Three-dimensional coculture provides an improved in vitro model for papillary renal cell carcinoma.

Papillary renal cell carcinoma (pRCC) represents the second most common kidney cancer and can be distinguished from other types based on its unique histological architecture and specific pattern of genomic alterations. Sporadic type 1 pRCC is almost universally driven by focal or chromosomal amplification of the receptor tyrosine kinase MET, although the specific mode of its activation is unclear. Although the MET receptors found in human tumor specimens appear highly active, those found on the surface of in vitro-cultured tumor cells are only weakly activated in the absence of exogenous hepatocyte growth factor ligand. Furthermore, pRCC cells cultured in standard two-dimensional conditions with serum fail to respond functionally to MET knockdown or the selective MET inhibitor capmatinib despite clear evidence of kinase inhibition at the molecular level. To better model pRCC in vitro, we developed a three-dimensional coculture system in which renal tumor cells are layered on top of primary fibroblasts in a fashion that mimics the papillary architecture of human tumors. In this three-dimensional spheroid model, the tumor cells survive and proliferate in the absence of serum due to trophic support of hepatocyte growth factor-producing fibroblasts. Unlike tumor cells grown in monoculture, the proliferation of cocultured tumor cells is sensitive to capmatinib and parallels inhibition of MET kinase activity. These findings demonstrate the importance of stromal fibroblasts in pRCC and indicate that accurate in vitro representation of this disease requires the presence of both tumor and fibroblast cells in a structured coculture model.NEW & NOTEWORTHY Two-dimensional monoculture of papillary renal cancer cells fails to replicate several features of the disease found in humans. We hypothesized that this discordance results from lack of trophic support from renal fibroblasts, which are involved in the architecture of human papillary renal tumors. We found that three-dimensional layering of renal cancer cells on top of a fibroblast core using magnetic bioprinting produces a structured spheroid that more faithfully mimics the behavior of human tumors.

Sox2 Expression Marks Castration-Resistant Progenitor Cells in the Adult Murine Prostate.

Identification of defined epithelial cell populations with progenitor properties is critical for understanding prostatic development and disease. Here, we demonstrate that Sox2 expression is enriched in the epithelial cells of the proximal prostate adjacent to the urethra. We use lineage tracing of Sox2-positive cells during prostatic development, homeostasis, and regeneration to show that the Sox2 lineage is capable of self-renewal and contributes to prostatic regeneration. Persisting luminal cells express Sox2 after castration, highlighting a potential role for Sox2 in cell survival and castration-resistance. In addition to revealing a novel progenitor population in the prostate, these data implicate Sox2 as a regulatory factor of adult prostate epithelial stem cells. Stem Cells 2019;37:690-700.

LRRK2 deficiency impairs trans-Golgi to lysosome trafficking and endocytic cargo degradation in human renal proximal tubule epithelial cells.

Defects in vesicular trafficking underlie a wide variety of human diseases. Genetic disruption of leucine-rich repeat kinase 2 (LRRK2) in rodents results in epithelial vesicular trafficking errors that can also be induced by treatment of animals with LRRK2 kinase inhibitors. Here we demonstrate that defects in human renal cells lacking LRRK2 phenocopy those seen in the kidneys of Lrrk2 knockout mice, characterized by accumulation of intracellular waste vesicles and fragmentation of the Golgi apparatus. This phenotype can be recapitulated by knockdown of N-ethylmaleimide-sensitive factor, which physically associates with LRRK2 in renal cells. Deficiency in either protein leads to a defect in trans-Golgi to lysosome protein trafficking, which compromises the capacity of lysosomes to degrade endocytic and autophagic cargo. In contrast, neither bulk endocytosis nor autophagic flux are impaired when LRRK2 is acutely knocked down in normal immortalized human kidney (HK2) cells. These data collectively suggest that the primary renal defect caused by LRRK2 deficiency is in protein trafficking between the Golgi apparatus and late endosome/lysosome, which leads to progressive impairments in lysosomal function.

MEIS-mediated suppression of human prostate cancer growth and metastasis through HOXB13-dependent regulation of proteoglycans.

The molecular roles of HOX transcriptional activity in human prostate epithelial cells remain unclear, impeding the implementation of new treatment strategies for cancer prevention and therapy. MEIS proteins are transcription factors that bind and direct HOX protein activity. MEIS proteins are putative tumor suppressors that are frequently silenced in aggressive forms of prostate cancer. Here we show that MEIS1 expression is sufficient to decrease proliferation and metastasis of prostate cancer cells in vitro and in vivo murine xenograft models. HOXB13 deletion demonstrates that the tumor-suppressive activity of MEIS1 is dependent on HOXB13. Integration of ChIP-seq and RNA-seq data revealed direct and HOXB13-dependent regulation of proteoglycans including decorin (DCN) as a mechanism of MEIS1-driven tumor suppression. These results define and underscore the importance of MEIS1-HOXB13 transcriptional regulation in suppressing prostate cancer progression and provide a mechanistic framework for the investigation of HOXB13 mutants and oncogenic cofactors when MEIS1/2 are silenced.

Decisions regarding the treatment of patients with early-stage prostate cancer are often based on the risk that the cancer could grow and spread quickly. However, it is not always straightforward to predict how the cancer will behave. Studies from 2017 and 2018 found that samples of less aggressive prostate cancer have higher levels of a group of proteins called MEIS proteins. MEIS proteins help control the production of numerous other proteins, which could affect the behavior of prostate cancer cells in many ways. VanOpstall et al. ­ including some of the researchers that performed the 2017 and 2018 studies ­ have investigated how MEIS proteins affect prostate cancer. When prostate cancer cells are implanted into mice, they result in tumors. VanOpstall et al. found that tumors that produced MEIS proteins grew more slowly. Next, MEIS proteins were extracted from the prostate cancer cells and were found to interact with another protein called HOXB13, which regulates the activity of numerous genes. When the cells were genetically modified to prevent HOXB13 being produced, the protective effect of MEIS proteins was lost. MEIS proteins work with HOXB13 to regulate the production of several other proteins, in particular a protein called Decorin that can suppress tumors. When MEIS proteins and HOXB13 are present, the cell produces more Decorin and the tumors grow more slowly and are less likely to spread. VanOpstall et al. found that blocking Decorin production rendered MEIS proteins less able to slow the spread of prostate cancer. These results suggest that MEIS proteins and HOXB13 are needed to stop tumors from growing and spreading, and some of this ability is by prompting production of Decorin. This study explains how MEIS proteins can reduce prostate cancer growth, providing greater confidence in using them to determine whether aggressive treatment is needed. A greater understanding of this pathway for tumor suppression could also provide an opportunity for developing anti-cancer drugs.

MEIS1 and MEIS2 Expression and Prostate Cancer Progression: A Role For HOXB13 Binding Partners in Metastatic Disease.

Purpose: Germline mutations within the MEIS-interaction domain of HOXB13 have implicated a critical function for MEIS-HOX interactions in prostate cancer etiology and progression. The functional and predictive role of changes in MEIS expression within prostate tumor progression, however, remain largely unexplored.Experimental Design: Here we utilize RNA expression datasets, annotated tissue microarrays, and cell-based functional assays to investigate the role of MEIS1 and MEIS2 in prostate cancer and metastatic progression.Results: These analyses demonstrate a stepwise decrease in the expression of both MEIS1 and MEIS2 from benign epithelia, to primary tumor, to metastatic tissues. Positive expression of MEIS proteins in primary tumors, however, is associated with a lower hazard of clinical metastasis (HR = 0.28) after multivariable analysis. Pathway and gene set enrichment analyses identified MEIS-associated networks involved in cMYC signaling, cellular proliferation, motility, and local tumor environment. Depletion of MEIS1 and MEIS2 resulted in increased tumor growth over time in vivo, and decreased MEIS expression in both patient-derived tumors and MEIS-depleted cell lines was associated with increased expression of the protumorigenic genes cMYC and CD142, and decreased expression of AXIN2, FN1, ROCK1, SERPINE2, SNAI2, and TGFß2.Conclusions: These data implicate a functional role for MEIS proteins in regulating cancer progression, and support a hypothesis whereby tumor expression of MEIS1 and MEIS2 expression confers a more indolent prostate cancer phenotype, with a decreased propensity for metastatic progression. Clin Cancer Res; 24(15); 3668-80. ©2018 AACR.

HOXB13 mutations and binding partners in prostate development and cancer: Function, clinical significance, and future directions.

The recent and exciting discovery of germline HOXB13 mutations in familial prostate cancer has brought HOX signaling to the forefront of prostate cancer research. An enhanced understanding of HOX signaling, and the co-factors regulating HOX protein specificity and transcriptional regulation, has the high potential to elucidate novel approaches to prevent, diagnose, stage, and treat prostate cancer. Toward our understanding of HOX biology in prostate development and prostate cancer, basic research in developmental model systems as well as other tumor sites provides a mechanistic framework to inform future studies in prostate biology. Here we describe our current understanding of HOX signaling in genitourinary development and cancer, current clinical data of HOXB13 mutations in multiple cancers including prostate cancer, and the role of HOX protein co-factors in development and cancer. These data highlight numerous gaps in our understanding of HOX function in the prostate, and present numerous potentially impactful mechanistic and clinical opportunities for future investigation.

Selective Glucocorticoid Receptor Modulators (SGRMs) Delay Castrate-Resistant Prostate Cancer Growth.

Increased glucocorticoid receptor (GR) expression and activity following androgen blockade can contribute to castration-resistant prostate cancer (CRPC) progression. Therefore, we hypothesized that GR antagonism will have therapeutic benefit in CRPC. However, the FDA-approved nonselective, steroidal GR antagonist, mifepristone, lacks GR specificity, reducing its therapeutic potential. Here, we report that two novel nonsteroidal and highly selective GR modulators (SGRM), CORT118335 and CORT108297, have the ability to block GR activity in prostate cancer and slow CRPC progression. In contrast to mifepristone, these novel SGRMs did not affect androgen receptor (AR) signaling, but potently inhibited GR transcriptional activity. Importantly, SGRMs decreased GR-mediated tumor cell viability following AR blockade. In vivo, SGRMs significantly inhibited CRPC progression in high GR-expressing, but not in low GR-expressing xenograft models. Transcriptome analysis following AR blockade and GR activation revealed that these SGRMs block GR-mediated proliferative gene expression pathways. Furthermore, GR-regulated proliferation-associated genes AKAP12, FKBP5, SGK1, CEBPD, and ZBTB16 are inhibited by CORT108297 treatment in vivo Together, these data suggest that GR-selective nonsteroidal SGRMs potently inhibit GR activity and prostate cancer growth despite AR pathway inhibition, demonstrating the therapeutic potential of SGRMs in GR-expressing CRPC. Mol Cancer Ther; 16(8); 1680-92. ©2017 AACR.

Determination of GLUT1 Oligomerization Parameters using Bioluminescent Förster Resonance Energy Transfer.

The facilitated glucose transporter GLUT1 (SLC2A1) is an important mediator of glucose homeostasis in humans. Though it is found in most cell types to some extent, the level of GLUT1 expression across different cell types can vary dramatically. Prior studies in erythrocytes-which express particularly high levels of GLUT1-have suggested that GLUT1 is able to form tetrameric complexes with enhanced transport activity. Whether dynamic aggregation of GLUT1 also occurs in cell types with more modest expression of GLUT1, however, is unclear. To address this question, we developed a genetically encoded bioluminescent Förster resonance energy transfer (BRET) assay using the luminescent donor Nanoluciferase and fluorescent acceptor mCherry. By tethering these proteins to the N-terminus of GLUT1 and performing saturation BRET analysis, we were able to demonstrate the formation of multimeric complexes in live cells. Parallel use of flow cytometry and immunoblotting further enabled us to estimate the density of GLUT1 proteins required for spontaneous oligomerization. These data provide new insights into the physiological relevance of GLUT1 multimerization as well as a new variant of BRET assay that is useful for measuring the interactions among other cell membrane proteins in live cells.