THE p63 ISOFORM ∆Np63α INHIBITS EPITHELIAL – MESENCHYMAL TRANSITION BY PROMOTING THE EXPRESSION OF MIR-205 IN HUMAN BLADDER CANCER CELLS

p63, a p53 family member, is a transcription factor that has complex roles in cancer. This study focuses on the role of the ∆Np63α isoform in bladder cancer (BC). Epithelial – mesenchymal transition (EMT) is a physiological process that plays an important part in metastasis and drug resistance. At the molecular level, EMT is characterized by the loss of the epithelial marker E-cadherin, and the acquisition of the transcriptional repressors of E-cadherin (ZEB1, ZEB2, TWIST, SNAI1 and SNAI2). Recent publications highlight the role of microRNAs belonging to the miR-200 family and miR-205 in preventing EMT through suppression of ZEB1 and ZEB2. p53, the homologue of p63, is implicated in regulating EMT by modulating the expression of miR-200c; however, the mechanisms underlying miR-205 control remain unclear. Here we show that ∆Np63α regulates the transcription of miR-205 and controls EMT in human BC cells. We observed a strong correlation between the expression of ∆Np63α, miR-205 and E-cadherin in a panel of BC cell lines (n=28) and also in bladder primary tumors from a cohort of patients (n=98). A remarkably inverse correlation is observed between ∆Np63α and ZEB1/2 in cell lines. Stable knockdown (KD) ∆Np63α in UC6, an “epithelial” BC cell line, decreased the expression of miR-205 and induced ZEB1/2 expression, the effects that were reversed by expression of exogenous miR-205. Moreover, overexpressing ∆Np63α in UC3, a “messenchymal” BC cell line, brought about opposite results, an increase in miR-205 expression and a reduction in ZEB1/2 expression. Modulation of ∆Np63α expression resulted in a parallel change in the expression of miR-205 and miR-205 “host” gene (miR-205HG). Nuclear run-on and chromatin immunoprecipitation experiments demonstrated that ∆Np63α regulates the transcription of miR-205 through controlling the recruitment of RNA Polymerase II to the promoter of miR-205HG. Interestingly, high miR-205 expression correlated with poor clinical outcome in BC patients, consistent with our recent publication highlighting the enrichment of ∆Np63 in a lethal subset of muscle invasive BC. In summary, our data present the important roles of ∆Np63α in preventing EMT mediated by miR-205. Our study also identifies miR-205 as a potential molecular marker to predict clinical outcome in BC patients.

p21WAF1 is required for butyrate-mediated growth inhibition of human colon cancer cells

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Conservation of the Drosophila lateral inhibition pathway in human lung cancer: A hairy-related protein (HES-1) directly represses achaete-scute homolog-1 expression

The achaete-scute genes encode essential transcription factors in normal Drosophila and vertebrate nervous system development. Human achaete-scute homolog-1 (hASH1) is constitutively expressed in a human lung cancer with neuroendocrine (NE) features, small cell lung cancer (SCLC), and is essential for development of the normal pulmonary NE cells that most resemble this neoplasm. Mechanisms regulating achaete-scute homolog expression outside of Drosophila are presently unclear, either in the context of the developing nervous system or in normal or neoplastic cells with NE features. We now provide evidence that the protein hairy-enhancer-of-split-1 (HES-1) acts in a similar manner as its Drosophila homolog, hairy, to transcriptionally repress achaete-scute expression. HES-1 protein is detected at abundant levels in most non-NE human lung cancer cell lines which lack hASH1 but is virtually absent in hASH1-expressing lung cancer cells. Moreover, induction of HES-1 in a SCLC cell line down-regulates endogenous hASH1 gene expression. The repressive effect of HES-1 is directly mediated by binding of the protein to a class C site in the hASH1 promoter. Thus, a key part of the process that determines neural fate in Drosophila is conserved in human lung cancer cells. Furthermore, modulation of this pathway may underlie the constitutive hASH1 expression seen in NE tumors such as SCLC, the most virulent human lung cancer.

Protective anti-tumor immunity induced by vaccination with recombinant adenoviruses encoding multiple tumor-associated cytotoxic T lymphocyte epitopes in a string-of-beads fashion

Vaccines harboring genes that encode functional oncoproteins are intrinsically hazardous, as their application may lead to introduction of these genes into normal cells and thereby to tumorigenesis. On the other hand, oncoproteins are especially attractive targets for immunotherapy of cancer, as their expression is generally required for tumor growth, making the arisal of tumor variants lacking these antigens unlikely. Using murine tumor models, we investigated the efficacy of polyepitope recombinant adenovirus (rAd) vaccines, which encode only the immunogenic T cell epitopes derived from several oncogenes, for the induction of protective anti-tumor immunity. We chose to employ rAd, as these are safe vectors that do not induce the side effects associated with, for example, vaccinia virus vaccines. A single polyepitope rAd was shown to give rise to presentation of both H-2 and human leukocyte antigen-restricted cytotoxic T lymphocyte (CTL) epitopes. Moreover, vaccination with a rAd encoding H-2-restricted CTL epitopes, derived from human adenovirus type 5 early region 1 and human papilloma virus type 16-induced tumors, elicited strong tumor-reactive CTL and protected the vaccinated animals against an otherwise lethal challenge with either of these tumors. The protection induced was superior compared with that obtained by vaccination with irradiated tumor cells. Thus, vaccination with polyepitope rAd is a powerful approach for the induction of protective anti-tumor immunity that allows simultaneous immunization against multiple tumor-associated T cell epitopes, restricted by various major histocompatibility complex haplotypes.

Increased expression of preprotachykinin-I and neurokinin receptors in human breast cancer cells: Implications for bone marrow metastasis

Neuropeptides are implicated in many tumors, breast cancer (BC) included. Preprotachykinin-I (PPT-I) encodes multiple neuropeptides with pleiotropic functions such as neurotransmission, immune/hematopoietic modulation, angiogenesis, and mitogenesis. PPT-I is constitutively expressed in some tumors. In this study, we investigated a role for PPT-I and its receptors, neurokinin-1 (NK-1) and NK-2, in BC by using quantitative reverse transcription–PCR, ELISA, and in situ hybridization. Compared with normal mammary epithelial cells (n = 2) and benign breast biopsies (n = 21), BC cell lines (n = 7) and malignant breast biopsies (n = 25) showed increased expression of PPT-I and NK-1. NK-2 levels were high in normal and malignant cells. Specific NK-1 and NK-2 antagonists inhibited BC cell proliferation, suggesting autocrine and/or intercrine stimulation of BC cells by PPT-I peptides. NK-2 showed no effect on the proliferation of normal cells but mediated the proliferation of BC cells. Cytosolic extracts from malignant BC cells enhanced PPT-I translation whereas extracts from normal mammary epithelial cells caused no change. These enhancing effects may be protein-specific because a similar increase was observed for IL-6 translation and no effect was observed for IL-1α and stem cell factor. The data suggest that PPT-I peptides and their receptors may be important in BC development. Considering that PPT-I peptides are hematopoietic modulators, these results could be extended to understand early integration of BC cells in the bone marrow, a preferred site of metastasis. Molecular signaling transduced by PPT-I peptides and the mechanism that enhances translation of PPT-I mRNA could lead to innovative strategies for BC treatments and metastasis.

Effect of 2′-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2′-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5′ upstream cis-acting regulatory element (nucleotides 80–109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of β-actin, α-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.

Estrogens increase the expression of fibulin-1, an extracellular matrix protein secreted by human ovarian cancer cells.

Ovarian cancers have a high ability to invade the peritoneal cavity and some are stimulated by estrogens. In an attempt to understand the mode of action of estrogens on these cancer cells and to develop new markers, we have characterized estrogen-regulated proteins. This study was aimed at identifying a protein secreted by ovarian cancer cells whose level was increased by estradiol [Galtier-Dereure, F., Capony, F., Maudelonde, T. & Rochefort, H. (1992) J. Clin. Endocrinol. Metab. 75, 1497-1502]. By using microprotein sequencing, the 110-kDa protein was identified as fibulin-1, a protein of the extracellular matrix that binds to fibronectin, laminin, and nidogen. The amount of immunoprecipitated fibulin-1 secreted into the medium and present in the cell extract was increased up to 10-fold by estradiol in three estrogen-responsive ovarian cancer cell lines. By immunohistochemistry fibulin-1 was located in the stroma of several ovarian cancers and cysts. The findings highlight a potential role for fibulin-1 in the spread of ovarian cancer in the peritoneal cavity and/or in distal metastases.

Ligand-directed retroviral targeting of human breast cancer cells.

We explored the feasibility of designing retroviral vectors that can target human breast cancer cells with characteristic receptors via ligand-receptor interaction. The ecotropic Moloney murine leukemia virus envelope was modified by insertion of sequences encoding human heregulin. Ecotropic virus, which normally does not infect human cells, when pseudotyped with the modified envelope protein now crosses species to infect human breast cancer cell lines that overexpress HER-2 (human epidermal growth factor receptor; also called ERBB2) and HER-4 (also called ERBB4), while human breast cancer cell lines expressing low levels of these receptors remain resistant to infection. Since about 20% of human breast cancers overexpress HER-2 and some of breast cancer cell lines overexpress both HER-2 and HER-4, cell-specific targeting of retroviral vectors may provide a different approach for in vivo gene therapy of this type of breast cancer.

De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells.

The rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth.

ETS1 suppresses tumorigenicity of human colon cancer cells.

We have ectopically expressed transcription factor ETS1 in two different highly tumorigenic human colon cancer cell lines, DLD-1 and HCT116, that do not express endogenous ETS1 protein and have obtained several independent clones. The expression of wild-type ETS1 protein in these colon cancer cells reverses the transformed phenotype and tumorigenicity in a dose-dependent manner. By contrast, expression in DLD-1 cells of a variant form of ETS1, lacking transcriptional activity, did not alter the tumorigenic properties of the cells, suggesting that the reduction in tumorigenicity in these clones was specific for the wild-type ETS1 gene products. Since these colon cancer cells have multiple genetic alterations, the system described in this paper could be a good model to study the suppression of tumorigenicity at a transcriptional level, which could lead to the design and development of novel drugs for cancer treatment.

Prostate cancer in Illinois, 1986-1992 /

"December 1994."

Acceptability of short term neo-adjuvant androgen deprivation in patients with locally advanced prostate cancer

Purpose: To determine the acceptability of short term neo-adjuvant maximal androgen deprivation (MAD) to patients treated with external beam radiation for locally advanced prostate cancer. Methods: Between 1996 and 2000, 818 patients with locally advanced, but non-metastatic, prostate cancer were entered into a randomised clinical trial (TROG 96.01), which compared radiation treatment alone with the same radiation treatment and 3 or 6 months neo-adjuvant MAD with goserelin and flutamide. Relevant symptoms, and how troublesome they were to the patient, were scored using a self-assessment questionnaire. This was completed by the patient at registration, and at specified times during and after treatment. Patients taking flutamide had liver function tests checked at regular intervals. Results: All patients have completed at least 12 months follow-up after treatment. Nearly all patients completed planned treatment with goserelin, but 27% of patients in the 6-month MAD treatment arm, and 20% in the 3-month arm, had to stop flutamide early. This was mainly due to altered liver function (up to 17% patients) and bowel side effects (up to 8% patients). However, although flutamide resulted in more bowel symptoms for patients on MAD, there was significant reduction in some urinary symptoms on this treatment. Acute bowel and urinary side effects at the end of radiation treatment were similar in all treatment arms. Side effect severity was unrelated to radiation target volume size, which was reduced by MAD, but symptomatology prior to any treatment was a powerful predictor. Of the 36% of patients who were sexually active before any treatment, the majority became inactive whilst on MAD. However, sexual activity at 12 months after radiation treatment was similar in all treatment arms, indicating that the effects of short term MAD on sexual function are reversible. Conclusion: Despite temporary effects on sexual activity, and compliance difficulties with flutamide, short-term neo-adjuvant MAD was not perceived by patients to be a major inconvenience. If neo-adjuvant MAD in the way tested can be demonstrated to lead to improved biochemical control and/or survival, then patients would view these therapeutic gains as worthwhile. Compliance with short-term goserelin was excellent, confirming that LH-RH analogues have a potential role in more long-term adjuvant treatment. However, for more protracted androgen deprivation, survival advantages and deleterious effects need to be assessed in parallel, in order to determine the optimal duration of treatment. (C) 2003 Elsevier Ireland Ltd. All fights reserved.