Michael L. Lu

SOX9 Is Expressed in Human Fetal Prostate Epithelium and Enhances Prostate Cancer Invasion

SOX9 is a transcription factor that plays a critical role in the development of multiple tissues. We previously reported that SOX9 in normal human adult prostate was restricted to basal epithelium. SOX9 was also expressed in a subset of prostate cancer (PCa) cells and was increased in relapsed hormone-refractory PCa. Moreover, SOX9 expression in PCa cell lines enhanced tumor cell proliferation and was beta-catenin regulated. Here we report additional in vivo results showing that SOX9 is highly expressed during fetal prostate development by epithelial cells expanding into the mesenchyme, suggesting it may contribute to invasive growth in PCa. Indeed, SOX9 overexpression in LNCaP PCa xenografts enhanced growth, angiogenesis, and invasion. Conversely, short hairpin RNA-mediated SOX9 suppression inhibited the growth of CWR22Rv1 PCa xenografts. These results support important functions of SOX9 in both the development and maintenance of normal prostate, and indicate that these functions contribute to PCa tumor growth and invasion.

Increased PAK6 expression in prostate cancer and identification of PAK6 associated proteins

PAK6 is a member of the p21‐activated kinase (PAK) family of serine/threonine kinases that was originally cloned from prostate cancer (PCa) cells as an androgen receptor interacting protein, but its cellular distribution and functions have not been established.

Direct Interaction between AR and PAK6 in Androgen-Stimulated PAK6 Activation

A p21-activated kinase 6 (PAK6) was previously identified to be an androgen receptor (AR) interacting protein through a yeast two-hybrid screening. We used hormone responsive prostate cancer LAPC4 and LNCap cell lines as models to study the signaling events associated with androgen stimulation and PAK6. An androgen-stimulated PAK6 kinase activation was observed in LAPC4 cells expressing endogenous PAK6 and in LNCap cells ectopically expressing a wild type PAK6. This activation was likely mediated through a direct interaction between AR and PAK6 since siRNA knock-down of AR in LAPC4 cells downregulated androgen-stimulated PAK6 activation. In addition, LNCap cells expressing a non-AR-interacting PAK6 mutant exhibited dampened androgen-stimulated kinase activation. As a consequence of androgen-stimulated activation, PAK6 was phosphorylated at multiple serine/threonine residues including the AR-interacting domain of PAK6. Furthermore, androgen-stimulation promoted prostate cancer cell motility and invasion were demonstrated in LNCap cells ectopically expressing PAK6-WT. In contrast, LNCap expressing non-AR-interacting mutant PAK6 did not respond to androgen stimulation with increased cell motility and invasion. Our results demonstrate that androgen-stimulated PAK6 activation is mediated through a direct interaction between AR and PAK6 and PAK6 activation promotes prostate cancer cells motility and invasion.

Abstract 2153: Active PAK6 induces aneuploidy in breast cancer cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL The natural history of breast cancer remains largely undefined. More than 50% of the clinical specimen exhibits aneuploidy. Among these aneuploid cancers, 50% has chromosome copy number near tetraploid. The high incidence of aneupoidy in advanced breast cancer is thought to be a byproduct of chromosome mis-segregation resulting from mutations of checkpoint-regulating tumor suppressor genes associated with disease progression. However, new large-scale sequencing projects reveal that most tumors contain approximately 14-20 different mutant genes, suggesting that genomes and karyotypes of cancer cells are equally heterogeneous. This notion revives the old concept that aneuploidy resulting from chromosome mis-segregation or other similar mechanisms may play an active role leading to transformation and disease progression. A novel p21-activated kinase 6 (PAK6) was recently identified to be an androgen receptor (AR) and estrogen receptor (ER) interacting protein. An estrogen-stimulated PAK6 activation was observed in breast cancer BT474 cells. This activation is mediated by estrogen-stimulated activation of PKA. At the cellular level, active PAK6 promotes breast cancer MCF7 cell metastatic phenotypes including increased cell motility, matrigel invasion and soft agar growth. Further characterization of breast cancer MCF7 cells expressing PAK6 constitutive active mutant revealed an accumulation of cells with higher ploidy as compared to that of parental MCF7. Using a proteomic approach to determine the PAK6 downstream target, we identified that Cdt1, a DNA pre-replicative complex (pre-RC) subunit, is overexpressed in response to PAK6 activation. In eukaryotes, the expression and stability of Cdt1 are strictly regulated to ensure only one round of DNA replication in each cell cycle. Deregulated overexpression of Cdt1 has previously been demonstrated to induce aneuploidy and malignant transformation due to re-initiation of DNA replication within the same S-phase. The identification of Cdt1 overexpression in response to PAK6 activation provides a plausible mechanism for the observed accumulation of higher ploidy cells in MCF7 expressing active PAK6. These findings directly link the hormonal signals to the DNA replication process and maintenance of genomic integrity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2153. doi:1538-7445.AM2012-2153

Abstract 4287: Intracellular trafficking of androgen receptor splice variant AR8.

In a classic paradigm, the intracellular trafficking of a ligand-stimulated androgen receptor involves receptor dimerization, dissociating from chaperones (such as HSPs), interact with nuclear membrane importin and Ran-GTPase to gain access to nucleus; once there, it complexes with transcriptional regulators and binding to target sites. Despite the compelling evidence for AR non-genomic signaling events, the molecular mechanism of how does AR signaling from cytoplasmic or membrane remains undetermined. Several recent studies suggested that membrane association of AR is mediated by palmitoylation of AR at cysteine-806. Two plamitoylation enzymes, DHHC7 and DHHC21, have been implicated in the process of AR palmitoylation. In the current study, we determined the intracellular trafficking of a newly identified AR isoform, AR8. AR8 is a splice variant of the original full-length AR. AR8 was demonstrated to be up-regulated in castration-resistant prostate cancer cells. Structurally, AR8 protein sequence is different from other AR variants with a substitution of all the amino acids after N-terminal domain (NTD) of a conventional AR with an unique C-terminal sequence. Coincidentally, two cysteine residues within the unique c-terminus, positions 558 and 560, were identified to be palmitoylated. The palmitoylation was determined to be responsible for the localization of AR8 to plasma membrane. Using a fluorescent protein mCherry-tagged AR8 and confocal microscopy, we confirmed the plasma membrane localization of AR8 as previously described. We determined that AR8 co-localized with a Golgi marker GM-130 at peri-nuclear regions by immunofluorescence microscopy. The result was further substantiated by co-localizing mCherry-AR8 with galactosyltransferase (GalT) golgi-targeting sequence-fused GFP (GalT-GFP; green fluorescent protein) at Golgi compartments. We also demonstrated that mCherry-AR8 co-localized with EGFR-GFP (epidermal growth factor receptor-GFP) on the plasma membrane within in the cholesterol rich raft domain. Using time-lapse confocal microscopy, we observed that AR8 co-migrated with EGFR in a retrograde fashion and become associated with endosomal compartments at the onset of EGF (5ng/ml) stimulation. Part of these endosomes was later fused with lysosomes as evident from co-localizations of mCherry-AR8 with GFP-LAMP-1. Although the significance of AR8 expression in the progression of prostate cancer remains to be determined, our observations underline a potential intracellular trafficking pathway associated with AR8 are consistent with the original report that EGF stimulation promotes the interactions between AR8 and EGFR. Citation Format: Ciny John, Michael L. Lu. Intracellular trafficking of androgen receptor splice variant AR8. [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 4287. doi:10.1158/1538-7445.AM2013-4287