Abstract Taxanes exert anticancer activity by binding beta-tubulin on the luminal surface of microtubules, stabilizing protofilaments, suppressing GTP hydrolysis, and thereby promoting drug-target engagement (DTE) and apoptotic cell death. Access of taxanes to luminal binding sites depends on protofilament organization and microtubule lattice integrity, including entry via plus-end openings, small lattice pores, and stress-induced lattice defects. Because microtubules are tightly coupled to the actin cytoskeleton through crosslinking and mechanical support, we hypothesized that kinases controlling actin-microtubule architecture modulate taxane DTE and sensitivity. We investigated the PAK6-LIMK1 signaling axis in human prostate cancer PC3 and DU145 cells using CRISPR/Cas9 PAK6 knockout and small-molecule inhibitors of PAK6 or LIMK1. Taxane chemosensitivity was quantified by cell viability assays, and cytoskeletal organization and DTE were assessed by immunofluorescence microscopy of F-actin, alpha-tubulin, and the +TIP protein CLIP170. Consistent with prior shRNA data, genetic ablation of PAK6 markedly increased taxane sensitivity in both cell lines. PAK6 loss or pharmacologic inhibition produced concordant, distinctive remodeling of the actin-microtubule network, characterized by loss of dorsal, ventral, and transverse stress fibers and the emergence of crooked, banded microtubule cables, indicating compromised cytoskeletal integrity. These structural changes were accompanied by enhanced taxane-induced microtubule bundling and a robust increase in CLIP170 comet number and size, identifying a novel cytoskeletal signature associated with heightened taxane responsiveness. Similar phenotypes and chemosensitization were observed following LIMK1 inhibition, supporting a shared PAK6-LIMK1 pathway that dually regulates actin and microtubule dynamics. Together, these data suggest that disruption of PAK6-LIMK1 signaling weakens cytoskeletal resilience, facilitates taxane access to intraluminal binding sites, and thereby amplifies DTE and cell killing in prostate cancer cells, nominating this axis as a tractable target to overcome taxane resistance in advanced disease. Citation Format: Felipe Carvolho, Tanvi Desai, Keira Ly, Joubin Jebelli, Ajay Bommareddy, Jianning Wei, Michael L. Lu. Target actin-microtubule network integrity to enhance taxane drug-target engagement and chemosensitivity in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2945.

BACKGROUND/AIM:α-Santalol, a major component of sandalwood oil, has been shown to have chemopreventive and antitumor effects in different pre-clinical cancer models. The present study was undertaken to determine the in vitro efficacy of α-santalol on SK-MEL2 human melanoma cells and an immortalized human keratinocyte cell line (HaCaT). MATERIALS AND METHODS:In this study, we employed 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, Trypan blue, wound-healing, and annexin V apoptosis assays, as well as confocal microscopy for imaging F-actin rhodamine phalloidin/4',6-diamidino-2-phenylindole-stained cells to investigate the cytotoxicity, cell viability, migratory potential and apoptotic cell death, respectively, of cells treated with different concentrations of α-santalol or dimethyl sulfoxide for different time periods. RESULTS:Results showed that α-santalol treatment significantly reduced SK-MEL2 cell viability and wound-healing ability, while only affecting HaCaT cells at higher concentrations. α-Santalol treatment also disrupted cytoskeletal structure and F-actin in SK-MEL2 cells, whereas HaCaT cells were more resistant to this effect. CONCLUSION:The selective growth-inhibitory and anti-migratory effects of α-santalol on human melanoma cells warrants future studies to systemically explore the mechanistic details involved.

We previously identified solute carrier family 7 member 2 (SLC7A2) as one of the top upregulated genes when normal Huntingtin was deleted. SLC7A2 has a high affinity for l-arginine. Arginine is implicated in inflammatory responses, and SLC7A2 is an important regulator of innate and adaptive immunity in macrophages. Although neuroinflammation is clearly demonstrated in animal models and patients with Huntington’s disease (HD), the question of whether neuroinflammation actively participates in HD pathogenesis is a topic of ongoing research and debate. Here, we studied the role of SLC7A2 in mediating the neuroinflammatory stress response in HD cells. RNA sequencing (RNA-seq), quantitative RT-PCR and data mining of publicly available RNA-seq datasets of human patients were performed to assess the levels of SLC7A2 mRNA in different HD cellular models and patients. Biochemical studies were then conducted on cell lines and primary mouse astrocytes to investigate arginine metabolism and nitrosative stress in response to neuroinflammation. The CRISPR–Cas9 system was used to knock out SLC7A2 in STHdhQ7 and Q111 cells to investigate its role in mediating the neuroinflammatory response. Live-cell imaging was used to measure mitochondrial dynamics. Finally, exploratory studies were performed using the Enroll-HD periodic human patient dataset to analyze the effect of arginine supplements on HD progression. We found that SLC7A2 is selectively upregulated in HD cellular models and patients. HD cells exhibit an overactive response to neuroinflammatory challenges, as demonstrated by abnormally high iNOS induction and NO production, leading to increased protein nitrosylation. Depleting extracellular Arg or knocking out SLC7A2 blocked iNOS induction and NO production in STHdhQ111 cells. We further examined the functional impact of protein nitrosylation on a well-documented protein target, DRP-1, and found that more mitochondria were fragmented in challenged STHdhQ111 cells. Last, analysis of Enroll-HD datasets suggested that HD patients taking arginine supplements progressed more rapidly than others. Our data suggest a novel pathway that links arginine uptake to nitrosative stress via upregulation of SLC7A2 in the pathogenesis and progression of HD. This further implies that arginine supplements may potentially pose a greater risk to HD patients.

P21 activated kinase 6 (PAK6) is a serine-threonine kinase with physiological expression enriched in the brain and overexpressed in a number of human tumors. While the role of PAK6 in cancer cells has been extensively investigated, the physiological function of the kinase in the context of brain cells is poorly understood. Our previous work uncovered a link between PAK6 and the Parkinson's disease (PD)-associated kinase LRRK2, with PAK6 controlling LRRK2 activity and subcellular localization via phosphorylation of 14-3-3 proteins. Here, to gain more insights into PAK6 physiological function, we performed protein-protein interaction arrays and identified a subgroup of PAK6 binders related to ciliogenesis. We confirmed that endogenous PAK6 localizes at both the centrosome and the cilium, and positively regulates ciliogenesis not only in tumor cells but also in neurons and astrocytes. Strikingly, PAK6 rescues ciliogenesis and centrosomal cohesion defects associated with the G2019S but not the R1441C LRRK2 PD mutation. Since PAK6 binds LRRK2 via its GTPase/Roc-COR domain and the R1441C mutation is located in the Roc domain, we used microscale thermophoresis and AlphaFold2-based computational analysis to demonstrate that PD mutations in LRRK2 affecting the Roc-COR structure substantially decrease PAK6 affinity, providing a rationale for the differential protective effect of PAK6 toward the distinct forms of mutant LRRK2. Altogether, our study discloses a novel role of PAK6 in ciliogenesis and points to PAK6 as the first LRRK2 modifier with PD mutation-specificity.

The lack of physiologically relevant human esophageal cancer models has as a result that many esophageal cancer studies are encountering major bottleneck challenges in achieving breakthrough progress. To address the issue, here we engineered a 3D esophageal tumor tissue model using a biomimetic decellularized esophageal matrix in a customized bioreactor. To obtain a biomimetic esophageal matrix, we developed a detergent-free, rapid decellularization method to decellularize porcine esophagus. We characterized the decellularized esophageal matrix (DEM) and utilized the DEM for the growth of esophageal cancer cell KYSE30 in well plates and the bioreactor. We then analyzed the expression of cancer-related markers of KYSE30 cells and compared them with formalin-fixed, paraffin-embedded (FFPE) esophageal squamous cell carcinoma (ESCC) tissue biospecimens. Our results show that the detergent-free decellularization method preserved the esophageal matrix components and effectively removed cell nucleus. KYSE30 cancer cells proliferated well on and inside the DEM. KYSE30 cells cultured on the DEM in the dynamic bioreactor show different cancer marker expressions than those in the static well plate, and also share some similarities to the FFPE-ESCC biospecimens. These findings built a foundation with potential for further study of esophageal cancer behavior in a biomimetic microenvironment using this new esophageal cancer model.

Huntingtin (Htt) is a large protein without clearly defined molecular functions. Mutation in this protein causes Huntington's disease (HD), a fatal inherited neurodegenerative disorder. Identification of Htt-interacting proteins by the traditional approaches including yeast two-hybrid systems and affinity purifications has greatly facilitated the understanding of Htt function. However, these methods eliminated the intracellular spatial information of the Htt interactome during sample preparations. Moreover, the temporal changes of the Htt interactome in response to acute cellular stresses cannot be easily resolved with these approaches. Ascorbate peroxidase (APEX2)-based proximity labeling has been used to spatiotemporally investigate protein-protein interactions in living cells. In this study, we generated stable human SH-SY5Y cell lines expressing full-length Htt23Q and Htt145Q with N-terminus tagged Flag-APEX2 to quantitatively map the spatiotemporal changes of Htt interactome to a mild acute proteotoxic stress. Our data revealed that normal and mutant Htt (muHtt) are associated with distinct intracellular microenvironments. Specifically, mutant Htt is preferentially associated with intermediate filaments and myosin complexes. Furthermore, the dynamic changes of Htt interactomes in response to stress are different between normal and mutant Htt. Vimentin is identified as one of the most significant proteins that preferentially interacts with muHtt in situ. Further functional studies demonstrated that mutant Htt affects the vimentin's function of regulating proteostasis in healthy and HD human neural stem cells. Taken together, our data offer important insights into the molecular functions of normal and mutant Htt by providing a list of Htt-interacting proteins in their natural cellular context for further studies in different HD models.

Insufficient nutrition exchange and limited transportation of blood supply in a porous only scaffold often hinder bone formation, even though the porous scaffold is loaded with cells or growth factors. To overcome these issues, we developed a cell- and growth factor-free approach to induce bone formation in a critical-size bone defect by using an interconnected porous beta-tricalcium phosphate (β-TCP) scaffold with multiple channels. In vitro cell experimental results showed that multiple channels significantly promoted cell attachment and proliferation of human bone marrow mesenchymal stem cells, stimulated their alkaline phosphatase activity, and up-regulated the osteogenic gene expression. Multiple channels also considerably stimulated the expression of various mechanosensing markers of the cells, such as focal adhesion kinase, filamentous actin, and Yes-associated protein-1 at both static and dynamic culturing conditions. The in vivo bone defect implantation results demonstrated more bone formation inside multiple-channeled scaffolds compared to non-channeled scaffolds. Multiple channels prominently accelerated collagen type I, bone sialoprotein and osteocalcin protein expression. Fluorochrome images and angiogenic marker CD31 staining exhibited more mineral deposition and longer vasculature structures in multiple-channeled scaffolds, compared to non-channeled scaffolds. All the findings suggested that the creation of interconnected multiple channels in the porous β-TCP scaffold is a very promising approach to promote bone tissue regeneration.

Background Huntingtin (Htt) protein is the product of the gene mutated in Huntington's disease (HD), a fatal, autosomal dominant, neurodegenerative disorder. Normal Htt is essential for early embryogenesis and the development of the central nervous system. However, the role of Htt in adult tissues is less defined. Following the recent promising clinical trial in which both normal and mutant Htt mRNA were knocked down in HD patients, there is an urgent need to fully understand the molecular consequences of knocking out/down Htt in adult tissues. Htt has been identified as an important transcriptional regulator. Unbiased investigations of transcriptome changes with RNA-sequencing (RNA-Seq) have been done in multiple cell types in HD, further confirming that transcriptional dysregulation is a central pathogenic mechanism in HD. However, there is lack of direct understanding of the transcriptional regulation by normal Htt. Methods To investigate the transcriptional role of normal Htt, we first knocked out Htt in the human neuroblastoma SH-SY5Y cell line using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR-associated protein 9) gene editing approach. We then performed RNA-seq analysis on Htt-null and wild type SH-SY5Y cells to probe the global transcriptome changes induced by Htt deletion. Results In general, Htt has a widespread effect on gene transcription. Functional analysis of the differentially expressed genes (DEGs) using various bioinformatic tools revealed irregularities in pathways related to cell communication and signaling, and more specifically those related to neuron development, neurotransmission and synaptic signaling. We further examined the transcription factors that may regulate these DEGs. Consistent with the disrupted pathways associated with cellular development, we showed that Htt-null cells exhibited slower cell proliferation than wild type cells. We finally validated some of the top DEGS with quantitative RT-PCR. Conclusions The widespread transcriptome changes in Htt-null cells could be directly caused by the loss of Htt-mediated transcriptional regulation or due to the secondary consequences of disruption in the gene regulatory network. Our study therefore provides valuable information about key genes associated with Htt-mediated transcription and improves our understanding of the molecular mechanisms underlying the cellular functions of normal and mutant Htt.

Many decellularized extracellular matrix-derived whole organs have been widely used in studies of tissue engineering and cancer models. However, decellularizing porcine esophagus to obtain decellularized esophageal matrix (DEM) for potential biomedical applications has not been widely investigated. In this study a modified decellularization protocol was employed to prepare a porcine esophageal DEM for the study of cancer cell growth. The cellular removal and retention of matrix components in the porcine DEM were fully characterized. The microstructure of the DEM was observed using scanning electronic microscopy. Human esophageal squamous cell carcinoma (ESCC) and human primary esophageal fibroblast cells (FBCs) were seeded in the DEM to observe their growth. Results show that the decellularization process did not cause significant loss of mechanical properties and that blood ducts and lymphatic vessels in the submucosa layer were also preserved. ESCC and FBCs grew on the DEM well and the matrix did not show any toxicity to cells. When FBS and ESCC were cocultured on the matrix, they secreted more periostin, a protein that supports cell adhesion on matrix. This study shows that the modified decellularization protocol can effectively remove the cell materials and maintain the microstructure of the porcine esophageal matrix, which has the potential application of studying cell growth and migration for esophageal cancer models.

Neurons are susceptible to different cellular stresses and this vulnerability has been implicated in the pathogenesis of Huntington’s disease (HD). Accumulating evidence suggest that acute or chronic stress, depending on its duration and severity, can cause irreversible cellular damages to HD neurons, which contributes to neurodegeneration. In contrast, how normal and HD neurons respond during the resolution of a cellular stress remain less explored. In this study, we challenged normal and HD cells with a low-level acute ER stress and examined the molecular and cellular responses after stress removal. Using both striatal cell lines and primary neurons, we first showed the temporal activation of p-eIF2α-ATF4-GADD34 pathway in response to the acute ER stress and during recovery between normal and HD cells. HD cells were more vulnerable to cell death during stress recovery and were associated with increased number of apoptotic/necrotic cells and decreased cell proliferation. This is also supported by the Gene Ontology analysis from the RNA-seq data which indicated that “apoptosis-related Biological Processes” were more enriched in HD cells during stress recovery. We further showed that HD cells were defective in restoring global protein synthesis during stress recovery and promoting protein synthesis by an integrated stress response inhibitor, ISRIB, could attenuate cell death in HD cells. Together, these data suggest that normal and HD cells undergo distinct mechanisms of transcriptional reprogramming, leading to different cell fate decisions during the stress recovery.

Proteotoxic stress plays an important role in the pathogenesis of Huntington's disease (HD). Autophagy is proposed as a compensatory mechanism to remove protein aggregates under proteotoxic stress by up-regulating p62 expression. In the present study, we investigated the molecular action of p62 to proteotoxic stress in HD cells. Using two different HD cellular models, STHdhQ7 and STHdhQ111 cells derived from wild type and HD knock-in mice and human fibroblasts from healthy and HD patients, we found that HD cells are more vulnerable to cell death under proteotoxic stress and during stress recovery. We further showed that P62 was up-regulated in both STHdhQ7 and STHdhQ111 cells in response to the stress with distinct subcellular localization patterns. While dispersed p62 puncti were found in STHdhQ7 cells, p62 bodies were initially present in the lysosomes and accumulated to the juxtanuclear regions of STHdhQ111 cells as MG132 incubation continued. Unlike in STHdhQ7 cells, p62 puncti were not associated with K48-linked polyubiquitinated protein aggregates or proteasomal components in STHdhQ111. Interestingly, addition of cysteine during MG132 incubation rescued cell death in STHdhQ111 cells caused by stress recovery and altered the subcellular distribution of p62. Our data suggest that aberrant positioning of p62 affects the proteasomal clearance of protein aggregates and may contribute to the increased vulnerability to proteotoxic stress-induced cell death in HD cells.

Abstract Centrosome abnormalities are linked to genomic instability and are considered one possible cause of cancer progression. In prostate and breast cancer, centrosome over-duplication is correlated with lymph node and distant metastasis. In prostate cancers, centrosome amplification is a useful predictor of tumor recurrence, highlighting its potential role in promoting advanced disease. PAK6 was identified as an androgen receptor (AR) interacting protein. It has been determined that activated PAK6 plays an active role in promoting hormone-regulated prostate cancer metastatic phenotypes such as upregulating cell motility and invasiveness. Several lines of evidence indicate that PAK6 is overexpressed in advanced cancers including prostate, colon and liver cancers. The up-regulated expression of PAK6 in advanced diseases underscores its potential role in disease progression. Using a novel anti-PAK6 antibody readily recognizes the native PAK6 protein, we determined that PAK6 localizes at centrosome and in some cell types also at plasma membrane. This observation revised our previous view of PAK6 as a soluble cytoplasmic protein. Ectopically expression of PAK6 leads to hyperploidy in LNCap cells. We hypothesized that aberrant PAK6 expression plays a critical role in the development of hyperploidy. In current study, we characterized the role of PAK6 in the dysregulation of centrosome-triggered hyperploidy in cancer cells. PAK6 centrosome localization was determined by co-staining of PAK6 with centrosome marker γ-tubulin using immunofluorescence microscopy of a pair of mouse embryo fibroblasts (MEF) derived either from WT mice (MEF-WT) or from PAK6-null mice (MEF-KO). PAK6-centrosome association was also confirmed biochemically by co-fractionation of PAK6 with γ-tubulin in centrosomal subcellular-fractions using a sucrose gradient. Down-regulation of PAK6 expression by lentivirus mediated shRNA promotes centrosome amplification with increased centrosome size in LNCap and MCF7 cells. Accelerated microtubule aster regrowth following nocodazole-induced depolarization was observed in MEF KO cells in as compared to MEF WT cells. Reciprocally, the inhibitory effects of PAK6 overexpression on aster growth was demonstrated in tetracycline inducible U2OS cells. Our results indicate that PAK6 on one hand promotes aneuploidy, but on the other hand its expression suppresses centrosome amplification and microtubule dynamics. These apparent incompatible results suggest that PAK6 functions at multiple levels that in one hand it regulates centrosome amplification and microtubule dynamics, and on the other hand it may be involved in regulating the chromosome segregation and cytokinesis. The net effects of PAK6 expression may be context dependent. Citation Format: Houjun Jia, Danda Chapagai, John Mallow, Michael D. Perttunen, Vijaya Iragavarapu, Jianning Wei, Michael L. Lu. PAK6 localizes to centrosome and modulates centrosome homeostasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3453.

Huntington's disease (HD) is a hereditary and devastating neurodegenerative disorder caused by a mutation in the huntingtin protein. Understanding the functions of normal and mutant huntingtin protein is the key to revealing the pathogenesis of HD and developing therapeutic targets. Huntingtin plays an important role in vesicular and organelle trafficking. Lysosomes are dynamic organelles that integrate several degradative pathways and regulate the activity of mammalian target of rapamycin complex 1 (mTORC1). In the present study, we found that the perinuclear accumulation of lysosomes was increased in a cellular model of HD derived from HD knock-in mice and primary fibroblasts from an HD patient. This perinuclear lysosomal accumulation could be reversed when normal huntingtin was overexpressed in HD cells. When we further investigated the functional significance of the increased perinuclear lysosomal accumulation in HD cells, we demonstrated that basal mTORC1 activity was increased in HD cells. In addition, autophagic influx was also increased in HD cells in response to serum deprivation, which leads to premature fusion of lysosomes with autophagosomes. Taken together, our data suggest that the increased perinuclear accumulation of lysosomes may play an important role in HD pathogenesis by altering lysosomal-dependent functions.

Replication of the integrated HIV-1 genome is tightly regulated by a series of cellular factors. In previous work we showed that transactivation of the HIV-1 promoter is regulated by the cellular splicing factor SRSF1. Here we report that SRSF1 can downregulate the replication of B, C, and D subtype viruses by >200-fold in a cell culture system. We show that viral transcription and splicing are inhibited by SRSF1 expression. Furthermore, SRSF1 deletion mutants containing the protein RNA-binding domains but not the arginine serine-rich activator domain can downregulate viral replication by >2,000-fold with minimal impact on cell viability and apoptosis. These data suggest a therapeutic potential for SRSF1 and its RNA-binding domains.

Heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) is one of the most abundant RNA binding proteins. hnRNP A1 is localized prevalently in the nucleus but it can relocate to the cytoplasm in response to specific stimuli shuttling between nuclear and cytoplasmic compartments. The cellular localization of this protein is regulated by a short C-terminus motif (M9) and other less defined sequences. The RNA binding specificity of this protein is dependent on multiple RNA binding domains (RBDs), which regulate its role in RNA processing and expression. hnRNP A1 plays multiple roles in gene expression by regulating the biogenesis and translation of messengers RNAs, the processing of miRNAs, affecting transcription and controlling telomere maintenance. The multiple functions of this protein correlate with diverse roles in genetic disease, cancer and the replication of viral pathogens. Utilizing a tagged hnRNP A1 deletion library we have shown that the three hnRNP A1 RBDs contribute to the prevalent nuclear distribution of the protein. Our data also indicate that a truncated form of the protein, lacking one of the RBDs, the RGG-box, can regulate splicing of a splicing reporter minigene and down-regulate replication of the HIV-1 virus with efficiency comparable to the wild-type protein. This functional hnRNP A1 deletion mutant is similar to a predicted hnRNP A1 isoform, which had not been previously experimentally characterized.

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 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

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

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.