Prostate cancer is the most common cancer among men in the world. About 60% of prostate cancer patients exhibit a chromosomal rearrangement involving the promoter region of an androgen-regulated gene and a member of the ETS transcription factor family; this molecular alteration results in the overexpression of the ETS transcriptional factor. The oncogenic role of ETV4 has been previously demonstrated in several human prostate cancer cells: ETV4 promotes cell proliferation, anchorage independent growth, migration and cell invasion. In addition, in a transgenic mouse model overexpressing ETV4 at prostate level, the overexpression of ETV4 induces prostatic intraepithelial lesion and promotes tumor cell proliferation. The oncogenic mechanisms of ETV4 over-expression have been further investigated by microarray gene expression profiling of prostate tissue comparing wild-type and ETV4 mice. The microarray data have shown that the secretory leukocyte peptidase inhibitor (SLPI) is downregulated in ETV4 mice and this ETV4-mediated down-regulation has been confirmed also in human prostate cell lines. SLPI is a serine protease inhibitor involved in inflammatory processes that plays a role in oncogenesis. It is intriguing, that the negative regulation of SLPI expression in ETV4 mice is in keeping with the peculiar pattern of SLPI expression in prostate cancer patients: SLPI expression is reduced in the early stage of prostate cancer whereas it is increased in a subset of metastatic prostate cancer patients after androgen deprivation therapy. The first aim was to investigate if ETV1, another ETS transcriptional factor, behaves as ETV4 in the regulation of SLPI expression, considering that also ETV1 is translocated in about 8-10% of prostate cancer patients. The effect of ETV1 on SLPI expression has been investigated in 2 human prostate cell lines (LNCaP and RWPE cells) by 2 complementary approaches: the stable silencing of ETV1 in LNCaP and its stable over-expression in RWPE demonstrated that also ETV1, as ETV4, regulates negatively SLPI expression. In addition, the role of androgenic system in the regulation of SLPI expression has been studied using as cellular model the androgen-sensitive and androgen receptor (AR)-positive LNCaP cells. In this cell line the androgenic stimulus has been able to regulate positively SLPI expression, despite the confounding effect of the androgen-mediated ETV1 over-expression that should reduce SLPI expression. The effect of androgens on the regulation of SLPI expression has been confirmed in another androgen-competent human prostate cancer cell line, 22RV1, that does not express any ETS proteins that could represent a confounding factor. These findings about the androgen/AR axis exert a positive regulation of both SLPI and ETS transcriptional factors combined with the negative regulation of SLPI exerted by the ETS transcriptional factors (ETV4, ETV1) have brought us to hypothesize a model that could explain the peculiar behavior of SLPI expression during the prostate cancer progression. The second aim was to investigate the contribution of SLPI expression in the determination of the neoplastic features of prostate cancer. We performed several functional analyses in human normal prostate cell line (RWPE) stably transduced with shRNA against SLPI and human prostate cancer cell lines (LNCaP and PC3) stably transduced with a vector expressing SLPI. In RWPE cells, SLPI was found to be able to increase cell migration and invasion ability, and to contribute the epithelial-mesenchymal transition (EMT) by promoting the “cadherin-switch” and by increasing the expression of several transcriptional factors involved in EMT process. Furthermore, SLPI could play a role in cancer-promotion by inhibiting apoptosis. However, in the cancerous LNCaP and PC3 cells the overexpression of SLPI was not able to provide any additional neoplastic features, likely because the neoplastic phenotype of these metastatic cancerous cell lines is already fully developed. The last aim was to study the mechanism by which ETS proteins regulates SLPI, since chromatin immuno-precipitation experiments have shown that ETV4 does not bind SLPI promoter. In RWPE cells stably transduced with an expressing vector encoding either ETV1 or ETV4, and in PC3 cells stably transduced with shRNA against ETV4, it has been found that ETV1 and ETV4 downregulate STAT1 expression. STAT1 has been reported to be a positive regulator of SLPI: we observed that in PC3 cells the overexpression of STAT1 increases the SLPI protein level supporting the notion that the ETV1/ETV4 mediated regulation of SLPI could be mediated by the downregulation of STAT1.

Moccia, A. (2022). SLPI: a novel target of ETS proteins in prostate cancer.

SLPI: a novel target of ETS proteins in prostate cancer

Moccia, Annalisa
2022

Abstract

Prostate cancer is the most common cancer among men in the world. About 60% of prostate cancer patients exhibit a chromosomal rearrangement involving the promoter region of an androgen-regulated gene and a member of the ETS transcription factor family; this molecular alteration results in the overexpression of the ETS transcriptional factor. The oncogenic role of ETV4 has been previously demonstrated in several human prostate cancer cells: ETV4 promotes cell proliferation, anchorage independent growth, migration and cell invasion. In addition, in a transgenic mouse model overexpressing ETV4 at prostate level, the overexpression of ETV4 induces prostatic intraepithelial lesion and promotes tumor cell proliferation. The oncogenic mechanisms of ETV4 over-expression have been further investigated by microarray gene expression profiling of prostate tissue comparing wild-type and ETV4 mice. The microarray data have shown that the secretory leukocyte peptidase inhibitor (SLPI) is downregulated in ETV4 mice and this ETV4-mediated down-regulation has been confirmed also in human prostate cell lines. SLPI is a serine protease inhibitor involved in inflammatory processes that plays a role in oncogenesis. It is intriguing, that the negative regulation of SLPI expression in ETV4 mice is in keeping with the peculiar pattern of SLPI expression in prostate cancer patients: SLPI expression is reduced in the early stage of prostate cancer whereas it is increased in a subset of metastatic prostate cancer patients after androgen deprivation therapy. The first aim was to investigate if ETV1, another ETS transcriptional factor, behaves as ETV4 in the regulation of SLPI expression, considering that also ETV1 is translocated in about 8-10% of prostate cancer patients. The effect of ETV1 on SLPI expression has been investigated in 2 human prostate cell lines (LNCaP and RWPE cells) by 2 complementary approaches: the stable silencing of ETV1 in LNCaP and its stable over-expression in RWPE demonstrated that also ETV1, as ETV4, regulates negatively SLPI expression. In addition, the role of androgenic system in the regulation of SLPI expression has been studied using as cellular model the androgen-sensitive and androgen receptor (AR)-positive LNCaP cells. In this cell line the androgenic stimulus has been able to regulate positively SLPI expression, despite the confounding effect of the androgen-mediated ETV1 over-expression that should reduce SLPI expression. The effect of androgens on the regulation of SLPI expression has been confirmed in another androgen-competent human prostate cancer cell line, 22RV1, that does not express any ETS proteins that could represent a confounding factor. These findings about the androgen/AR axis exert a positive regulation of both SLPI and ETS transcriptional factors combined with the negative regulation of SLPI exerted by the ETS transcriptional factors (ETV4, ETV1) have brought us to hypothesize a model that could explain the peculiar behavior of SLPI expression during the prostate cancer progression. The second aim was to investigate the contribution of SLPI expression in the determination of the neoplastic features of prostate cancer. We performed several functional analyses in human normal prostate cell line (RWPE) stably transduced with shRNA against SLPI and human prostate cancer cell lines (LNCaP and PC3) stably transduced with a vector expressing SLPI. In RWPE cells, SLPI was found to be able to increase cell migration and invasion ability, and to contribute the epithelial-mesenchymal transition (EMT) by promoting the “cadherin-switch” and by increasing the expression of several transcriptional factors involved in EMT process. Furthermore, SLPI could play a role in cancer-promotion by inhibiting apoptosis. However, in the cancerous LNCaP and PC3 cells the overexpression of SLPI was not able to provide any additional neoplastic features, likely because the neoplastic phenotype of these metastatic cancerous cell lines is already fully developed. The last aim was to study the mechanism by which ETS proteins regulates SLPI, since chromatin immuno-precipitation experiments have shown that ETV4 does not bind SLPI promoter. In RWPE cells stably transduced with an expressing vector encoding either ETV1 or ETV4, and in PC3 cells stably transduced with shRNA against ETV4, it has been found that ETV1 and ETV4 downregulate STAT1 expression. STAT1 has been reported to be a positive regulator of SLPI: we observed that in PC3 cells the overexpression of STAT1 increases the SLPI protein level supporting the notion that the ETV1/ETV4 mediated regulation of SLPI could be mediated by the downregulation of STAT1.
Moccia, A. (2022). SLPI: a novel target of ETS proteins in prostate cancer.
Moccia, Annalisa
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11365/1211854