Marc A. Thomas

Transcriptional regulation of the homeobox gene NKX3.1 by all-trans retinoic acid in prostate cancer cells

NKX3.1 is a homeobox gene, expression of which is largely restricted to the adult prostatic epithelium. Loss of NKX3.1 expression has been linked to prostate carcinogenesis and disease progression and occurs in the absence of mutations in the coding region of the NKX3.1 gene. In this study, we have characterized regulation of NKX3.1 expression by all‐trans retinoic acid (tRA), a naturally occurring vitamin A metabolite that is accumulated at high levels in the prostate. Using the prostate cancer cell line LNCaP, Western blot analysis revealed a ∼twofold induction of NKX3.1 protein levels following tRA exposure, with sequential analysis of NKX3.1 protein levels in cycloheximide co‐treated cells indicating that tRA does not alter NKX3.1 protein turnover. The ∼1.6‐fold increase in NKX3.1 mRNA levels detected in tRA‐treated LNCaP cells also occurred independently of new protein synthesis and was not mediated by changes in NKX3.1 mRNA stability. In contrast, nuclear run‐on assays indicated that tRA treatment increased NKX3.1 transcription. To identify retinoid responsive regions of the NKX3.1 gene, DNA sequences encompassing ∼2 kb of the NKX3.1 promoter or the entire 3′untranslated region (UTR) were cloned into luciferase reporter plasmids. Analysis of induced luciferase activity following transfection of these constructs into prostate cancer cells did not identify tRA responsiveness, however the 3′UTR was found to be strongly androgen responsive. These studies demonstrate that the NKX3.1 gene is a direct target of retinoid receptors and suggest that androgen regulation of NKX3.1 expression is mediated in part by the 3′UTR. J. Cell. Biochem. 99: 1409–1419, 2006.

ETS1 Regulates NKX3.1 5' Promoter Activity and Expression in Prostate Cancer Cells

NKX3.1 controls the differentiation and proliferation of prostatic epithelial cells both during development and in the adult, while its expression is frequently downregulated in prostate cancers. Transcriptional control of NKX3.1 expression and in particular, factors that function via the NKX3.1 5′ proximal promoter are poorly characterized.

EGFR mutation testing in NSCLC: Patterns of care and outcomes in Western Australia

Aims:  This study evaluated the EGFR mutation status, administration of gefitinib or erlotinib and outcomes of patients assessed for EGFR mutations since the commencement of testing in Western Australia.

Activity of ABCG2 Is Regulated by Its Expression and Localization in DHT and Cyclopamine-Treated Breast Cancer Cells

Elevated expression of the efflux transporter, ATP‐binding cassette subfamily G isoform 2 (ABCG2) on the plasma membrane of cancer cells contributes to the development of drug resistance and is a key characteristic of cancer stem cells. In this study, gene expression analysis identified that treatment of the MCF‐7 and T‐47D breast cancer cell lines with the androgen, 5α‐dihydrotestosterone (DHT), and the Hedgehog signaling inhibitor, cyclopamine downregulated ABCG2 mRNA levels. In MCF‐7 cells, and in Hoechst 33342lo/CD44hi/CD24lo breast cancer stem‐like cells isolated from MCF‐7 cultures, ABCG2 was accumulated in cell‐to‐cell junction complexes and in large cytoplasmic aggresome‐like vesicles. DHT treatments, which decreased cellular ABCG2 protein levels, led to diminished ABCG2 localization in both cell‐to‐cell junction complexes and in cytoplasmic vesicles. In contrast, cyclopamine, which did not alter ABCG2 protein levels, induced accumulation of ABCG2 in cytoplasmic vesicles, reducing its localization in cell‐to‐cell junction complexes. The reduced localization of ABCG2 at the plasma membrane of MCF‐7 cells was associated with decreased efflux of the ABCG2 substrate, mitoxantrone, and increased sensitivity of cyclopamine‐treated cultures to the cytotoxic effects of mitoxantrone. Together, these findings indicate that DHT and cyclopamine reduce ABCG2 activity in breast cancer cells by distinct mechanisms, providing evidence to advocate the adjunct use of analogous pharmaceutics to increase or prolong the efficacy of breast cancer treatments. J. Cell. Biochem. 117: 2249–2259, 2016.

Erdheim–Chester disease associated with a novel, complex BRAF p.Thr599_Val600delinsArgGlu mutation

BRAF mutation testing to determine eligibility for treatment with vemurafenib was performed on archival skin lesions of a 54-year-old patient diagnosed with Erdheim–Chester disease (ECD) in 1999. Sanger sequencing of DNA extracted from a 2008 skin lesion identified two non-contiguous base substitutions in BRAF, which were shown by next-generation sequencing (NGS) to be located in the same allele. Due to its long-standing duration, molecular evolution of disease was possible; however, both Sanger and NGS of a 2000 skin lesion were unsuccessful due to the poor quality of DNA. Finally, droplet digital PCR using a probe specific for this novel mutation detected the complex BRAF mutation in both the 2000 and 2008 lesions, indicating this case to be ECD with a novel underlying BRAF p.Thr599_Val600delinsArgGlu mutation. Although well at present, molecular modelling of the mutant BRAF suggests suboptimal binding of vemurafenib and hence reduced therapeutic effectiveness.

ETS1 induces transforming growth factor β signaling and promotes epithelial-to-mesenchymal transition in prostate cancer cells: RODGERS et al.

Expression of the transcriptional regulator, E26 transformation‐specific 1 (ETS1), is elevated in human prostate cancers, and this is associated with more aggressive tumor behavior and a rapid progression to castrate‐resistant disease. Multiple ETS1 isoforms with distinct biological activities have been characterized and in 44 matched nonmalignant and malignant human prostate specimens, messenger RNAs for two ETS1 isoforms, ETS1p51 and ETS1p42, were detected, with ETS1p51 levels significantly lower in prostate tumor compared to matched nonmalignant prostate tissues. In contrast, ETS1p51 protein, the only ETS1 isoform detected, was expressed at significantly higher levels in malignant prostate. Analysis of epithelial‐to‐mesenchymal transition (EMT)–associated genes regulated following overexpression of ETS1p51 in the LNCaP prostate cancer cell line predicted promotion of transforming growth factor β (TGFβ) signaling and of EMT. ETS1p51 overexpression upregulated cellular levels of the EMT transcriptional regulators, ZEB1 and SNAIL1, resulted in reduced expression of the mesenchymal marker vimentin with concomitantly elevated levels of claudin 1, an epithelial tight junction protein, and increased prostate cancer cell migration and invasion. ETS1p51‐induced activation of the pro‐EMT TGFβ signaling pathway that was predicted in polymerase chain reaction arrays was verified by demonstration of elevated SMAD2 phosphorylation following ETS1p51 overexpression. Attenuation of ETS1p51 effects on prostate cancer cell migration and invasion by inhibition of TGFβ pathway signaling indicated that ETS1p51 effects were in part mediated by induction of TGFβ signaling. Thus, overexpression of ETS1p51, the predominant ETS1 isoform expressed in prostate tumors, promotes an EMT program in prostate cancer cells in part via activation of TGFβ signaling, potentially accounting for the poor prognosis of ETS1‐overexpressing prostate tumors.

Abstract 1124: ETS1 regulates epithelial-to-mesenchymal transition (EMT) in prostate cancer cells

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: The transcription factor ETS1 is overexpressed in up to 70% of prostate tumors. Three ETS1 isoforms which arise due to alternative splicing have been reported, ETS1p51, ETS1p42 and ETS1p27, each with distinct transcriptional activator or repressor functions. Elevated ETS1 levels in prostate tumors are correlated with more rapid progression to castrate-resistant disease and a poor prognosis, while ETS1 overexpression in vitro is correlated with increased migration and invasion of prostate cancer cells. Aim: To characterise ETS1 isoform expression in prostate tumors and to determine the effects of ETS1 overexpression on the regulation of epithelial-to-mesenchymal transition (EMT). Methods: ETS1 isoform expression was characterised in 45 malignant prostate and adjacent non-malignant prostatic tissues using isoform-specific RT-qPCR and western blotting. Expression of genes encoding regulators of EMT was screened using a Human EMT PCR array (SABiosciences) in the human prostate cancer cell line, LNCaP that had been transiently transfected to overexpress ETS1p51. RT-qPCR and western blotting were used to confirm the ETS1-induced levels of differentially expressed genes/proteins and western blotting for phospho-SMAD2 (pSMAD2)/total SMAD2 (tSMAD2) was used to investigate activation of TGF-β signalling. Results: ETS1p51 was the predominant ETS1 isoform expressed in both the normal prostate and prostate tumors, with significantly decreased mRNA (p 1.5 fold) levels of pro-EMT regulators including members of the WNT (WNT5B, β-catenin) and TGF-β (TGF-β1/2/3) signalling pathways. Phosphorylation of the TGF-β signalling intermediate, SMAD2 was increased and mRNA/protein levels of the EMT transcriptional regulators, TWIST1, SNAIL1, SLUG and ZEB1 were also elevated. In ETS1p51 overexpressing cells, expression of the mesenchymal marker vimentin was increased, while expression of the epithelial tight-junction molecule, Claudin-1 was decreased. Together with increased expression of effectors of migration and invasion (e.g. SPARC, MMP2/9), the findings indicate induction of an EMT programme. Conclusions: Levels of ETS1p51, the predominant ETS1 isoform expressed in the prostate are upregulated by post-transcriptional mechanisms in prostate tumors. Increased ETS1p51 expression promotes TGF-β signalling and EMT, processes that are likely to contribute to the accelerated disease progression and poorer outcomes associated with ETS1 overexpressing prostate tumors. Citation Format: Jamie J. Rodgers, Michael Epis, Ronald Cohen, Peter Leedman, Jennet Harvey, Marc Thomas, Jacqueline Bentel. ETS1 regulates epithelial-to-mesenchymal transition (EMT) in prostate cancer cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1124. doi:10.1158/1538-7445.AM2014-1124

Abstract 4075: Regulation of NKX3.1 by RMND5 proteins.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Expression of the prostatic tumour suppressor, NKX3.1 is reduced or undetectable in a large proportion of advanced human prostate tumours. Although loss of heterozygosity (LOH) is common at the 8p21.2 locus which encompasses the NKX3.1 gene, mutations of the remaining NKX3.1 allele that would account for loss of NKX3.1 expression have not been identified. Indeed, the documented discordance between NKX3.1 mRNA and protein expression suggests that translational or posttranslational mechanisms contribute to the loss of NKX3.1 expression in prostate tumour tissues. We have identified two novel NKX3.1 binding proteins in LNCaP cells, RMND5A and RMND5B. The RMND5A gene locus at 2p11.1 is deleted or amplified in a variety of cancers, while the RMND5B locus at 5q35.3 undergoes frequent LOH in breast tumours from BRCA1 and BRCA2 mutation carriers and is located within an uncharacterised prostate cancer heritability locus. RMND5 proteins are widely expressed, multidomain proteins that each contain a putative RING domain, suggesting that they are able to function as E3 ubiquitin ligases. In in vitro ubiquitin assays, the RING domain of RMND5A was able to associate with UbcH2, UbcH5b and UbcH5c, while the RING domain of RMND5B interacted with UbcH5b and UbcH5c to mediate ubiquitin transfer. Both RMND5A and RMND5B were associated with ubiquitinated proteins in vivo and the increased expression of RMND5A or RMND5B augmented cellular levels of ubiquitinated proteins, providing further evidence of their E3 ubiquitin ligase activity. Overexpression of either RMND5A or RMND5B in LNCaP cells resulted in dose-dependent declines in endogenous NKX3.1 levels, which were attenuated following proteasome inhibition. In addition, RMND5A or RMND5B overexpression enhanced cellular levels of ubiquitinated NKX3.1. In LNCaP cells, RMND5 proteins were diffusely distributed in the cytoplasm and nucleus, with RMND5B occasionally exhibiting a punctate cytoplasmic distribution. When NKX3.1 and RMND5A or RMND5B were overexpressed, RMND5 proteins became predominantly nuclear, while the nuclear levels of NKX3.1 were markedly reduced. Together, these findings indicate that RMND5 proteins promote NKX3.1 ubiquitination and proteasome-dependent degradation and additionally modify the intracellular localisation of NKX3.1, potentially regulating its function as a transcription factor. Citation Format: Alison Louw, Marc A. Thomas, Jennet Harvey, Jacqueline M. Bentel. Regulation of NKX3.1 by RMND5 proteins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4075. doi:10.1158/1538-7445.AM2013-4075

Abstract B7: ETS1 overexpression in prostate cancer cells

Abstract The ETS1 transcription factor is overexpressed in human prostate cancers in comparison to nonmalignant prostate and exhibits nuclear, in addition to aberrant cytoplasmic localisation. A previous report has identified direct interaction between ETS1 and the androgen receptor (AR) in the prostate cancer cell line, LNCaP and we have demonstrated that this ligand-dependent interaction is mediated via the ETS (DNA binding) domain of ETS1 and the ligand-binding domain of the AR. In a cohort of 44 prostate cancer cases, mRNA for the full-length ETS1 isoform, p54ETS1 was detected in all prostate tumour and adjacent nonmalignant prostate tissues, while expression of the internally truncated p42 ETS1 isoform was very low or undetectable. In protein lysates extracted from the same cases, p54 ETS1 was detected in all samples, with expression of a lower molecular weight ETS1 corresponding in size to the dominant negative p27 ETS1 isoform also observed. Increased expression of one or both of the ETS1 isoforms was identified in 38 (86%) cases, supporting the elevated expression of ETS1 in human prostate tumours. Both ETS1 and the AR upregulate expression of the prostatic tumour suppressor, NKX3.1 via sequences located in the 5′ promoter and 3′ untranslated region (3′UTR), respectively. Although unresponsive to androgens, the proximal ∼2kb of the NKX3.1 promoter, and in particular a 76bp sequence surrounding the ETS binding site (EBS) become androgen responsive following ETS1 overexpression. In addition, in LNCaP cells that stably overexpress p54 ETS1 , the biphasic proliferative responses to androgens, where low (≤10 −10 M) DHT concentrations stimulate proliferation and higher (>10 −10 M) DHT levels inhibit proliferation, were altered and DHT concentrations as low as 10 −12 M inhibited proliferation. The findings indicate that the increased expression of ETS1 commonly observed in human prostate tumours may modify the androgen responsiveness of the cancer cells, augmenting AR-mediated regulation of androgen target genes and potentially sensitising prostate cancer cells to the very low androgen levels in men treated with androgen ablation therapies. Citation Format: Jacqueline M. Bentel, Jennet Harvey, Marc A. Thomas, Jamie J. Rodgers, Ebony J. Rouse, Darren M. Preece, Siaw M. Chai, Michael R. Epis, Ronald J. Cohen, Peter J. Leedman, Kimberley Roehrig. ETS1 overexpression in prostate cancer cells [abstract]. In: Proceedings of the AACR Special Conference on Advances in Prostate Cancer Research; 2012 Feb 6-9; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(4 Suppl):Abstract nr B7.

Abstract 1707: Androgen responsiveness of the NKX3.1 gene is regulated by the proto-oncogene ETS1

Expression of the prostatic homeodomain protein NKX3.1 is androgen-regulated in both the normal prostate and in prostate cancer cells. Androgen responsiveness of NKX3.1 is mediated in part by the 3’ untranslated region (39UTR) of the NKX3.1 gene, within which we have isolated 3 discrete regions of androgen-induced transcriptional regulatory activity including 2 androgen response elements (AREs). The proximal ∼2kb of the 5’ promoter of the NKX3.1 gene is incompletely characterised, however it is not androgen-responsive using conventional luciferase reporter assays. Investigation of the NKX3.1 proximal promoter has identified regulation of promoter activity by the ETS transcription factor, ETS1, which is mediated by an ETS binding site (EBS) located at −1008 relative to the NKX3.1 transcription start site. Overexpression of ETS1 in the LNCaP prostate cancer cell line results in upregulation of NKX3.1 protein levels, supporting a functional role of ETS1 in the regulation of NKX3.1 expression. ETS1 has been reported previously to transactivate the androgen receptor (AR) in a ligand-dependent manner on a subset of androgen-responsive promoters, suggesting cooperativity or co-regulation of AR and ETS1 signalling in prostate cancer cells. Treatment of LNCaP cells with 10 −8 M 5α-dihydrotestosterone (DHT) results in upregulation of endogenous ETS1 protein levels, while overexpression of exogenous ETS1 causes a concomitant increase in AR expression. A cooperative interaction between ETS1 and the AR on the NKX3.1 promoter is evident in LNCaP cells whereby overexpression of ETS1 is able to induce androgen responsiveness of the initial ∼2kb of the NKX3.1 promoter sequence and in particular the 76bp region surrounding the EBS. Reversal of DHT effects by the antiandrogen bicalutamide indicates an AR-mediated mechanism. ETS1 is overexpressed in a high proportion of prostate cancers with its expression aberrantly localised in the cytoplasm of a subset of prostate tumour cells. Findings of the present study support a complex mechanism of AR-mediated regulation of NKX3.1 expression via multiple regions of the NKX3.1 gene and modified by additional transcription factors in prostate cancer cells. Abnormal expression and/or function of these factors may contribute to the transcriptional deregulation of NKX3.1 expression and the downregulation of NKX3.1 protein levels that is frequently observed in advanced prostate tumours. Similarly, it is also feasible that upregulation of ETS1 contributes to the deregulation of AR signalling in advanced prostate cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1707.