with invasive and migratory potential

Vimentin is an intermediate filament protein normally expressed in mesenchymal cells, but evidence is accumulating in the literature which suggests that the aberrant expression of vimentin in epithelial cancer cells might be related to local invasiveness and metastatic potential. Vimentin expression has previously been associated with invasive properties in an in vitro model consisting of a set of HPV-33-transformed cervical keratinocyte cell lines. In the present study, in order to emphasize those in vitro findings, the expression of vimentin has been investigated in cervical neoplasms of different grades, using immunohistochemistry. A clear association is reported between vimentin expression and metastatic progression, since vimentin was detected in all invasive carcinomas and lymph node metastases, but not in CIN III lesions. These in vivo results are compared with present and previous data obtained in vitro on cervical keratinocyte cell lines, where vimentin expression also correlated with in vitro invasiveness.

The expression profiles of the galectin gene family in primary and metastatic papillary thyroid carcinoma with particular emphasis on galectin-1 and galectin-3 expression

INTRODUCTION: Galectin family members have been demonstrated to be abnormally expressed in cancer at the protein and mRNA level. This study investigated the levels of galectin proteins and mRNA expression in a large cohort of patients with papillary thyroid carcinoma and matched lymph node metastases with particular emphasis on galectin-1 and galectin-3. METHODS: mRNA expression of galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were analysed by real-time polymerase chain reaction in 65 papillary thyroid carcinomas, 30 matched lymph nodes with metastatic papillary thyroid carcinoma and 5 non-cancer thyroid tissues. Galectin-1 and 3 protein expression was determined by immunohistochemistry in these samples. RESULTS: Significant expression differences in all tested galectin family members (1, 2, 3, 4, 7, 8, 9, 10 and 12) were noted for mRNA in papillary thyroid carcinomas, with and without lymph node metastasis. Galectin-1 protein was more strongly expressed than galectin-3 protein in papillary thyroid carcinoma. Galectin-1 protein was found to be overexpressed in 32% of primary papillary thyroid carcinomas. A majority of lymph nodes with metastatic papillary thyroid carcinoma (53%) had significantly increased expression of galectin-1 protein, as did 47% of primaries with metastases. Galectin-1 mRNA levels were decreased in the vast majority (94%) of primary thyroid carcinomas that did not have metastases present. Galectin-3 protein levels were noted to be overexpressed in 15% of primary papillary thyroid carcinomas. In primary papillary thyroid carcinoma with lymph node metastases, 32% had over expression of galectin-3 protein. Overexpression of galectin-3 mRNA was noted in 58% of papillary thyroid carcinomas and 64% of lymph nodes bearing metastatic papillary thyroid carcinoma. Also, primary papillary thyroid carcinoma with lymph node metastases had significantly higher expression of galectin-3 mRNA compared to those without lymph node metastases. CONCLUSION: Galectin family members show altered expression at the mRNA level in papillary thyroid cancers. Overexpression of galectin-1 and 3 proteins were noted in papillary thyroid carcinoma with lymph node metastases. The results presented here demonstrated that galectin-1 and galectin-3 expression have important roles in clinical progression of papillary thyroid carcinoma.

JK1 (FAM134B) gene and colorectal cancer : a pilot study on the gene copy number alterations and correlations with clinicopathological parameters

AIMS The aims of the study are to characterize changes in JK-1 (FAM134B) at the DNA level in colorectal adenocarcinoma and adenoma and exploring the possible correlations with clinical and pathological features. METHOD JK-1 gene DNA copy number changes were studied in 211 colorectal carcinomas, 32 colorectal adenoma and 20 colorectal non-cancer colorectal tissue samples by real-time quantitative polymerase chain reaction. The results were correlated with clinical and pathological parameters. RESULTS Colorectal adenomas were more likely to be amplified than deleted with regard to JK-1 (FAM134B) DNA copy number change. The copy number level of JK-1 (FAM134B) DNA in colorectal adenocarcinomas was significantly lower in comparison to colorectal adenomas. Changes in JK-1 (FAM134B) DNA copy number were associated with histological subtypes, and cancer stage. Lower copy numbers were associated with higher tumor stage, lymph node stage and overall pathological stage of cancer. Conversely, higher DNA copy numbers were detected more often in the mucinous adenocarcinoma. CONCLUSIONS This is the first study showing significant correlations of the JK-1 (FAM134B) gene copy number alterations with clinical and pathological features in a large cohort of pre-invasive and invasive colorectal malignancies. The changes in DNA copy number associated with progression of colorectal malignancies reflect that JK-1 (FAM134B) gene could play a role in controlling some steps in development of the invasive phenotypes.

Expression profile of endothelin 1 and its receptor endothelin receptor A in papillary thyroid carcinoma and their correlations with clinicopathologic characteristics

The endothelin axis is a group of signaling molecules and their receptors that have been implicated in vascularization of cancers, with their expression being observed to change in different cancer types. In this research, we examined the expression of endothelin 1 and endothelin receptor A at the protein and messenger RNA (mRNA) levels in 123 papillary thyroid carcinomas and 40 matched lymph nodes with metastatic papillary thyroid carcinomas. We found altered endothelin axis mRNA expression in several clinicopathologic parameters with increased endothelin 1 expression in thyroid papillary carcinoma showing stromal calcification, cancers in men, and primary cancers with lymph node metastases. Increased endothelin receptor A mRNA expression was noted in the larger cancers. There is a significant correlation between expression of endothelin receptor A and endothelin 1 in papillary thyroid carcinoma. Both endothelin receptor A and endothelin 1 mRNA expressions were significantly higher in metastatic carcinoma in the lymph node than in primary thyroid cancer. The metastatic carcinoma in the lymph node had increased expression compared with matched primary thyroid carcinoma. Expressions of endothelin 1 and endothelin receptor A were also documented as being high at the protein level. Our results indicate that in thyroid cancer, endothelin 1 and endothelin receptor A are associated with growth in advanced stages and lymph node metastases, likely through known angiogenic linkages. Targeting the endothelin axis may be useful in planning angiogenesis therapy for thyroid cancer.

Co-regulatory potential of vascular endothelial growth factor–A and vascular endothelial growth factor–C in thyroid carcinoma

Vascular endothelial growth factor (VEGF) promotes growth of blood or lymphatic vessels. The aim of the current study is to identify relationships between VEGF-A and VEGF-C, and their impact in angiogenesis and metastases in thyroid cancers. VEGF-A and VEGF-C mRNA and protein expression was investigated in 136 thyroid cancers (123 papillary thyroid carcinomas and 13 undifferentiated thyroid carcinomas) and 40 matched lymph node metastases with papillary thyroid carcinoma using reverse transcription polymerase chain reaction and immunohistochemistry. VEGF-A and VEGF-C mRNA expression was significantly different between conventional papillary thyroid carcinoma, follicular variant of papillary thyroid carcinoma, and undifferentiated thyroid carcinomas (P = 1 x 10(-6) and 1 x 10(-5), respectively). In undifferentiated carcinoma, VEGF-A and VEGF-C protein overexpression was noted in all cases. VEGF-A and VEGF-C mRNA overexpression was noted in 51% (n = 62) and 27% (n = 33) of the papillary thyroid carcinomas, whereas VEGF-A and VEGF-C protein overexpression was also identified in 70% (n = 86) and 62% (n = 76) of the carcinomas. VEGF-A mRNA was significantly higher in cancers with lymph node metastases compared with nonmetastatic cancers (P = .001), whereas most metastatic cancers underexpressed VEGF-C (P = .0002), with a similar trend for protein. The expression of VEGF-A and VEGF-C correlated with each other at both mRNA and protein levels (P = .00004 and .003, respectively). In summary, VEGF-A and -C expressions correlate with the pathological parameters and metastatic status of thyroid carcinomas. The significant correlations between the expressions of these genes add weight to hypotheses concerning VEGF-A and -C interaction in cancer progression.

Clinical impacts of mammalian target of rapamycin expression in human colorectal cancers

This study investigated the clinicopathologic roles of mammalian target of rapamycin (mTOR) expression and its relationship to carcinogenesis and tumor progression in a colorectal adenoma-adenocarcinoma model. Two colon cancer cell lines with different pathologic stages (SW480 and SW48) and 1 normal colonic epithelial cell line (FHC) were used, in addition to 119 colorectal adenocarcinomas and 32 adenomas. mTOR expression profiles at messenger RNA (mRNA) and protein levels were investigated in the cells and tissues using real-time quantification polymerase chain reaction and immunohistochemistry. The findings were correlated with the clinicopathologic features of the tumors. The colon cell line from stage III cancer (SW48) showed higher expression of mTOR mRNA than that from stage II cancer (SW480). At the tissue level, mTOR showed higher mRNA and protein expression in colorectal carcinoma than in adenoma. The mRNA and protein expression was correlated with each other in approximately one-third of the carcinomas and adenomas. High levels of mTOR mRNA expression were noted more in carcinoma or adenoma arising from the distal portion of the large intestine (P = .025 and .019, respectively). Within the colorectal cancer population, a high level of expression of mTOR mRNA was related to the presence of lymph node metastases (P = .031), advanced pathologic stage (P = .05), and presence of persistent disease or tumor recurrence (P = .035). To conclude, the study has indicated that mTOR is likely to be involved in the development and progression of colorectal cancer and is linked to cancer initiation, invasiveness, and progression.

Single nucleotide polymorphisms and mRNA expression of VEGF-A in papillary thyroid carcinoma: Potential markers for aggressive phenotypes

BACKGROUND AND OBJECTIVES Polymorphisms of the VEGF gene are known to affect the biological behaviour of cancers but have seldom been studied in thyroid cancer. The aim of the current study is to evaluate the prevalence and relevance of VEGF-A polymorphisms and mRNA expression in papillary thyroid carcinoma (PTC). MATERIALS AND METHODS Genomic DNA and total RNA were isolated from paraffin-embedded tissue from 91 PTC (51 conventional PTC and 40 follicular variant) and 78 control thyroid tissues. Three DNA polymorphisms (+936C > T, +405C > G and -141A > C) in the 3' and 5' untranslated region (3'-UTR, 5'-UTR) of VEGF-A were studied using PCR and RFLP. Also, the mRNA expression of VEGF-A in these tissues was studied by real-time PCR. RESULTS Distribution of polymorphisms in the 5'-UTR (VEGF-VEGF -141A > C and +405C > G) and 3'-UTR (VEGF +936C > T) were all significantly different in PTC and benign thyroid tissue (p = 0.0001, 0.001 and 0.028 respectively). The VEGF -141 C allele was more common in PTC with lymph node metastases (p = 0.026). VEGF + 405 Galleles andVEGF +936 CC genotype were more common in PTC of advanced pathological staging (p = 0.018 and 0.017 respectively). Also, increased expression of VEGF-A mRNA was noted in PTC compared to control (p = 0.009). Within the group of patients with conventional PTC, those with lymph nodal metastases had a higher level of VEGF-A mRNA expression than other patients (p = 0.0003). CONCLUSION These findings suggest that VEGF polymorphisms and mRNA expression may predict the aggressiveness behaviour of thyroid cancer.

CT Anatomy for Radiotherapy

Knowledge of CT anatomy is increasingly vital in daily radiotherapy practice, especially with more widespread use of cross-sectional image-guided radiotherapy (IGRT) techniques. Existing CT anatomy texts are predominantly written for the diagnostic practitioner and do not always address the radiotherapy issues while emphasising structures that are not common to radiotherapy practice. CT Anatomy for Radiotherapy is a new radiotherapy-specific text that is intended to prepare the reader for CT interpretation for both IGRT and treatment planning. It is suitable for undergraduate students, qualified therapy radiographers, dosimetrists and may be of interest to oncologists and registrars engaged in treatment planning. All essential structures relevant to radiotherapy are described and depicted on 3D images generated from radiotherapy planning systems. System-based labelled CT images taken in relevant imaging planes and patient positions build up understanding of relational anatomy and CT interpretation. Images are accompanied by comprehensive commentary to aid with interpretation. This simplified approach is used to empower the reader to rapidly gain image interpretation skills. The book pays special attention to lymph node identification as well as featuring a unique section on Head and Neck Deep Spaces to help understanding of common pathways of tumour spread. Fully labelled CT images using radiotherapy-specific views and positioning are complemented where relevant by MR and fusion images. A brief introduction to image interpretation using IGRT devices is also covered. The focus of the book is on radiotherapy and some images of common tumour pathologies are utilised to illustrate some relevant abnormal anatomy. Short self-test questions help to keep the reader engaged throughout.

Elevated level of inhibin-α subunit is pro-tumourigenic and pro-metastatic and associated with extracapsular spread in advanced prostate cancer

The biological function of inhibin-a subunit (INHa) in prostate cancer (PCa) is currently unclear. A recent study associated elevated levels of INHa in PCa patients with a higher risk of recurrence. This prompted us to use clinical specimens and functional studies to investigate the pro-tumourigenic and pro-metastatic function of INHa. We conducted a cross-sectional study to determine a link between INHa expression and a number of clinicopathological parameters including Gleason score, surgical margin, extracapsular spread, lymph node status and vascular endothelial growth factor receptor-3 expression, which are well-established prognostic factors of PCa. In addition, using two human PCa cell lines (LNCaP and PC3) representing androgen-dependent and -independent PCa respectively, we investigated the biological function of elevated levels of INHa in advanced cancer. Elevated expression of INHa in primary PCa tissues showed a higher risk of PCa patients being positive for clinicopathological parameters outlined above. Overexpressing INHa in LNCaP and PC3 cells demonstrated two different and cell-type-specific responses. INHa-positive LNCaP demonstrated reduced tumour growth whereas INHa-positive PC3 cells demonstrated increased tumour growth and metastasis through the process of lymphangiogenesis. This study is the first to demonstrate a pro-tumourigenic and pro-metastatic function for INHa associated with androgen-independent stage of metastatic prostate disease. Our results also suggest that INHa expression in the primary prostate tumour can be used as a predictive factor for prognosis of PCa.

Vascular endothelial growth factor receptor-3 directly interacts with phosphatidylinositol 3-kinase to regulate lymphangiogenesis

Background Dysfunctional lymphatic vessel formation has been implicated in a number of pathological conditions including cancer metastasis, lymphedema, and impaired wound healing. The vascular endothelial growth factor (VEGF) family is a major regulator of lymphatic endothelial cell (LEC) function and lymphangiogenesis. Indeed, dissemination of malignant cells into the regional lymph nodes, a common occurrence in many cancers, is stimulated by VEGF family members. This effect is generally considered to be mediated via VEGFR-2 and VEGFR-3. However, the role of specific receptors and their downstream signaling pathways is not well understood. Methods and Results Here we delineate the VEGF-C/VEGF receptor (VEGFR)-3 signaling pathway in LECs and show that VEGF-C induces activation of PI3K/Akt and MEK/Erk. Furthermore, activation of PI3K/Akt by VEGF-C/VEGFR-3 resulted in phosphorylation of P70S6K, eNOS, PLCc1, and Erk1/2. Importantly, a direct interaction between PI3K and VEGFR-3 in LECs was demonstrated both in vitro and in clinical cancer specimens. This interaction was strongly associated with the presence of lymph node metastases in primary small cell carcinoma of the lung in clinical specimens. Blocking PI3K activity abolished VEGF-C-stimulated LEC tube formation and migration. Conclusions Our findings demonstrate that specific VEGFR-3 signaling pathways are activated in LECs by VEGF-C. The importance of PI3K in VEGF-C/VEGFR-3-mediated lymphangiogenesis provides a potential therapeutic target for the inhibition of lymphatic metastasis.

Differential effects of tissue culture coating substrates on prostate cancer cell adherence, morphology and behavior

Weak cell-surface adhesion of cell lines to tissue culture surfaces is a common problem and presents technical limitations to the design of experiments. To overcome this problem, various surface coating protocols have been developed. However, a comparative and precise real-time measurement of their impact on cell behavior has not been conducted. The prostate cancer cell line LNCaP, derived from a patient lymph node metastasis, is a commonly used model system in prostate cancer research. However, the cells’ characteristically weak attachment to the surface of tissue culture vessels and cover slips has impeded their manipulation and analysis and use in high throughput screening. To improve the adherence of LNCaP cells to the culture surface, we compared different coating reagents (poly-L-lysine, poly-L-ornithine, collagen type IV, fibronectin, and laminin) and culturing conditions and analyzed their impact on cell proliferation, adhesion, morphology, mobility and gene expression using real-time technologies. The results showed that fibronectin, poly-L-lysine and poly-L-ornithine improved LNCaP cells adherence and provoked cell morphology alterations, such as increase of nuclear and cellular area. These coating reagents also induced a higher expression of F-actin and reduced cell mobility. In contrast, laminin and collagen type IV did not improve adherence but promoted cell aggregation and affected cell morphology. Cells cultured in the presence of laminin displayed higher mobility than control cells. All the coating conditions significantly affected cell viability; however, they did not affect the expression of androgen receptor-regulated genes. Our comparative findings provide important insight for the selection of the ideal coating reagent and culture conditions for the cancer cell lines with respect to their effect on proliferation rate, attachment, morphology, migration, transcriptional response and cellular cytoskeleton arrangement.

Weight and weight change following breast cancer : evidence from a prospective, population-based, breast cancer cohort study

Background: While weight gain following breast cancer is considered common, results supporting these findings are dated. This work describes changes in body weight following breast cancer over 72 months, compares weight with normative data and explores whether weight changes over time are associated with personal, diagnostic, treatment or behavioral characteristics. Methods: A population-based sample of 287 Australian women diagnosed with early-stage invasive breast cancer was assessed prospectively at six, 12, 18 and 72 months post-surgery. Weight was clinically measured and linear mixed models were used to explore associations between weight and participant characteristics (collected via self-administered questionnaire). Those with BMI changes of one or more units were considered to have experienced clinically significant changes in weight. Results: More than half (57%) of participants were overweight or obese at 6 months post-surgery, and by 72 months post-surgery 68% of women were overweight or obese. Among those who gained more weight than age-matched norms, clinically significant weight gain between 6 and 18 months and 6 and 72 months post-surgery was observed in 24% and 39% of participants, respectively (median [range] weight gain: 3.9kg [2.0-11.3kg] and 5.2kg [0.6-28.7], respectively). Clinically-significant weight losses were observed in up to 24% of the sample (median [range] weight loss between 6 and 72 months post-surgery: -6.4kg [-1.9--24.6kg]). More extensive lymph node removal, being treated on the non-dominant side, receiving radiation therapy and lower physical activity levels at 6 months was associated with higher body weights post-breast cancer (group differences >3kg; all p<0.05). Conclusions: While average weight gain among breast cancer survivors in the long-term is small, subgroups of women experience greater gains linked with adverse health and above that experienced by age-matched counterparts. Weight change post-breast cancer is a contemporary public health issue and the integration of healthy weight education and support into standard breast cancer care has potential to significantly improve the length and quality of cancer survivorship.

Cytokine mRNA expression and IFN-γ production of immunised nematode resistant and susceptible lambs against natural poly-generic challenge

IL-2, IL-4 and IFN-γ mRNA expression, and production of IFN-γ was examined in mesenteric lymph node cells (MLNC) and CD4+ enriched T cell populations of nematode resistant (R) and susceptible (S) line lambs by use of RT-PCR and ELISA. Five R and S line lambs that were immunised by repeated oxfendazole-abbreviated infections and 5 non-immunised R and S line lambs were used. All lambs grazed nematode infected pasture for 107 days. Immunisation enhanced the resistant status in both R and S lambs. MLNC obtained from slaughtered animals were stimulated with Con A or T. colubriformis specific antigen. Non-stimulated MLNC of immunised lambs expressed higher levels of IL-4 mRNA and lower levels of IL-2 mRNA than non-immunised lambs. MLNC of immunised R and S line lambs stimulated with antigen for 24 h expressed detectable amounts of IL-4 mRNA that was not seen in non-immunised controls. CD4+ T cell enriched cell populations of immunised R and S lambs and non-immunised R lambs expressed moderate to high levels of IL-4 mRNA. Con A stimulated MLNC of immunised R and S lambs expressed high levels IFN-γ mRNA and produced high amounts of IFN-γ. Lower levels were present in non-immunised controls. The results indicate that R line lambs and immunised S line lambs respond to natural nematode challenge with a predominating IL-4 cytokine response when compared to non-immunised S lambs.