Genetic variants in PDSS1 and SLC16A6 of the ketone body metabolic pathway predict cutaneous melanoma-specific survival

A few single-nucleotide polymorphisms (SNPs) have been identified to be associated with cutaneous melanoma (CM) survival though genome-wide association studies, but stringent multiple testing corrections required for the hypothesis-free testing may have masked some true associations. Using a hypothesis-driven analysis approach, we sought to evaluate associations between SNPs in ketone body metabolic pathway genes and CM survival. We comprehensively assessed associations between 4,196 (538 genotyped and 3,658 imputed) common SNPs in ketone body metabolic pathway genes and CM survival, using a dataset of 858 patients of a case-control study from The University of Texas M.D. Anderson Cancer Center as the discovery set and another dataset of 409 patients from the Nurses’ Health Study and the Health Professionals Follow-up Study as the replication set. There were 95/858 (11.1%) and 48/409 (11.5%) patients who died of CM, respectively. We identified two independent SNPs (i.e., PDSS1 rs12254548 G>C and SLC16A6 rs71387392 G>A) that were associated with CM survival, with allelic hazards ratios of 0.58 (95% confidence interval [CI]=0.44-0.76, P=9.00×10−5) and 1.98 (95% CI=1.34-2.94, P=6.30×10−4), respectively. Additionally, associations between genotypes of the SNPs and mRNA expression levels of their corresponding genes support the biologic plausibility of a role for these two variants in CM tumor progression and survival. Once validated by larger studies, PDSS1 rs12254548 and SLC16A6 rs71387392 may be biomarker for CM survival

Molecular evidence of adenosine deaminase linking adenosine A2A receptor and CD26 proteins

Adenosine is an endogenous purine nucleoside that acts in all living systems as a homeostatic network regulator through many pathways, which are adenosine receptor (AR)-dependent and -independent. From a metabolic point of view, adenosine deaminase (ADA) is an essential protein in the regulation of the total intracellular and extracellular adenosine in a tissue. In addition to its cytosolic localization, ADA is also expressed as an ecto-enzyme on the surface of different cells. Dipeptidyl peptidase IV (CD26) and some ARs act as binding proteins for extracellular ADA in humans. Since CD26 and ARs interact with ADA at opposite sites, we have investigated if ADA can function as a cell-to-cell communication molecule by bridging the anchoring molecules CD26 and A2AR present on the surfaces of the interacting cells. By combining site-directed mutagenesis of ADA amino acids involved in binding to A2AR and a modification of the bioluminescence resonance energy transfer (BRET) technique that allows detection of interactions between two proteins expressed in different cell populations with low steric hindrance (NanoBRET), we show direct evidence of the specific formation of trimeric complexes CD26-ADA-A2AR involving two cells. By dynamic mass redistribution assays and ligand binding experiments, we also demonstrate that A2AR-NanoLuc fusion proteins are functional. The existence of this ternary complex is in good agreement with the hypothesis that ADA could bridge T-cells (expressing CD26) and dendritic cells (expressing A2AR). This is a new metabolic function for ecto-ADA that, being a single chain protein, it has been considered as an example of moonlighting protein, because it performs more than one functional role (as a catalyst, a costimulator, an allosteric modulator and a cell-to-cell connector) without partitioning these functions in different subunits

A new murine model of osteoblastic/osteolytic lesions from human androgen-resistant prostate cancer

BACKGROUND: Up to 80% of patients dying from prostate carcinoma have developed bone metastases that are incurable. Castration is commonly used to treat prostate cancer. Although the disease initially responds to androgen blockade strategies, it often becomes castration-resistant (CRPC for Castration Resistant Prostate Cancer). Most of the murine models of mixed lesions derived from prostate cancer cells are androgen sensitive. Thus, we established a new model of CRPC (androgen receptor (AR) negative) that causes mixed lesions in bone. METHODS: PC3 and its derived new cell clone PC3c cells were directly injected into the tibiae of SCID male mice. Tumor growth was analyzed by radiography and histology. Direct effects of conditioned medium of both cell lines were tested on osteoclasts, osteoblasts and osteocytes. RESULTS: We found that PC3c cells induced mixed lesions 10 weeks after intratibial injection. In vitro, PC3c conditioned medium was able to stimulate tartrate resistant acid phosphatase (TRAP)-positive osteoclasts. Osteoprotegerin (OPG) and endothelin-1 (ET1) were highly expressed by PC3c while dikkopf-1 (DKK1) expression was decreased. Finally, PC3c highly expressed bone associated markers osteopontin (OPN), Runx2, alkaline phosphatase (ALP), bone sialoprotein (BSP) and produced mineralized matrix in vitro in osteogenic conditions. CONCLUSIONS: We have established a new CRPC cell line as a useful system for modeling human metastatic prostate cancer which presents the mixed phenotype of bone metastases that is commonly observed in prostate cancer patients with advanced disease. This model will help to understand androgen-independent mechanisms involved in the progression of prostate cancer in bone and provides a preclinical model for testing the effects of new treatments for bone metastases

Identifying Mediators of Androgen-independent Prostate Cancer Using Mass Spectrometry-based Proteomics

Androgen-deprivation remains the principal therapy for advanced and metastatic prostate cancers. However, some cancer cells are able to survive this treatment and transform themselves to a more aggressive androgen-independent prostate cancer (AIPC). An understanding of the molecular alterations that occur during the progression to androgen-independence is of utmost importance in order to generate effective targeted therapies. Using two different in vitro approaches coupled to high through-put mass spectrometry, we were able to identify numerous potential mediators of AIPC and aggressive prostate cancer. Our first approach using an in vitro cell line model of androgen-independence allowed us to identify enzymes of the ketogenic pathway as elevated in cases of aggressive prostate cancer. Specifically, ACAT1, an enzyme in this pathway, was further validated and shown to be substantially elevated in clinical cases of castration-resistant metastatic prostate cancer. In addition, ACAT1 was found to be an independent tissue-based prognostic marker of biochemical recurrence-free survival. In our second approach we compared the secreted proteomes of androgen independent cell lines (PC3, DU145, PPC1, LNCaP-SF, 22Rv1) to androgen-dependent (LNCaP, VCaP) and normal prostate epithelial (RWPE) cell lines. Of the over 3000 proteins identified in the secretomes, we found more than 100 proteins that were differentially secreted in the androgen independent cell lines. Of these, Protein S (PROS1) was elevated in the secretomes of all the AIPC cell lines and was not detected in the normal or androgen-dependent prostate cancer cell lines. Next, using RT-PCR and immunohistochemistry, we observed significantly higher tissue expression levels of PROS1 in localized high-grade and castrate-resistant metastatic prostate cancer samples compared to normal and low-grade prostate cancer, further indicating its importance in prostate cancer progression. Finally, functional validation revealed that PROS1 increases cell proliferation, migration and viability and thus may play a direct role on prostate cancer biology.Taken together, the results of this research will help in not only increasing the understanding of key molecular alterations and mechanisms by which prostate cancer cells can utilize to gain androgen-independence, and more importantly, aid in the development of new targeted therapies by potentially focusing on blocking or altering specific critical signalling cascades.Ph.D