[Show abstract][Hide abstract] ABSTRACT: Vascular endothelial growth factor (VEGF)–targeted antiangiogenic therapy significantly inhibits the growth of clear cell renal cell carcinoma (RCC). Eventually, therapy resistance develops in even the most responsive cases, but the mechanisms of resistance remain unclear. Herein, we developed two tumor models derived from an RCC cell line by conditioning the parental cells to two different stresses caused by VEGF-targeted therapy (sunitinib exposure and hypoxia) to investigate the mechanism of resistance to such therapy in RCC. Sunitinib-conditioned Caki-1 cells in vitro did not show resistance to sunitinib compared with parental cells, but when tested in vivo, these cells appeared to be highly resistant to sunitinib treatment. Hypoxia-conditioned Caki-1 cells are more resistant to hypoxia and have increased vascularity due to the upregulation of VEGF production; however, they did not develop sunitinib resistance either in vitro or in vivo. Human endothelial cells were more proliferative and showed increased tube formation in conditioned media from sunitinib-conditioned Caki-1 cells compared with parental cells. Gene expression profiling using RNA microarrays revealed that several genes related to tissue development and remodeling, including the development and migration of endothelial cells, were upregulated in sunitinib-conditioned Caki-1 cells compared with parental and hypoxia-conditioned cells. These findings suggest that evasive resistance to VEGF-targeted therapy is acquired by activation of VEGF-independent angiogenesis pathways induced through interactions with VEGF-targeted drugs, but not by hypoxia. These results emphasize that increased inhibition of tumor angiogenesis is required to delay the development of resistance to antiangiogenic therapy and maintain the therapeutic response in RCC.
No preview · Article · Nov 2015 · Neoplasia (New York, N.Y.)
[Show abstract][Hide abstract] ABSTRACT: Purpose:
The optimal extent of pelvic lymph node dissection (PLND) during radical cystectomy (RC) in patients with urothelial carcinoma of the bladder (UCB) is the subject of ongoing debate. In this study, we compared local recurrence-free and overall survival, in addition to complication rates, after extended PLND (ePLND) compared to standard PLND (sPLND).
We reviewed the charts of 314 patients who underwent RC for UCB between 2008 and 2013. ePLND was performed in 105 patients, and 105 matched patients who underwent standard PLND (sPLND) were selected based on clinical parameters. Local recurrence-free and overall survival rates were assessed using Kaplan-Meier survival analysis, and Cox proportional hazards models were used to assess potential determinants of these outcomes. Complications were assessed at 30 and 90 days using the Clavien-Dindo reporting system.
More lymph nodes were removed by ePLND (median 21) compared to sPLND (median 9; P < 0.001), but the rate of nodal involvement was not different. In multivariable analysis, ePLND was associated with a better local recurrence-free survival (HR = 0.63, P = 0.005), but was not an independent predictor of overall survival (HR = 1.06, P = 0.84). Estimated blood loss was greater with ePLND (1047.3 vs. 584.5 ml P < 0.001), but there was no significant difference in complications.
Extended PLND appears to reduce the risk of local recurrence, but was not an independent predictor of overall survival in this cohort. ePLND was associated with greater blood loss compared to sPLND, but not with other perioperative complications.
No preview · Article · Nov 2015 · World Journal of Urology
[Show abstract][Hide abstract] ABSTRACT: Purpose:
Clinical evidence suggests an increased CSC in tumor mass may contribute to the failure of conventional therapies since CSCs seem to be more resistant than differentiated tumor cells. Thus, unveiling the mechanism regulating CSCs and candidate target molecules will provide new strategy to cure the patients.
The stemness of prostate cancer cells was determined by a prostasphere assay, and dye exclusion assay. To find critical stem cell marker and reveal regulation mechanism, basic biochemical and molecular biological methods such as qRT-PCR, Western blot, reporter gene assay and chromatin immunoprecipitation assay were employed. In addition, to determine the effect of combination therapy targeting both CSCs and its progeny, in vitro MTT assay and in vivo xenograft model was used.
We demonstrate immortalized normal human prostate epithelial cells, appeared non-tumorigenic in vivo, become tumorigenic and acquire stem cell phenotype after knocking down a tumor suppressor gene. Also, those stem-like cells increase chemoresistance to conventional anti-cancer reagent. Mechanistically, we unveil that Wnt signaling is a key pathway regulating well-known stem cell marker CD44 by directly interacting to the promoter. Thus, by targeting CSCs using Wnt inhibitors synergistically enhances the efficacy of conventional drugs. Furthermore, the in vivo mice model bearing xenografts showed a robust inhibition of tumor growth after combination therapy.
Overall, this study provides strong evidence of CSC in CRPC. This new combination therapy strategy targeting CSC could significantly enhance therapeutic efficacy of current chemotherapy regimen only targeting non-CSC cells.
No preview · Article · Oct 2015 · Clinical Cancer Research
[Show abstract][Hide abstract] ABSTRACT: Tumor microenvironments are characterized by decreased oxygen and nutrition due to the rapid and progressive nature of tumors and also stresses induced by several anti-tumor therapies. These intense cell stressors trigger a protective cell survival mechanism heralded by the unfolded protein response (UPR). The UPR is induced by an accumulation of unfolded proteins in the endoplasmic reticulum (ER) following cell starvation. Although the ER stress response is implicated in cytoprotection, its precise role during anti-angiogenic therapy remains unclear. One of the major proteins involved in ER stress is glucose-regulated protein 78 (GRP78), which binds to unfolded proteins and dissociates from membrane-bound ER stress sensors. To determine the role of ER stress responses during anti-angiogenic therapy and the potential role of GRP78 in combined therapy in renal cell carcinoma (RCC), we used GRP78 overexpressing or knockdown RCC cells under hypoxic or hypoglycemic conditions in vitro and in animal models treated with sunitinib. Here, we report that GRP78 plays a crucial role in protecting RCC cells from hypoxic and hypoglycemic stress induced by anti-angiogenic therapy. Knockdown of GRP78 using siRNA inhibited cancer cell survival and induced apoptosis in RCC cells in vitro and also resulted in ER stress-induced apoptosis and hypoxic/hypoglycemic stress-induced apoptosis by inactivating the PERK/eIF-2α pathway. Finally, GRP78 knockdown showed potent suppression of tumor growth and enhanced the antitumor effect of sunitinib in RCC xenografts. Our findings suggest that GRP78 may serve as a novel therapeutic target in combination with anti-angiogenic therapy for the management of RCC.
[Show abstract][Hide abstract] ABSTRACT: Dry eye is a common disorder caused by inadequate hydration of the ocular surface that results in disruption of barrier function. The homeostatic protein clusterin (CLU) is prominent at fluid-tissue interfaces throughout the body. CLU levels are reduced at the ocular surface in human inflammatory disorders that manifest as severe dry eye, as well as in a preclinical mouse model for desiccating stress that mimics dry eye. Using this mouse model, we show here that CLU prevents and ameliorates ocular surface barrier disruption by a remarkable sealing mechanism dependent on attainment of a critical all-or-none concentration. When the CLU level drops below the critical all-or-none threshold, the barrier becomes vulnerable to desiccating stress. CLU binds selectively to the ocular surface subjected to desiccating stress in vivo, and in vitro to the galectin LGALS3, a key barrier component. Positioned in this way, CLU not only physically seals the ocular surface barrier, but it also protects the barrier cells and prevents further damage to barrier structure. These findings define a fundamentally new mechanism for ocular surface protection and suggest CLU as a biotherapeutic for dry eye.
[Show abstract][Hide abstract] ABSTRACT: Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked.
[Show abstract][Hide abstract] ABSTRACT: Understanding molecular mechanisms of response and resistance to anticancer therapies requires prospective patient follow-up and clinical and functional validation of both common and low-frequency mutations. We describe a whole-exome sequencing (WES) precision medicine trial focused on patients with advanced cancer.
To understand how WES data affect therapeutic decision making in patients with advanced cancer and to identify novel biomarkers of response.
Patients with metastatic and treatment-resistant cancer were prospectively enrolled at a single academic center for paired metastatic tumor and normal tissue WES during a 19-month period (February 2013 through September 2014). A comprehensive computational pipeline was used to detect point mutations, indels, and copy number alterations. Mutations were categorized as category 1, 2, or 3 on the basis of actionability; clinical reports were generated and discussed in precision tumor board. Patients were observed for 7 to 25 months for correlation of molecular information with clinical response.
Feasibility, use of WES for decision making, and identification of novel biomarkers.
A total of 154 tumor-normal pairs from 97 patients with a range of metastatic cancers were sequenced, with a mean coverage of 95X and 16 somatic alterations detected per patient. In total, 16 mutations were category 1 (targeted therapy available), 98 were category 2 (biologically relevant), and 1474 were category 3 (unknown significance). Overall, WES provided informative results in 91 cases (94%), including alterations for which there is an approved drug, there are therapies in clinical or preclinical development, or they are considered drivers and potentially actionable (category 1-2); however, treatment was guided in only 5 patients (5%) on the basis of these recommendations because of access to clinical trials and/or off-label use of drugs. Among unexpected findings, a patient with prostate cancer with exceptional response to treatment was identified who harbored a somatic hemizygous deletion of the DNA repair gene FANCA and putative partial loss of function of the second allele through germline missense variant. Follow-up experiments established that loss of FANCA function was associated with platinum hypersensitivity both in vitro and in patient-derived xenografts, thus providing biologic rationale and functional evidence for his extreme clinical response.
The majority of advanced, treatment-resistant tumors across tumor types harbor biologically informative alterations. The establishment of a clinical trial for WES of metastatic tumors with prospective follow-up of patients can help identify candidate predictive biomarkers of response.
[Show abstract][Hide abstract] ABSTRACT: SRC kinase is activated in castration resistant prostate cancer (CRPC), phosphorylates the androgen receptor (AR), and causes its ligand-independent activation as a transcription factor. However, activating SRC mutations are exceedingly rare in human tumors, and mechanisms of ectopic SRC activation therefore remain largely unknown. Performing a functional genomics screen, we found that downregulation of SRC inhibitory kinase CSK is sufficient to overcome growth arrest induced by depriving human prostate cancer cells of androgen. CSK knockdown led to ectopic SRC activation, increased AR signaling, and resistance to anti-androgens. Consistent with the in vitro observations, stable knockdown of CSK conferred castration resistance in mouse xenograft models, while sensitivity to the tyrosine kinase inhibitor dasatinib was retained. Finally, CSK was found downregulated in a distinct subset of CRPCs marked by AR amplification and ETS2 deletion but lacking PTEN and RB1 mutations. These results identify CSK downregulation as a principal driver of SRC activation and castration resistance and validate SRC as a drug target in a molecularly defined subclass of CRPCs.
[Show abstract][Hide abstract] ABSTRACT: Although the new generation of androgen receptor (AR) antagonists like enzalutamide (ENZ) prolong survival of metastatic castration-resistant prostate cancer (CRPC), AR-driven tumors eventually recur indicating that additional therapies are required to fully block AR function. Since DNA topoisomerase II (Topo II) was demonstrated to be essential for AR to initiate gene transcription, this study tested whether catalytic inhibitors of Topo II can block AR signaling and suppress ENZ-resistant CRPC growth. Using multiple prostate cancer cell lines, we showed that catalytic Topo II inhibitors, ICRF187 and ICRF193 inhibited transcription activities of the wild-type AR, mutant ARs (F876L and W741C) and the AR-V7 splice variant. ICRF187 and ICRF193 decreased AR recruitment to target promoters and reduced AR nuclear localization. Both ICRF187 and ICRF193 also inhibited cell proliferation and delayed cell cycling at the G2/M phase. ICRF187 inhibited tumor growth of castration-resistant LNCaP and 22RV1 xenografts as well as ENZ-resistant MR49F xenografts. We conclude that catalytic Topo II inhibitors can block AR signaling and inhibit tumor growth of CRPC xenografts, identifying a potential co-targeting approach using these inhibitors in combination with AR pathway inhibitors in CRPC.
[Show abstract][Hide abstract] ABSTRACT: In this study, we show that the transmembrane glycoprotein Trop-2 is up-regulated in human prostate cancer (PCa) with extracapsular extension (stages pT3/pT4) as compared to organ-confined (stage pT2) PCa. Consistent with this evidence, Trop-2 expression is found to be increased in metastatic prostate tumors of Transgenic Adenocarcinoma of Mouse Prostate mice and to strongly correlate with α5β1 integrin levels. Using PCa cells, we show that Trop-2 specifically associates with the α5 integrin subunit, as binding to α3 is not observed, and that Trop-2 displaces focal adhesion kinase from focal contacts. In support of the role of Trop-2 as a promoter of PCa metastatic phenotype, we observe high expression of this molecule in exosomes purified from Trop-2-positive PCa cells. These vesicles are then found to promote migration of Trop-2-negative PCa cells on fibronectin, an α5β1 integrin/focal adhesion kinase substrate, thus suggesting that the biological function of Trop-2 may be propagated to recipient cells. In summary, our findings show that Trop-2 promotes an α5β1 integrin-dependent pro-metastatic signaling pathway in PCa cells and that the altered expression of Trop-2 may be utilized for early identification of capsule-invading PCa.