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Pancreatic ductal adenocarcinoma (PDAC) is a disease with no effective therapeutics. We have developed a novel targeted therapy drug consisting of a tumor-targeting ligand, near-infrared (NIR) organic heptamethine carbocyanine dye (HMCD), and HMG-CoA inhibitor simvastatin (SIM), and assessed its efficacy in PDAC. PDAC cell specific targeting of DZ-SIM was measured by determining the fluorescence in cells and animals. Mitochondrial bioenergetics and functions were measured by Seahorse and flow cytometry, respectively. Apoptosis was assessed by DNA fragmentation, AnnexinV/Propidium Iodide staining, and TUNEL. Markers of cell invasion, epithelial-to-mesenchymal transition, and cancer stemness were measured. The effect of DZ-SIM on survival, tumor growth and metastasis was measured in the Kras

Figure S1: R1 does not affect contact inhibition during prostate cancer cell proliferation; Figure S2: R1 does not affect cell apoptosis in prostate cancer cells under both steady state and dynamic conditions; Figure S3: R1 promotes prostate cancer cell migration and invasion; Figure S4: R1/c-Myc physical interaction is not required for R1 regulation of c-Myc ubiquitination; Figure S5: R1 does not affect MIZ-1 and p53 protein expression in prostate cancer cells.

Background Keratins (KRTs) are intermediate filament proteins that interact with multiple regulatory proteins to initiate signaling cascades. Keratin 13 (KRT13) plays an important role in breast cancer progression and metastasis. The objective of this study is to elucidate the mechanism by which KRT13 promotes breast cancer growth and metastasis. Methods The function and mechanisms of KRT13 in breast cancer progression and metastasis were assessed by overexpression and knockdown followed by examination of altered behaviors in breast cancer cells and in xenograft tumor formation in mouse mammary fat pad. Human breast cancer specimens were examined by immunohistochemistry and multiplexed quantum dot labeling analysis to correlate KRT13 expression to breast cancer progression and metastasis. Results KRT13-overexpressing MCF7 cells displayed increased proliferation, invasion, migration and in vivo tumor growth and metastasis to bone and lung. Conversely, KRT13 knockdown inhibited the aggressive behaviors of HCC1954 cells. At the molecular level, KRT13 directly interacted with plakoglobin (PG, γ-catenin) to form complexes with desmoplakin (DSP). This complex interfered with PG expression and nuclear translocation and abrogated PG-mediated suppression of c-Myc expression, while the KRT13/PG/c-Myc signaling pathway increased epithelial to mesenchymal transition and stem cell-like phenotype. KRT13 expression in 58 human breast cancer tissues was up-regulated especially at the invasive front and in metastatic specimens (12/18) (p Conclusions This study reveals that KRT13 promotes breast cancer cell growth and metastasis via a plakoglobin/c-Myc pathway. Our findings reveal a potential novel pathway for therapeutic targeting of breast cancer progression and metastasis.

A, Nanog and Oct-3/4 expression in tumor and stromal areas of prostates expressing miR-409 compared to control normal prostates, using IHC analysis. B, IHC staining of cytokeratin 8 (CK-8) and cytokeratin 5 (CK-5) in normal control prostate, and tumors of control and miR-409 expressing prostates.

Invasion assay of ARCaPM-TBX2 and ARCaPM-Neo cells

Supplementary Data from β2-Microglobulin Signaling Blockade Inhibited Androgen Receptor Axis and Caused Apoptosis in Human Prostate Cancer Cells

Nuclear membrane blebbing in amoeboid cells. Time-lapse analysis of DU145 DIAPH3-depleted cells treated with EGF and IL6 (exacerbates amoeboid features (13)) and stained with lipid fluorescent dye CellMask Orange.

Legend to Supplementary Figures

Purpose: β2-Microglobulin (β2M) has been shown to promote osteomimicry and the proliferation of human prostate cancer cells. The objective of this study is to determine the mechanism by which targeting β2M using anti-β2M antibody inhibited growth and induced apoptosis in prostate cancer cells.Experimental Design: Polyclonal and monoclonal β2M antibodies were used to interrupt β2M signaling in human prostate cancer cell lines and the growth of prostate tumors in mice. The effects of the β2M antibody on a survival factor, androgen receptor (AR), and its target gene, prostate-specific antigen (PSA) expression, were investigated in cultured cells and in tumor xenografts.Results: The β2M antibody inhibited growth and promoted apoptosis in both AR-positive and PSA-positive, and AR-negative and PSA-negative, prostate cancer cells via the down-regulation of the AR in AR-positive prostate cancer cells and directly caused apoptosis in AR-negative prostate cancer cells in vitro and in tumor xenografts. The β2M antibody had no effect on AR expression or the growth of normal prostate cells.Conclusions: β2M downstream signaling regulates AR and PSA expression directly in AR-positive prostate cancer cells. In both AR-positive and AR-negative prostate cancer cells, interrupting β2M signaling with the β2M antibody inhibited cancer cell growth and induced its apoptosis. The β2M antibody is a novel and promising therapeutic agent for the treatment of human prostate cancers.

Plot showing relative protein expression of TBX2 and WNT3A in human PCa cell lines

Gleason scores of the cancer from which the TMA cores were obtained and the miR-409 intensity counts by ISH-QD. None of the TMA cores had any treatment.

Invasion assay of PC3 (TBX2DN+WNT3A, TBX2DN, and Neo)cells with the addition of SFRP-2

Supplementary Materials and Methods

Supplementary Methods, Figure Legends from miR-409-3p/-5p Promotes Tumorigenesis, Epithelial-to-Mesenchymal Transition, and Bone Metastasis of Human Prostate Cancer

Abnormalities in nuclear shape are a well-known feature of cancer, but their contribution to malignant progression remains poorly understood. Here, we show that depletion of the cytoskeletal regulator, Diaphanous-related formin 3 (DIAPH3), or the nuclear membrane–associated proteins, lamin A/C, in prostate and breast cancer cells, induces nuclear shape instability, with a corresponding gain in malignant properties, including secretion of extracellular vesicles that contain genomic material. This transformation is characterized by a reduction and/or mislocalization of the inner nuclear membrane protein, emerin. Consistent with this, depletion of emerin evokes nuclear shape instability and promotes metastasis. By visualizing emerin localization, evidence for nuclear shape instability was observed in cultured tumor cells, in experimental models of prostate cancer, in human prostate cancer tissues, and in circulating tumor cells from patients with metastatic disease. Quantitation of emerin mislocalization discriminated cancer from benign tissue and correlated with disease progression in a prostate cancer cohort. Taken together, these results identify emerin as a mediator of nuclear shape stability in cancer and show that destabilization of emerin can promote metastasis.Significance: This study identifies a novel mechanism integrating the control of nuclear structure with the metastatic phenotype, and our inclusion of two types of human specimens (cancer tissues and circulating tumor cells) demonstrates direct relevance to human cancer.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/21/6086/F1.large.jpg. Cancer Res; 78(21); 6086–97. ©2018 AACR.

Schematic depiction of WNT3A gene promoter, and ChIP analysis of LNCaP-TBX2 and LNCaP-Neo cells to assess the binding of TBX2 on WNT3A promoter

Emerin deregulation links nuclear shape instability to metastatic potential

Supplementary Fig S2. (A) Expression of miR-409-3p and miR-154* assayed by qRT- PCR in ARCaPM-C control PCa cells and ARCaPM-cluster inhibitor transduced cells. Data is normalized to RNU6B. (B) Morphological changes in ARCaPM-cluster inhibitor compared to ARCaPM-C control PCa cells. (C) E-cadherin and N-cadherin mRNA assayed in ARCaPM-C control PCa cells and ARCaPM cluster inhibitor expressing PCa cells by qRT-PCR, normalized to 18s RNA. (D) Invasion assay of in ARCaPM-C and ARCaPM-cluster inhibitor expressing PCa cells. *: p

Purpose: miR-409-3p/-5p is a miRNA expressed by embryonic stem cells, and its role in cancer biology and metastasis is unknown. Our pilot studies demonstrated elevated miR-409-3p/-5p expression in human prostate cancer bone metastatic cell lines; therefore, we defined the biologic impact of manipulation of miR-409-3p/-5p on prostate cancer progression and correlated the levels of its expression with clinical human prostate cancer bone metastatic specimens.Experimental Design: miRNA profiling of a prostate cancer bone metastatic epithelial-to-mesenchymal transition (EMT) cell line model was performed. A Gleason score human tissue array was probed for validation of specific miRNAs. In addition, genetic manipulation of miR-409-3p/-5p was performed to determine its role in tumor growth, EMT, and bone metastasis in mouse models.Results: Elevated expression of miR-409-3p/-5p was observed in bone metastatic prostate cancer cell lines and human prostate cancer tissues with higher Gleason scores. Elevated miR-409-3p expression levels correlated with progression-free survival of patients with prostate cancer. Orthotopic delivery of miR-409-3p/-5p in the murine prostate gland induced tumors where the tumors expressed EMT and stemness markers. Intracardiac inoculation (to mimic systemic dissemination) of miR-409-5p inhibitor–treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival compared with control vehicle–treated cells.Conclusion: miR-409-3p/-5p plays an important role in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding bears particular translational importance as miR-409-3p/-5p appears to be an attractive biomarker and/or possibly a therapeutic target to treat bone metastatic prostate cancer. Clin Cancer Res; 20(17); 4636–46. ©2014 AACR.

Micro-CT images of mouse tibiae injected with ARCaPM-TBX2 and ARCaPM-Neo cells

Emerin deregulation links nuclear shape instability to metastatic potential

Identification of factors that mediate visceral and bone metastatic spread and subsequent bone remodeling events is highly relevant to successful therapeutic intervention in advanced human prostate cancer. TBX2, a T-box family transcription factor that negatively regulates cell-cycle inhibitor p21, plays critical roles during embryonic development, and recent studies have highlighted its role in cancer. Here, we report that TBX2 is overexpressed in human prostate cancer specimens and bone metastases from xenograft mouse models of human prostate cancer. Blocking endogenous TBX2 expression in PC3 and ARCaPM prostate cancer cell models using a dominant-negative construct resulted in decreased tumor cell proliferation, colony formation, and invasion in vitro. Blocking endogenous TBX2 in human prostate cancer mouse xenografts decreased invasion and abrogation of bone and soft tissue metastasis. Furthermore, blocking endogenous TBX2 in prostate cancer cells dramatically reduced bone-colonizing capability through reduced tumor cell growth and bone remodeling in an intratibial mouse model. TBX2 acted in trans by promoting transcription of the canonical WNT (WNT3A) promoter. Genetically rescuing WNT3A levels in prostate cancer cells with endogenously blocked TBX2 partially restored the TBX2-induced prostate cancer metastatic capability in mice. Conversely, WNT3A-neutralizing antibodies or WNT antagonist SFRP-2 blocked TBX2-induced invasion. Our findings highlight TBX2 as a novel therapeutic target upstream of WNT3A, where WNT3A antagonists could be novel agents for the treatment of metastasis and for skeletal complications in prostate cancer patients. Cancer Res; 77(6); 1331–44. ©2017 AACR.

Quantitative Real-Time RT-PCR and Invasion assay of LNCaP-TBX2 and LNCaP-Neo cells

Cancer Outlier Profile Analysis (COPA) in Oncomine 4.4 showing TBX2 over-expression in PCa microarray data sets

Purpose: MicroRNAs in the delta-like 1 homolog–deiodinase, iodothyronine 3 (DLK1-DIO3) cluster have been shown to be critical for embryonic development and epithelial to mesenchymal transition (EMT). DLK1-DIO3 cluster miRNAs are elevated in the serum of patients with metastatic cancer. However, the biologic functions of these miRNAs in the EMT and metastasis of cancer cells are poorly understood. We previously demonstrated the oncogenic and metastatic role of miR-409-3p/5p, a member of this cluster, in prostate cancer. In this study, we defined the role of miR-154* and miR-379, two key members of this cluster, in prostate cancer progression and bone metastasis in both cell line models and clinical specimens.Experimental Design: Genetic manipulation of miR-154* and miR-379 was performed to determine their role in tumor growth, EMT, and bone metastasis in mouse models. We determined the expression of miR-154* in prostate cancer clinical samples and bone metastasis samples using in situ hybridization and quantum dot labeling.Results: Elevated expression of miR-154* and miR-379 was observed in bone metastatic prostate cancer cell lines and tissues, and miR-379 expression correlated with progression-free survival of patients with prostate cancer. Intracardiac inoculation (to mimic systemic dissemination) of miR-154* inhibitor-treated bone metastatic ARCaPM prostate cancer cells in mice led to decreased bone metastasis and increased survival.Conclusion: miR-154* and miR-379 play important roles in prostate cancer biology by facilitating tumor growth, EMT, and bone metastasis. This finding has particular translational importance because miRNAs in the DLK1-DIO3 cluster can be attractive biomarkers and possible therapeutic targets to treat bone metastatic prostate cancer. Clin Cancer Res; 20(24); 6559–69. ©2014 AACR.

During disease progression and bone metastasis, breast tumor cells interact with various types of bystander cells residing in the tumor microenvironment. Such interactions prompt tumor cell heterogeneity. We used successive co-culture as an experimental model to examine cancer–bystander cell interaction. RMCF7-2, a clone of the human breast cancer MCF-7 cells tagged with a red fluorescent protein, was tracked for morphologic, behavioral, and gene expression changes. Co-cultured with various types of hematopoietic cells, RMCF7-2 adopted stable changes to a rounded shape in suspension growth of red fluorescent cells, from which derivative clones displayed marked expressional changes of marker proteins, including reduced E-cadherin and estrogen receptor α, and loss of progesterone receptor. In a successive co-culture with bone marrow-derived mesenchymal stem/stromal cells, the red fluorescent clones in suspension growth changed once more, adopting an attachment growth, but in diversified shapes. Red fluorescent clones recovered from the second-round co-culture were heterogeneous in morphology, but retained the altered marker protein expression while displaying increased proliferation, migration, and xenograft tumor formation. Interaction with bystander cells caused permanent morphologic, growth behavioral, and gene expressional changes under successive co-culture, which is a powerful model for studying cancer cell heterogeneity during breast cancer progression and metastasis.

Prostate cancer (PCa) affects an estimated 250,000 men every year and causes 34,000 deaths annually. A high-fat diet and obesity are associated with PCa progression and mortality. This study’s premise was the novel observation of crosstalk between PCa epithelia and cancer-associated fibroblasts (CAF) in response to palmitate-mediated lineage plasticity. We found that cholesterol activated canonical Hedgehog (Hh) signaling by increasing cilium Gli activity in PCa cells, while palmitate activated Hh independent of Gli. Exogenous palmitate activated SOX2, a known mediator of lineage plasticity, in PCa cells cocultured with CAF. Stroma-derived Wnt5a was upregulated in CAF while cocultured with PCa cells and treated with palmitate. Wnt5a knockdown in CAF inhibited Hh and SOX2 expression in PCa cells from cocultures. These findings supported our proposed mechanism of a high-fat diet promoting Hh signaling-mediated transformation within the tumor microenvironment. SOX2 and Wnt5a expression were limited by the CD36 neutralizing antibody. Mice xenografted with PCa epithelia and CAF tumors were fed a high-fat diet, leading to elevated SOX2 expression and lineage plasticity reprogramming compared to mice fed an isocaloric rodent diet. CD36 inhibition with enzalutamide elevated apoptosis by TUNEL, but limited proliferation and SOX2 expression compared to enzalutamide alone. This study revealed a mechanism for a high-fat diet to affect prostate cancer progression. We found that saturated fat induced lineage plasticity reprogramming of PCa by interaction with CAF through Wnt5a and Hh signaling.

Chien-Chih Chen,1,2 Toshiaki Nakano,3,4 Li-Wen Hsu,3,4 Chia Yi Chu,1 Kuang-Tzu Huang4,5 1Department of Psychiatry, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan; 2School of Medicine, Chang Gung University, Taoyuan, Taiwan; 3Graduate Institute of Clinical Medical Sciences, Chang Gung University College of Medicine, Kaohsiung, Taiwan; 4Liver Transplantation Center, Kaohsiung Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Kaohsiung, Taiwan; 5Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, Kaohsiung, TaiwanCorrespondence: Kuang-Tzu Huang, Institute for Translational Research in Biomedicine, Kaohsiung Chang Gung Memorial Hospital, No. 123, DAPI Road Niaosong Dist, Kaohsiung, 833, Taiwan, Tel +886-7-731-7123 (Ext.8193), Email huangkt@cgmh.org.twBackground: Atypical antipsychotics such as olanzapine often cause metabolic side effects such as obesity and diabetes, leading to an increased risk of nonalcoholic fatty liver disease. The aim of the present study was to investigate the effects of olanzapine treatment on hepatic lipid metabolism and its possible relationship with adipose tissue status.Methods: Using a female rat model, we investigated the effects of chronic olanzapine administration on the regulation of carbohydrate and lipid metabolism including lipid biosynthesis, oxidation, efflux, and lipolysis in liver and adipose tissue.Results: The body weight, liver mass and visceral adiposity after olanzapine treatment (2 mg/kg) for five weeks were not significantly different compared with vehicle controls. The serum level of triglycerides was higher in the vehicle controls than in olanzapine-treated rats. Unexpectedly, olanzapine treatment did not reduce glucose tolerance in our model. The expression of functional thermogenic protein uncoupling protein 1 (UCP1) was increased in brown adipose tissue (BAT) of the olanzapine group. Additionally, olanzapine treatment also reduced adipose inflammation in white adipose tissue (WAT). The transcription factor sterol regulatory element-binding protein (SREBP)-1c, a key early regulator of lipogenesis, was downregulated following olanzapine treatment. The expression of genes related to the triglycerides synthesis apparatus in the liver was upregulated in the olanzapine group. Olanzapine treatment induced genes involved in PPAR-α signaling and mitochondrial fatty acid oxidation in response to increased ATGL-mediated lipolysis in the liver.Conclusion: Together, our findings suggest a complicated link between olanzapine therapy and metabolic disturbance and may garner interest in assessing the action of antipsychotic-induced metabolic disturbances.Keywords: atypical antipsychotics, olanzapine, metabolic disturbances, weight gain, lipogenesis, lipolysis

Background Burkitt lymphoma is a fast‐growing mature B cell malignancy, whose genetic hallmark is translocation and activation of the c‐myc gene. Prompt multiagent immunochemotherapy regimens can have favorable outcomes, but prognosis is poor in refractory or relapsed disease. We previously identified a novel family of near‐infrared heptamethine carbocyanine fluorescent dyes (HMCD or DZ) with tumor‐homing properties via organic anion‐transporting peptides. These membrane carriers have uptake in tumor cells but not normal cells in cell culture, mouse and dog tumor models, patient‐derived xenografts, and perfused kidney cancers in human patients. Methods Here we report the cytotoxic effects of a synthesized conjugate of DZ with cisplatin (CIS) on B cell lymphoma CA46, Daudi, Namalwa, Raji, and Ramos cell lines in cell culture and in xenograft tumor formation. Impaired mitochondrial membrane permeability was examined as the mechanism of DZ‐CIS–induced lymphoma cell death. Results The new conjugate, DZ‐CIS, is cytotoxic against Burkitt lymphoma cell lines and tumor models. DZ‐CIS retains tumor‐homing properties to mitochondrial and lysosomal compartments, does not accumulate in normal cells and tissues, and has no nephrotoxicity in mice. DZ‐CIS accumulated in Burkitt lymphoma cells and tumors induces apoptosis and retards tumor cell growth in culture and xenograft tumor growth in mice. Conclusion DZ‐CIS downregulated c‐myc and overcame CIS resistance in myc‐driven TP53‐mutated aggressive B cell Burkitt lymphoma. We propose that DZ‐CIS could be used to treat relapsed/refractory aggressive Burkitt lymphomas., The cytotoxic effect of a synthesized conjugate between a tumor cell–specific heptamethine carbocyanine and cisplatin on Burkitt lymphoma cells is evaluated. The conjugate inhibits c‐myc expression, reduces mitochondrial membrane permeability, and induces lymphoma cell death in culture and in xenograft tumors in athymic mice.

This article describes cell signaling network of metastatic prostate cancer (PCa) to bone and visceral organs in the context of tumor microenvironment and for the development of novel therapeutics. The article focuses on our recent progress in the understanding of: 1) The plasticity and dynamics of tumor–stroma interaction; 2) The significance of epigenetic reprogramming in conferring cancer growth, invasion and metastasis; 3) New insights on altered junctional communication affecting PCa bone and brain metastases; 4) Novel strategies to overcome therapeutic resistance to hormonal antagonists and chemotherapy; 5) Genetic-based therapy to co-target tumor and bone stroma; 6) PCa-bone-immune cell interaction and TBX2-WNTprotein signaling in bone metastasis; 7) The roles of monoamine oxidase and reactive oxygen species in PCa growth and bone metastasis; and 8) Characterization of imprinting cluster of microRNA, in tumor–stroma interaction. This article provides new approaches and insights of PCa metastases with emphasis on basic science and potential for clinical translation. This article referenced the details of the various approaches and discoveries described herein in peer-reviewed publications. We dedicate this article in our fond memory of Dr. Donald S. Coffey who taught us the spirit of sharing and the importance of focusing basic science discoveries toward translational medicine. Keywords: Prostate cancer, Castration resistance, Metastasis, Cancer-stromal interaction, Targeted therapy

Prostate cancer is a prevalent public health problem, especially because noncutaneous advanced malignant forms significantly affect the lifespan and quality of life of men worldwide. New therapeutic targets and approaches are urgently needed. The current study reports elevated expression of R1 (CDCA7L/RAM2/JPO2), a c-Myc–interacting protein and transcription factor, in human prostate cancer tissue specimens. In a clinical cohort, high R1 expression is associated with disease recurrence and decreased patient survival. Overexpression and knockdown of R1 in human prostate cancer cells indicate that R1 induces cell proliferation and colony formation. Moreover, silencing R1 dramatically reduces the growth of prostate tumor xenografts in mice. Mechanistically, R1 increases c-Myc protein stability by inhibiting ubiquitination and proteolysis through transcriptional suppression of HUWE1, a c-Myc–targeting E3 ligase, via direct interaction with a binding element in the promoter. Moreover, transcriptional repression is supported by a negative coexpression correlation between R1 and HUWE1 in a prostate cancer clinical dataset. Collectively, these findings, for the first time, characterize the contribution of R1 to prostate cancer pathogenesis. Implications: These findings provide evidence that R1 is a novel regulator of prostate tumor growth by stabilizing c-Myc protein, meriting further investigation of its therapeutic and prognostic potential.

Tumor metastasis depends on the dynamic regulation of cell adhesion through β1-integrin. The Cub-Domain Containing Protein-1, CDCP1, is a transmembrane glycoprotein which regulates cell adhesion. Overexpression and loss of CDCP1 have been observed in the same cancer types to promote metastatic progression. Here, we demonstrate reduced CDCP1 expression in high-grade, primary prostate cancers, circulating tumor cells and tumor metastases of patients with castrate-resistant prostate cancer. CDCP1 is expressed in epithelial and not mesenchymal cells, and its cell surface and mRNA expression declines upon stimulation with TGFβ1 and epithelial-to-mesenchymal transition. Silencing of CDCP1 in DU145 and PC3 cells resulted in 3.4-fold higher proliferation of non-adherent cells and 4.4-fold greater anchorage independent growth. CDCP1-silenced tumors grew in 100% of mice, compared to 30% growth of CDCP1-expressing tumors. After CDCP1 silencing, cell adhesion and migration diminished 2.1-fold, caused by loss of inside-out activation of β1-integrin. We determined that the loss of CDCP1 reduces CDK5 kinase activity due to the phosphorylation of its regulatory subunit, CDK5R1/p35, by c-SRC on Y234. This generates a binding site for the C2 domain of PKCδ, which in turn phosphorylates CDK5 on T77. The resulting dissociation of the CDK5R1/CDK5 complex abolishes the activity of CDK5. Mutations of CDK5-T77 and CDK5R1-Y234 phosphorylation sites re-establish the CDK5/CDKR1 complex and the inside-out activity of β1-integrin. Altogether, we discovered a new mechanism of regulation of CDK5 through loss of CDCP1, which dynamically regulates β1-integrin in non-adherent cells and which may promote vascular dissemination in patients with advanced prostate cancer.

Background Global disparities in prostate cancer (PCa) incidence highlight the urgent need to identify genomic abnormalities in prostate tumors in different ethnic populations including Asian men. Objective To systematically explore the genomic complexity and define disease-driven genetic alterations in PCa. Design, setting, and participants The study sequenced whole-genome and transcriptome of tumor-benign paired tissues from 65 treatment-naive Chinese PCa patients. Subsequent targeted deep sequencing of 293 PCa-relevant genes was performed in another cohort of 145 prostate tumors. Outcome measurements and statistical analysis The genomic alteration landscape in PCa was analyzed using an integrated computational pipeline. Relationships with PCa progression and survival were analyzed using nonparametric test, log-rank, and multivariable Cox regression analyses. Results and limitations We demonstrated an association of high frequency of CHD1 deletion with a low rate of TMPRSS2-ERG fusion and relatively high percentage of mutations in androgen receptor upstream activator genes in Chinese patients. We identified five putative clustered deleted tumor suppressor genes and provided experimental and clinical evidence that PCDH9, deleted/loss in approximately 23% of tumors, functions as a novel tumor suppressor gene with prognostic potential in PCa. Furthermore, axon guidance pathway genes were frequently deregulated, including gain/amplification of PLXNA1 gene in approximately 17% of tumors. Functional and clinical data analyses showed that increased expression of PLXNA1 promoted prostate tumor growth and independently predicted prostate tumor biochemical recurrence, metastasis, and poor survival in multi-institutional cohorts of patients with PCa. A limitation of this study is that other genetic alterations were not experimentally investigated. Conclusions There are shared and salient genetic characteristics of PCa in Chinese and Caucasian men. Novel genetic alterations in PCDH9 and PLXNA1 were associated with disease progression. Patient summary We reported the first large-scale and comprehensive genomic data of prostate cancer from Asian population. Identification of these genetic alterations may help advance prostate cancer diagnosis, prognosis, and treatment.

e17008 Background: A morphological subset of prostate cancer (PCa) circulating tumor cells (CTCs) with particularly small nuclei (< 8.5 μm), named very-small-nuclear CTCs (vsnCTCs), were found to be correlated with the presence of visceral metastases. It is reported that the depletion of nuclear envelope protein emerin promotes PCa metastasis and is associated with nuclear shape instability. In this study, we hypothesize vsnCTCs as prognostic biomarkers in metastatic castration resistant PCa (mCRPC), and aim to investigate the correlation between emerin level and vsnCTCs. Methods: PCa CTCs were enriched using the NanoVelcro CTC Assay from 76 patients with mCRPC. The Kaplan-Meier analysis and log-rank test were used to estimate and compare the overall survival (OS) and progression free survival (PFS) of androgen receptor signaling inhibitor (ARSI), taxanes and other therapy in patients stratified by the presence of vsnCTCs. The correlation between the presence of vsnCTCs and OS and PFS were evaluated using the Cox proportional hazard regression. The expression level of emerin in patients with and without vsnCTC were compared using Mann-Whitney U test, and the correlation between emerin level and CTC nuclear size was tested by Pearson correlation coefficient. Results: Patients with vsnCTC (i.e, vsnCTC+) had significantly shortened OS and PFS compared with those without vsnCTC (i.e, vsnCTC-). The median OS was 34 (vsnCTC+, n= 49) vs. 149 (vsnCTC-, n= 27) weeks (hazard ratio [HR] = 2.6, 95% confidence interval [CI]: 1.5-4.5, P< 0.001). The median PFS was 12 (vsnCTC+, n= 32) vs. 26 (vsnCTC-, n= 18) weeks (HR = 2.2, 95% CI: 1.3 -4.0, P= 0.004). The emerin expression level was significantly higher in vsnCTC+ compared to vsnCTC- ( P= 0.009). In addition, we observed a significantly positive correlation between emerin expression and CTC nuclear size (r = 0.52, P< 0.001). Conclusions: This study casts light on the importance of the vsnCTCs in patients with mCRPC, as vsnCTC+ patients represented a group at risk for faster clinical progression who are at the highest risk for mortality. vsnCTC represents a new hallmark of an aggressive subtype of mCRPC and is related to emerin dysregulation, which promotes lethal progression and metastasis of PCa.

167 Background: Circulating tumor cells (CTCs) have arisen as contemporary noninvasive prognostic biomarkers for prostate cancer (PCa). Previously, a subgroup of PCa CTCs, with particularly small nuclei ( < 8.5 μm), were found to be correlated with the presence of visceral metastases. This subgroup was named very-small-nuclear CTCs (vsnCTCs). We hypothesized vsnCTCs as a putative biomarker of a lethal subtype associated with androgen receptor (AR) indifference and nuclear shape instability in metastatic castration resistant PCa (mCRPC). Methods: CTCs in blood from 76 patients with mCRPC were analyzed using NanoVelcro CTC assay for CTC nuclear size measurement and CTC RNA profiling of AR-indifferent pathways. Overall survival (OS) and progression free survival (PFS) of androgen receptor signaling inhibitor (ARSI), taxanes and other therapy were correlated with CTC nuclear size using Kaplan-Meier analysis and Cox proportional hazards model. Emerin fluorescence intensity and localization from patients with and without vsnCTC were compared. RNA profiles of CTCs were scored using Prostate Cancer Subtype (PCS) classification system. The CTC-PCS scores from patients with and without vsnCTC were compared using Mann-Whitney test. To investigate the underlying biology of vsnCTC phenotype, the nuclear size, the nuclear sizes of ARSI-resistant and lineage plasticity PCa cell lines were measured and correlated with the expression levels of RNA related to ARSI-indifferent pathways and nuclear envelope protein Emerin. Results: Patients with vsnCTC (i.e., vsnCTC+) had significantly shortened OS and PFS compared with patients without vsnCTC (i.e., vsnCTC-). The median OS was 34 (vsnCTC+, n = 49) vs. 149 (vsnCTC-, n = 27) weeks (HR = 2.6 with 95% CI 1.5 to 4.5, p < 0.001). The median PFS was 12 (vsnCTC+, n = 32) vs. 26 (vsnCTC-, n = 18) weeks (HR = 2.2 with 95% CI 1.3 to 4.0, p = 0.004). CTC nuclear sizes were significantly smaller in patients with prior ARSI therapy. CTC-RNA analysis revealed that vsnCTC+ patients had a significant higher CTC-PCS1 Z score(n = 19) compared with vsnCTC- patients(n = 26)(p = 0.01). In the cell line models, nuclear sizes were significantly smaller in cell lines with ARSI-resistance(p = 0.002) and lineage plasticity (p = 0.006). Emerin expression is significantly lower in vsnCTC+ patients(p = 0.009) and ARSI-resistant cell lines(p = 0.03). Conclusions: This study casts light on the importance of the vsnCTC in patients with mCRPC, as vsnCTC+ patients represented a group at risk for faster clinical progression who are at the highest risk for mortality. This has potential importance in optimizing therapeutic choices. We posit that the vsnCTC represents a new hallmark of an aggressive subtype of mCRPC and is related to the cellular mechanism of AR-indifference and Emerin dysregulation, which promotes lethal progression of metastatic PCa.

In this work, the electrostatic discharge (ESD) protection of Schottky diode with fluorine-based plasma treatment is proven by transmission-line pulse (TLP) measurement. And the low-frequency noise (LFN) is used to determine the activation energy (E a ) of a GaN Schottky diode with plasma treatment. This experiment compares the Schottky diode with CF 4 and CHF 3 plasma treatment, respectively with a standard Schottky diode to determine the characteristics and to assess the low-frequency noise and the ESD protection ability.

e17512 Background: Very-small-nuclear circulating tumor cells (vsnCTCs) as a subset of CTCs with nuclear size < 8.5 μm associated with visceral metastases (VM) in advanced metastatic, castration-resistant prostate cancer (mCRPC). As VM predicts foreshortened survival, we hypothesized that vsnCTCs would be directly associated with poor overall survival (OS). Methods: Clinically annotated, blood samples from patients with mCRPC with survival follow-up within the Translational Oncology Program Blood & Biospecimen Bank were selected for analysis. CTCs were isolated and analyzed using the NanoVelcro assay. The nuclear size from all CTCs from each patient sample was compared to OS and progression-free survival (PFS) from the time of the blood draw. Results: A total of 76 patients had samples suitable for analysis. Sixty-six (87%) had CTCs; 49 (64%) were vsnCTC+ (≥1 vsnCTC). vsnCTCs were more common in mCRPC patients with previous androgen receptor signaling inhibitor (ARSI) therapy and/or 2 or more lines of treatment. OS was significantly shorter for vsnCTC+ than vsnCTC- patients (median 34 vs. 149 weeks; log-rank HR = 2.6; 95% CI = 1.5 to 4.5; P = 0.0006). Fifty patient samples were available for PFS analysis (i.e. drawn within 4 weeks of starting therapy). vsnCTC+ patients experienced more rapid progression than vsnCTC- patients (median 12 vs. 26 weeks, log-rank HR = 2.2, 95% CI = 1.3 to 4.0; P = 0.004). Multivariable Cox regression revealed that vsnCTC status was independently associated with OS and PFS. P-spline plot analysis showed that the HR of OS increased as the minimum CTC nuclear size decreased. The minimum CTC nuclear size was also independently associated with OS and PFS in a multivariable Cox regression analysis. Patients with prior use of androgen receptor signaling inhibitor (ARSI) therapy had significantly smaller minimum CTC nuclear size compared to those without prior use of ARSI. Average and median nuclear size did not strongly associate with OS or PFS. Conclusions: vsnCTC+ patients are at risk for more rapid clinical progression and have greater risk of death than vsnCTC-. We posit that the vsnCTC may be a biomarker of aggressive mCRPC with foreshortened survival. Interestingly, the CTCs with the smallest nuclei appear to best reflect the observed clinical behavior. This has potential importance in optimizing therapeutic choices and may point toward a unique biology relating nuclear size to aggressive molecular features. Our group continues to explore the biology underlying the vsnCTC.

168 Background: Circulating tumor cells (CTCs) have arisen as contemporary noninvasive prognostic biomarkers for prostate cancer (PC). Previously, a subgroup of PC CTCs, with particularly small nuclei (

170 Background: Transcriptome-based analysis has begun to reshape the approach to prostate cancer (PC). Two different gene expression signatures have shown that PC can be divided into 3 subclasses reflecting luminal-basal biology. These subtypes point toward biological drivers that may strongly influence how care should be personalized including optimization of androgen receptor targeted therapy. The majority of work done in this area has been based on tissue-based gene expression. With the advent of newer nanotechnology platforms for isolation of circulating tumor cells (CTCs), profiling of PC gene expression from blood is now possible. Methods: We recruited 34 patients with metastatic castration resistant PC at Cedars-Sinai Medical Center who had available blood specimens prior to initiation of androgen receptor signaling inhibitor (ARSI, e.g. abiraterone, enzalutamide and apalutamide) therapy.We utilized the NanoVelcro Assays which allow for capture and release of CTCs with intact mRNA. Gene sets from the PCS and PAM50 signatures were re-reviewed to optimize signal detection in the blood and enriched for genes upregulated in PC. The NanoString nCounter platform was used for RNA profiling. Results: The final assay was tested in banked blood samples and provided classifications of patients that associated with clinical responsiveness to therapy. Validation was conducted to examine the performance of the CTC-specific PCS/PAM50 panel in public databases (including Prostate Cancer Transcriptome Atlas and GenomeDx). Our pilot study showed that the median overall survival was significantly worse in PCS1 patients. Conclusions: This study shows initial proof of principle that genomic classification in blood is possible using contemporary tool for blood component isolation and RNA profiling. Additional technical and clinical validations are needed prior to widespread implementation, but these methods may make it possible to increase the utilization of genomic classifiers in clinical studies and in practice.