Y-box binding protein-1 is crucial in acquired drug resistance development in metastatic clear-cell renal cell carcinoma.

BACKGROUND: Renal cell carcinoma (RCC) is a highly vascular tumor and patients with low risk metastatic RCC of clear-cell histological sub-type (mccRCC) are treated with tyrosine-kinase inhibitors (TKIs), sunitinib, as the first-line of treatment. Unfortunately, TKI resistance eventually develops, and the underlying molecular mechanism is not well understood. METHODS: RCC cell-line with metastatic clear-cell histology (Caki-1), and patient samples were analysed to identify the role of Y-box binding protein 1 (YB-1) and ATP-binding cassette sub-family B member 1 (ABCB-1) in acquired sunitinib-resistance development. Caki-1 was conditioned with increasing sunitinib doses to recapitulate acquired resistance development in clinics. Sunitinib-conditioned and wild-type Caki-1 were subjected to cell viability assay, scratch assay, chicken embryo chorioallantoic membrane engraftment and proteomics analysis. Classical biochemical assays like flow cytometry, immunofluorescent staining, immunohistochemical staining, optical coherence tomography imaging, Western Blot and RT-PCR assays were applied to determine the possible mechanism of sunitinib-resistance development and the effect of drug treatments. Publicly available data was also used to determine the role of YB-1 upregulation in ccRCC and the patients' overall survival. RESULTS: We demonstrate that YB-1 and ABCB-1 are upregulated in sunitinib-resistant in vitro, ex vivo, in vivo and patient samples compared to the sensitive samples. This provides evidence to a mechanism of acquired sunitinib-resistance development in mccRCC. Furthermore, our results establish that inhibiting ABCB-1 with elacridar, in addition to sunitinib, has a positive impact on reverting sunitinib-resistance development in in vitro, ex vivo and in vivo models. CONCLUSION: This work proposes a targeted therapy (elacridar and sunitinib) to re-sensitize sunitinib-resistant mccRCC and, possibly, slow disease progression.

Identification of gene signature for treatment response to guide precision oncology in clear-cell renal cell carcinoma.

Clear-cell renal cell carcinoma (ccRCC) is a common therapy resistant disease with aberrant angiogenic and immunosuppressive features. Patients with metastatic disease are treated with targeted therapies based on clinical features: low-risk patients are usually treated with anti-angiogenic drugs and intermediate/high-risk patients with immune therapy. However, there are no biomarkers available to guide treatment choice for these patients. A recently published phase II clinical trial observed a correlation between ccRCC patients' clustering and their response to targeted therapy. However, the clustering of these groups was not distinct. Here, we analyzed the gene expression profile of 469 ccRCC patients, using featured selection technique, and have developed a refined 66-gene signature for improved sub-classification of patients. Moreover, we have identified a novel comprehensive expression profile to distinguish between migratory stromal and immune cells. Furthermore, the proposed 66-gene signature was validated using a different cohort of 64 ccRCC patients. These findings are foundational for the development of reliable biomarkers that may guide treatment decision-making and improve therapy response in ccRCC patients.

Inhibition of GLI2 with antisense-oligonucleotides: A potential therapy for the treatment of bladder cancer.

The sonic hedgehog (SHH) signaling pathway plays an integral role in the maintenance and progression of bladder cancer (BCa) and SHH inhibition may be an efficacious strategy for BCa treatment. We assessed an in-house human BCa tissue microarray and found that the SHH transcription factors, GLI1 and GLI2, were increased in disease progression. A panel of BCa cell lines show that two invasive lines, UM-UC-3 and 253J-BV, both express these transcription factors but UM-UC-3 produces more SHH ligand and is less responsive in viability to pathway stimulation by recombinant human SHH or smoothened agonist, and less responsive to inhibitors including the smoothened inhibitors cyclopamine and SANT-1. In contrast, 253J-BV was highly responsive to these manipulations. We utilized a GLI1 and GLI2 antisense oligonucleotide (ASO) to bypass pathway mechanics and target the transcription factors directly. UM-UC-3 decreased in viability due to both ASOs but 253J-BV was only affected by GLI2 ASO. We utilized the murine intravesical orthotopic human BCa (mio-hBC) model for the establishment of noninvasive BCa and treated tumors with GLI2 ASO. Tumor size, growth rate, and GLI2 messenger RNA and protein expression were decreased. These results suggest that GLI2 ASO may be a promising new targeted therapy for BCa.

Development of a murine intravesical orthotopic human bladder cancer (mio-hBC) model.

We have developed a murine intravesical orthotopic human bladder cancer (mio-hBC) model for the establishment of superficial urothelial cell carcinomas. In this model we catheterize female atyhmic nude mice and pre-treat the bladder with poly-L-lysine for 15 minutes, followed by intravesical instillation of luciferase-transfected human UM-UC-3 cells. Cancer cells are quantified by bioluminescent imaging which has been validated by small animal ultrasound. Poly-L-lysine pre-treatment increased engraftment rate (84.4%) and resulted in faster growing tumors than trypsin pre-treatment. In addition, tumors respond through a decrease in growth and increase in apoptosis to chemotherapy with mitomycin C. Previous intravesical models utilized KU7 cells which have been later determined to be of non-bladder origin. They display markers consistent with HeLa cells, requiring a need for a true intravesical bladder model. Efficient engraftment and rapid superficial growth patterning of the human bladder tumor differentiate this in vivo orthotopic model from previous bladder models.

Magnetically-actuated drug delivery device (MADDD) for minimally invasive treatment of prostate cancer: An in vivo animal pilot study.

BACKGROUND: The vast majority of prostate cancer presents clinically localized to the prostate without evidence of metastasis. Currently, there are several modalities available to treat this particular disease. Despite radical prostatectomy demonstrating a modest prostate cancer specific mortality benefit in the PIVOT trial, several novel modalities have emerged to treat localized prostate cancer in patients that are either not eligible for surgery or that prefer an alternative approach. METHODS: Athymic nude mice were subcutaneously inoculated with prostate cancer cells. The mice were divided into four cohorts, one cohort untreated, two cohorts received docetaxel (10 mg/kg) either subcutaneously (SC) or intravenously (IV) and the fourth cohort was treated using the magnetically-actuated docetaxel delivery device (MADDD), dispensing 1.5 µg of docetaxel per 30 min treatment session. Treatment in all three therapeutic arms (SC, IV, and MADDD) was administered once weekly for 6 weeks. Treatment efficacy was measured once a week according to tumor volume using ultrasound. In addition, calipers were used to assess tumor volume. RESULTS: Animals implanted with the device demonstrated no signs of distress or discomfort, neither local nor systemic symptoms of inflammation and infection. Using an independent sample t-test, the tumor growth rate of the treated tumors was significant when compared to the control. Post hoc Tukey HSD test results showed that the mean tumor growth rate of our device cohort was significantly lower than SC and control cohorts. Moreover, IV cohort showed slight reduction in mean tumor growth rates than the ones from the device cohort, however, there was no statistical significance in tumor growth rate between these two cohorts. Furthermore, immunohistochemistry demonstrated an increased cellular apoptosis in the MADDD treated tumors and a decreased proliferation when compared to the other cohorts. In addition, IV cohort showed increased treatment side effects (weight loss) when compared to the device cohort. Finally, MADDD showed minimal expression of CD45 comparable to the control cohort, suggesting no signs of chronic inflammation. CONCLUSIONS: In conclusion, this study showed for the first time that MADDD, clearly suppressed tumor growth in local prostate cancer tumors. This could potentially be a novel clinical treatment approach for localized prostate cancer.