Joyce Yamashiro

Prostate stem cell antigen (PSCA) expression increases with high gleason score, advanced stage and bone metastasis in prostate cancer.

Prostate stem cell antigen (PSCA) is a recently defined homologue of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA mRNA is expressed in the basal cells of normal prostate and in more than 80% of prostate cancers. The purpose of the present study was to examine PSCA protein expression in clinical specimens of human prostate cancer. Five monoclonal antibodies were raised against a PSCA-GST fusion protein and screened for their ability to recognize PSCA on the cell surface of human prostate cancer cells. Immunohistochemical analysis of PSCA expression was performed on paraffin-embedded sections from 25 normal tissues, 112 primary prostate cancers and nine prostate cancers metastatic to bone. The level of PSCA expression in prostate tumors was quantified and compared with expression in adjacent normal glands. The antibodies detect PSCA expression on the cell surface of normal and malignant prostate cells and distinguish three extracellular epitopes on PSCA. Prostate and transitional epithelium reacted strongly with PSCA. PSCA staining was also seen in placental trophoblasts, renal collecting ducts and neuroendocrine cells in the stomach and colon. All other normal tissues tested were negative. PSCA protein expression was identified in 105/112 (94%) primary prostate tumors and 9/9 (100%) bone metastases. The level of PSCA expression increased with higher Gleason score (P=0.016), higher tumor stage (P=0.010) and progression to androgen-independence (P=0.021). Intense, homogeneous staining was seen in all nine bone metastases. PSCA is a cell surface protein with limited expression in extraprostatic normal tissues. PSCA expression correlates with tumor stage, grade and androgen independence and may have prognostic utility. Because expression on the surface of prostate cancer cells increases with tumor progression, PSCA may be a useful molecular target in advanced prostate cancer.

Monoclonal antibody targeting of N-cadherin inhibits prostate cancer growth, metastasis and castration resistance

The transition from androgen-dependent to castration-resistant prostate cancer (CRPC) is a lethal event of uncertain molecular etiology. Comparing gene expression in isogenic androgen-dependent and CRPC xenografts, we found a reproducible increase in N-cadherin expression, which was also elevated in primary and metastatic tumors of individuals with CRPC. Ectopic expression of N-cadherin in nonmetastatic, androgen-dependent prostate cancer models caused castration resistance, invasion and metastasis. Monoclonal antibodies against the ectodomain of N-cadherin reduced proliferation, adhesion and invasion of prostate cancer cells in vitro. In vivo, these antibodies slowed the growth of multiple established CRPC xenografts, blocked local invasion and metastasis and, at higher doses, led to complete regression. N-cadherin–specific antibodies markedly delayed the time to emergence of castration resistance, markedly affected tumor histology and angiogenesis, and reduced both AKT serine-threonine kinase activity and serum interleukin-8 (IL-8) secretion. These data indicate that N-cadherin is a major cause of both prostate cancer metastasis and castration resistance. Therapeutic targeting of this factor with monoclonal antibodies may have considerable clinical benefit.

Prostate Stem Cell Antigen Is Overexpressed in Prostate Cancer Metastases

Purpose: Prostate stem cell antigen (PSCA) is expressed by a majority of prostate cancers and is a promising therapeutic target. PSCA protein and mRNA expression was examined in prostate cancer bone, lymph node, and visceral metastases to assess the potential of PSCA as an immunotherapeutic target in advanced prostate cancer. Experimental Design: Immunohistochemical analysis of PSCA protein expression and quantitative mRNA expression analysis of PSCA was done on clinical specimens of prostate cancer bone, lymph node, and visceral metastases. PSCA protein and mRNA expression levels were quantified and compared between available matched pairs of bone and lymph node or visceral metastases. Results: Bone metastases stained with higher intensity of PSCA compared with lymph node or liver metastases in seven of eight (87.5%) matched pairs (P = 0.035). PSCA mRNA expression was equal or greater than that of LAPC-9, a PSCA expressing xenograft, in 12 of 24 (50%) cases of prostate cancer metastases and was significantly correlated with PSCA protein expression (σ = 0.84, P = 0.0019). Overall, PSCA protein expression was detected in 41 of 47 (87.2%), four of six (66.7%), and two of three (66.7%) cases of bone, lymph node, and liver metastases, respectively. Mean PSCA staining intensity was significantly higher in prostate cancer bone metastases compared with lymph node metastases (2.0 ± 0.02 versus 0.83 ± 0.31, P = 0.014). Conclusions: Prostate cancer metastases express PSCA. However, greater PSCA staining intensity and level of PSCA mRNA expression was associated with bone metastases compared with lymph node metastases. This study suggests that PSCA is a promising tumor marker and potential therapeutic target for patients with metastatic prostate cancer.

Modulation of N-myc expression alters the invasiveness of neuroblastoma.

N-myc oncogene expression plays a pivotal role in the biology of neuroblastoma, a common childhood tumor. High N-myc expression is associated with advanced disease stage, and in animal models, increased expression results in increased metastatic potential. In normal embryologic development, N-myc expression is associated with neuroblast migration out from the neural crest. To further define the relationship between N-myc and metastasis, an in vitro assay was adapted to measure tumor cell attachment, motility, and proteolytic ability in neuroblastoma cell lines. These parameters were examined in a non-amplified, uniformly N-myc overexpressing cell line and its anti-sense N-myc expressing clones. These lines have been characterized previously, and have a decrease in N-myc expression, growth rate, and tumorigenicity relative to the parent line and vector-only control transfectant. Decrease in N-myc expression resulted in a non-proportional increase of tumor cell attachment, and a proportional decrease in both tumor cell motility and proteolytic ability. In further experiments, assay of a N-myc-amplified overexpressing cell line with an intrinsic heterogeneous pattern of expression demonstrated that motile cells expressed higher amounts of N-myc relative to the general population. Together these relationships indicate that N-myc plays a causative role in the invasive phenotype, and suggest that metastasis may, in part, result from the disruption of a developmentally important normal process.

Vitamin D3 analogs inhibit growth and induce differentiation in LA-N-5 human neuroblastoma cells

The physiologically active metabolite of vitamin D3, 1,25-dihydroxycholecalciferol (D3), plays an important role in embryonic development and cell differentiation. Previously, we have demonstrated that D3 significantly induces differentiation and inhibits growth of LA-N-5 human neuroblastoma cells at concentrations of 24 nm and higher. In this study, we compared two D3 analogs, 20-epi-22oxa-25a,26a,27a-tri-homo-1,25-D3 (KH 1060) and 1,25-dihydroxy-22,24-diene, 24,26,27-trihomo (EB 1089), with D3 with respect to their effects on differentiation and growth inhibition. We report an inhibition of growth by 45–55% in cells treated with 0.24 nm EB 1089 and 0.24 nm KH 1060, similar to that seen in cells treated with 24 nM D3. At these concentrations, both EB 1089 and KH 1060 stimulate the differentiation of LA-N-5 neuroblastoma cells as shown by increased neurite outgrowth, decreased N-myc expression and decreased invasiveness in vitro. An increase in acetylcholinesterase activity, a functional measure of differentiation, was also exhibited. Previous reports have shown that treatment doses needed to achieve 24 nm serum concentrations of D3 in patients would result in hypercalcemia. EB 1089 and KH 1060 can cause the same in vitro effects on LA-N-5 human neuroblastoma cells at 1/100 of the concentration required of D3. These data suggest a potential clinical efficacy of EB 1089 and KH 1060 as biological response modifiers.

Enrichment of putative prostate cancer stem cells after androgen deprivation: upregulation of pluripotency transactivators concurs with resistance to androgen deprivation in LNCaP cell lines.

Prostate cancer stem cells (PCSC) offer theoretical explanations to many clinical and biological behaviors of the disease in human. In contrast to approaches of using side populations and cell‐surface markers to isolate and characterize the putative PCSC, we hypothesize that androgen deprivation leads to functional enrichment of putative PCSC.

Differentiating effects of 1,25-dihydroxycholecalciferol (D3) on LA-N-5 human neuroblastoma cells and its synergy with retinoic acid.

Purpose 1,25-Dihydroxycholecalciferol (D3) plays an important role in embryonic development and cell differentiation. It has previously been reported to decrease c-myc expression by HL-60 cells and downregulate c-myc expression by breast and ovarian cancer cells. We report the results of our investigations into the differentiating effects of D3 on LA-N-5 human neurob-lastoma cells. Methods LA-N-5 human neuroblastoma cell line was treated with D3, retinoic acid (RA), D3 and RA, or solvent control. Growth inhibitory effects, neurite extension, acetylcholinesterase activity, invasiveness, motility, and N-myc protein expression were examined following treatment. Results Growth inhibition was observed at concentrations of >24 nM. D3 stimulated the differentiation of LA-N-5 cells as demonstrated by increased neurite outgrowth, increased acetyl-cholinesterase activity, and decreased invasiveness. A decrease in N-myc expression was observed in immunostained cells treated with either agent alone, with a more profound effect appreciated with the combination. Conclusion Vitamin D3 decreases N-myc expression in LA-N-5 human neuroblastoma cells, with extended treatment causing growth inhibition and differentiation. When used in combination with RA, these effects are more profound than with either agent alone. The therapeutic use of differentiating agent combinations such as D3 and RA may provide a relatively nontoxic means of treating susceptible tumor types.

Abstract A52: Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer

Introduction: Recent advances improved survival in men with metastatic castration-resistant prostate cancer (CRPC) after chemotherapy when treated with androgen ablation therapies, including the well-known androgen receptor (AR) antagonist enzalutamide. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next-generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance. Targeting N-cadherin positive cells with specific monoclonal antibodies (mAb) affect tumor growth in both AR-positive and -negative prostate cancers. Here we report the further development of effective N-cadherin specific monoclonal antibodies against CRPC and their value in combination therapies. Method: Endogenously expressing N-cadherin prostate cancer cell lines (PC3, LAPC9) and androgen-dependent prostate cancer cells ectopically expressing N-cadherin (LNCaP, MDA-PCa-2b, VCaP) were evaluated in vitro for invasion, growth, and self-renewal in the presence of monoclonal antibodies raised against various extracellular domains of N-cadherin and their effects in combination with enzalutamide. Further, enzalutamide-resistant cell lines were also generated and evaluated. In vivo studies were performed using castration-resistant N-cadherin and AR positive, as well as androgen-dependent tumors implanted subcutaneously in mice as xenografts. The tumors were analyzed for response to enzalutamide or mAb alone or in combination. When the tumors reached 100-200 mm3 in volume the mice were castrated and treated with 5-10 mg/kg enzalutamide and/or 5-10 mg/kg mAb candidates. Tumors were monitored for growth, local invasion, and metastasis. At endpoint, tumors were harvested and further analyzed. Promising mAb candidates, as well as new epitope targets, were then selected and reformatted into full humanized N-cadherin monoclonal antibodies and further analyzed. Results: Ectopic expression of N-cadherin in N-cadherin negative cells enhanced cell growth and invasion in androgen-deprived conditions. In vitro studies showed that combination treatment of N-cadherin positive and AR-positive cells reduced invasion and cell proliferation than either treatment alone. Certain enzalutamide-resistant cell lines generated showed an increase in N-cadherin expression. Such cells responded to N-cadherin antibodies in androgen-deprived conditions, suggesting a role for N-cadherin in a subset of enzalutamide resistance. In vivo studies using CRPC xenograft models expressing N-cadherin and AR showed inhibition of tumor growth when the animals were castrated and treated with a combination of enzalutamide and N-cadherin mAb than either treatment alone. Androgen-dependent xenograft models in castrated mice also significantly delayed castration-resistant tumor growth when treated in combination than either treatment alone. The newly generated humanized mAbs showed similar outcomes. Conclusion: Previous studies suggested that therapeutic targeting of N-cadherin with monoclonal antibodies have significant effect in inhibiting tumor progression. We have verified that humanized N-cadherin mAb exhibit significant efficacy, fostering our search for increasingly effective immunotherapy in the clinic. Further, the antibodies showed synergy in combination with enzalutamide, suggesting promise in potential combination therapies for CRPC. Other androgen receptor antagonists and other drugs targeting those potentially involved in downstream pathways may prove to have additional clinical benefit. Citation Format: Evelyn A. Kono, Naoko Kobayashi, Kirstin Zettlitz, Joyce Yamashiro, Wang Chun, Della Z. Hu, Anna M. Wu, Robert E. Reiter. Efficacy of new developed N-cadherin monoclonal antibodies in combination with enzalutamide against castration-resistant prostate cancer [abstract]. In: Proceedings of the AACR International Conference held in cooperation with the Latin American Cooperative Oncology Group (LACOG) on Translational Cancer Medicine; May 4-6, 2017; Sao Paulo, Brazil. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(1_Suppl):Abstract nr A52.

Abstract LB-279: N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer cells

New generation of anti-androgen treatments such as enzalutamide and abiraterone are improving survival of men with advanced and metastatic prostate cancers, but resistance to these inevitably arises. The molecular mechanisms governing the emergence of treatment resistance in castration resistant prostate cancer (CRPC) patients are not well understood. Recent experience suggests that tumor regeneration from castration-resistant stem-like cells induce resistance to hormonal therapy. Therefore, elucidating novel targets essential for driving stem-like activity is critical to prevent and defeat CRPC. It has been shown that normal and cancer stem cells exploit normal development process of epithelial-mesenchymal transition (EMT) to survive and metastasize, and that EMT confer stem cell properties to more differentiated cancer cell progeny in breast and other cancers. However, it is unclear if EMT is linked with stem cells in normal and malignant prostate. We previously showed that N-cadherin, a marker of EMT, promotes CRPC progression and that there was a correlation between N-cadherin expression and the expression of stem cell associated markers. This leads to hypothesis that N-cadherin may promote CRPC by enhancing tumorige esis/tumor initiating properties of cancer cells in a castrate environment. N-cadherin may confer tumor initiating or stem-like properties to prostate cancer cells perhaps uniquely or specific to in a castrate environment. Here, we verified that EMT is linked to stem cells in both normal prostate and CRPC. We further demonstrate that N-cadherin is involved in increased stem cell properties in normal mouse and human prostates from clinical samples in an androgen-deprived environment. Castration elevates the expression of N-cadherin and stem cell marker Trop2, accompanied with enhanced stem cell properties of luminal population in normal mouse prostates. N-cadherin upregulates stem cell properties in prostate cancer cells in the absence of androgen and CRPC tumors. N-cadherin-positive cells from CRPC tumors behave as stem cells. Overexpression of N-cadherin enhances ALDH activity in a panel of prostate cancer cell lines. Our findings reveal an important role of N-cadherin in prostate cancer and stem cells, and provide useful information of EMT-related biomarkers for preventing and developing efficient therapeutics to combat the treatment resistance to next-generation AR antagonists. Citation Format: Shu Lin, Tanya Stonyanova, Evelyn Kono, Jaclyn Matsuura, Naoko Kobayashi, Joyce Yamashiro, Dai Hong, Owen N. Witte, Andrew S. Goldstein, Robert E. Reiter. N-cadherin promotes castration-resistant prostate cancer progression by enhancing stem cell properties of prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-279.

Abstract 677: Co-targeting of AR and N-cadherin with Enzalutamide and 2A9 monoclonal antibody to treat castration resistant prostate cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Introduction: Enzalutamide (MDV3100) is an androgen receptor (AR) antagonist that targets various stages of the AR signaling pathway, and has been approved for the treatment of castration resistant prostate cancer (CRPC). Studies show improved survival in men with metastatic CRPC after chemotherapy when treated with Enzalutamide. However, emergence of potential AR-independent mechanisms of castration resistance and resistance to these next generation AR inhibitors has proven to be a challenge. Previously, we reported an association of N-cadherin, a mesenchymal cadherin expressed on the cell surface, with tumor progression and castration resistance. Targeting N-cadherin positive cells with specific monoclonal antibodies affect tumor growth in both AR positive and negative prostate cancers. Here we investigate the effects of co-targeting AR with Enzalutamide and N-cadherin with the N-cadherin specific monoclonal antibody 2A9 in CRPC. Method: N-cadherin was ectopically expressed in androgen dependent prostate cancer cells (LNCaP, MDA-PCa-2b, VCaP) and evaluated in vitro for invasion, growth, and self-renewal in the presence of Enzalutamide and 2A9 monoclonal antibody, raised against extracellular domain 4 of N-cadherin. Enzalutamide-resistant cell lines were generated and characterized. For in vivo studies, castration resistant N-cadherin and AR positive, as well as androgen-dependent tumors were implanted in mice as xenografts. When the tumors reached 100mm^3 in volume, mice were castrated and treated with 5-10mg/kg Enzalutamide and/or 5-10mg/kg 2A9 and monitored for tumor growth, local invasion, and metastasis. The tumors were harvested and analyzed for cell proliferation, apoptosis, and other markers. Results: Ectopic expression of N-cadherin in N-cadherin negative cells showed increase in cell growth and invasion in androgen-deprived conditions. In vitro studies show single or combination treatment of N-cadherin positive and AR positive cells reduce invasion and cell proliferation. Enzalutamide-resistant cell lines show response to 2A9 in androgen-deprived conditions. In vivo studies using CRPC xenograft models expressing N-cadherin and AR show inhibition of tumor growth when the animals are castrated and treated with a combination of Enzalutamide and 2A9 than either treatment alone. Androgen dependent xenograft models in castrated mice also showed significant delay in progression to castration resistance when treated in combination than either treatment alone. Conclusion: Previous studies suggested that therapeutic targeting of N-cadherin with monoclonal antibodies have significant effect in inhibiting tumor progression. The antibody in combination with Enzalutamide showed synergy. Other androgen receptor antagonists and other drugs targeting those potentially involved in downstream pathways may prove to have additional clinical benefit. Citation Format: Evelyn A. Kono, Sean Hyung-Kwon Lee, Tatsuya Shimomura, Shu Lin, Joyce Yamashiro, Robert E. Reiter. Co-targeting of AR and N-cadherin with Enzalutamide and 2A9 monoclonal antibody to treat castration resistant prostate cancer. [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 677. doi:10.1158/1538-7445.AM2014-677