Efficient generation of patient-matched malignant and normal primary cell cultures from clear cell renal cell carcinoma patients: clinically relevant models for research and personalized medicine.

BACKGROUND: Patients with clear cell renal cell carcinoma (ccRCC) have few therapeutic options, as ccRCC is unresponsive to chemotherapy and is highly resistant to radiation. Recently targeted therapies have extended progression-free survival, but responses are variable and no significant overall survival benefit has been achieved. Commercial ccRCC cell lines are often used as model systems to develop novel therapeutic approaches, but these do not accurately recapitulate primary ccRCC tumors at the genomic and transcriptional levels. Furthermore, ccRCC exhibits significant intertumor genetic heterogeneity, and the limited cell lines available fail to represent this aspect of ccRCC. Our objective was to generate accurate preclinical in vitro models of ccRCC using tumor tissues from ccRCC patients. METHODS: ccRCC primary single cell suspensions were cultured in fetal bovine serum (FBS)-containing media or defined serum-free media. Established cultures were characterized by genomic verification of mutations present in the primary tumors, expression of renal epithelial markers, and transcriptional profiling. RESULTS: The apparent efficiency of primary cell culture establishment was high in both culture conditions, but genotyping revealed that the majority of cultures contained normal, not cancer cells. ccRCC characteristically shows biallelic loss of the von Hippel Lindau (VHL) gene, leading to accumulation of hypoxia-inducible factor (HIF) and expression of HIF target genes. Purification of cells based on expression of carbonic anhydrase IX (CA9), a cell surface HIF target, followed by culture in FBS enabled establishment of ccRCC cell cultures with an efficiency of >80 %. Culture in serum-free conditions selected for growth of normal renal proximal tubule epithelial cells. Transcriptional profiling of ccRCC and matched normal cell cultures identified up- and down-regulated networks in ccRCC and comparison to The Cancer Genome Atlas confirmed the clinical validity of our cell cultures. CONCLUSIONS: The ability to establish primary cultures of ccRCC cells and matched normal kidney epithelial cells from almost every patient provides a resource for future development of novel therapies and personalized medicine for ccRCC patients.

Clonal evolution and tumor-initiating cells: New dimensions in cancer patient treatment.

Human cancer is not a uniform disease but a plethora of disparate tumor types and subtypes. The differences that exist between individual tumors (intertumoral heterogeneity) present a significant roadblock to the eradication of cancer. It has also become increasingly clear that variations across individual tumors (intratumoral heterogeneity) have important implications to cancer progression and treatment efficacy. Therefore, in order to improve patient care and develop novel chemotherapeutics, the evolving tumor landscape needs to be further explored. Next-generation sequencing (NGS) technologies are revolutionizing the cancer research arena by providing state-of-the-art, high-speed methods of genome sequencing at single-nucleotide resolution, thus enabling an unprecedented detection of tumor-specific genetic abnormalities. These anomalies can be quantified to reveal specific frequencies of DNA alterations that correspond to distinct clonal populations within a given tumor. As such, NGS approaches have also been utilized to explore the heterogeneous landscape of patient tumors as well as to match metastatic and/or recurrent growths and patient-derived engrafts. By sequencing in this manner--through time so to speak--cancer researchers can track shifting clonal populations, make important inferences about tumor evolution and potentially identify tumor subclones that could be viably targeted. This exciting new territory has important implications for the competing clonal evolution and cancer stem cell models of tumor heterogeneity, and also offers a new dimension for cancer treatment and profound hope for patients in the coming years.

Opposing roles for mammary epithelial-specific PPARγ signaling and activation during breast tumour progression.

BACKGROUND: Among women worldwide, breast cancer is the most commonly diagnosed cancer, and the second leading cause of cancer-related deaths. Improved understanding of breast tumourigenesis may facilitate the development of more effective therapies. Peroxisome proliferator-activated receptor (PPAR)γ is a transcription factor that regulates genes involved in insulin sensitivity and adipogenesis. Previously, we showed, using 7,12-dimethylbenz [a] anthracene (DMBA)-treated haploinsufficient PPARγ mice, that PPARγ suppresses breast tumour progression; however, the PPARγ expressing cell types and mechanisms involved remain to be clarified. Here, the role of PPARγ expression and activation in mammary epithelial cells (MG) with respect to DMBA-mediated breast tumourigenesis was investigated. METHODS: PPARγ MG knockout (PPARγ-MG KO) mice and their congenic, wild-type controls (PPARγ-WT) were treated once a week for six weeks by oral gavage with 1 mg DMBA dissolved in corn oil and maintained on a normal chow diet. At week 7, mice were randomly divided into those maintained on a normal chow diet (DMBA Only; PPARγ-WT: n = 25 and PPARγ-MG KO: n = 39) or those receiving a diet supplemented with the PPARγ ligand, rosiglitazone (ROSI, 4 mg/kg/day) (DMBA + ROSI; PPARγ-WT: n = 34 and PPARγ-MG KO: n = 17) for the duration of the 25-week study. RESULTS: Compared to DMBA Only-treated PPARγ-WTs, both breast tumour susceptibility and serum levels of proinflammatory and chemotactic cytokines, namely IL-4, eotaxin, GM-CSF, IFN-γ, and MIP-1α, were decreased among PPARγ-MG KOs. Cotreatment with ROSI significantly reduced breast tumour progression among PPARγ-WTs, correlating with increased BRCA1 and decreased VEGF and COX-2 protein expression levels in breast tumours; whereas, surprisingly DMBA + ROSI-treated PPARγ-MG KOs showed increased breast tumourigenesis, correlating with activation of COX-2. CONCLUSION: These novel data suggest MG-specific PPARγ expression and signaling is critical during breast tumourigenesis, and may serve as a strong candidate predictive biomarker for response of breast cancer patients to the use of therapeutic strategies that include PPARγ ligands.

Loss of PPARγ expression in mammary secretory epithelial cells creates a pro-breast tumorigenic environment.

Breast cancer is the leading cause of new cancer diagnoses among women. Using peroxisome proliferator-activated receptor (PPAR)γ((+/-)) mice, we showed normal expression of PPARγ was critical to stop 7,12-dimethylbenz[a]anthracene (DMBA)-induced breast tumorigenesis. PPARγ is expressed in many breast cell types including mammary secretory epithelial (MSE) cells. MSEs proliferate as required during pregnancy, and undergo apoptosis or reversible transdifferentiation during involution once lactation is complete. Thus, MSE-specific loss of PPARγ was hypothesized to enhance DMBA-mediated breast tumorigenesis. To test this, MSE cell-specific PPARγ knockout (PPARγ-MSE KO) and control (PPARγ-WT) mice were generated, mated and allowed to nurse for three days. One week after involution, dams were treated with DMBA to initiate breast tumors, and randomized on week 7 to continue receiving a normal chow diet (DMBA Only: PPARγ-WT, n = 15; PPARγ-MSE KO, n = 25) or one supplemented with a PPARγ activating drug (DMBA + ROSI: PPARγ-WT, n = 17; PPARγ-MSE KO, n = 24), and monitored for changes in breast tumor outcomes. PPARγ-MSE KOs had significantly lower overall survival and decreased mammary tumor latency as compared to PPARγ-WT controls. PPARγ activation significantly reduced DMBA-mediated malignant mammary tumor volumes irrespective of genotype. MSE-specific PPARγ loss resulted in decreased mammary gland expression of PTEN and Bax, increased superoxide anion production, and elevated serum eotaxin and RANTES, creating a protumorigenic environment. Moreover, PPARγ activation in MSEs delayed mammary tumor growth in part by down-regulating Cox-1, Cox-2 and cyclin D1. Collectively, these studies highlight a protective role of MSE-specific PPARγ during breast tumorigenesis, and support a novel chemotherapeutic role of PPARγ activation in breast cancer.

PPAR Medicines and Human Disease: The ABCs of It All.

ATP-dependent binding cassette (ABC) transporters are a family of transmembrane proteins that pump a variety of hydrophobic compounds across cellular and subcellular barriers and are implicated in human diseases such as cancer and atherosclerosis. Inhibition of ABC transporter activity showed promise in early preclinical studies; however, the outcomes in clinical trials with these agents have not been as encouraging. Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that regulate genes involved in fat and glucose metabolism, and inflammation. Activation of PPAR signaling is also reported to regulate ABC gene expression. This suggests the potential of PPAR medicines as a novel means of controlling ABC transporter activity at the transcriptional level. This paper summarizes the advances made in understanding how PPAR medicines affect ABC transporters, and the potential implications for impacting on human diseases, in particular with respect to cancer and atherosclerosis.

Stromal adipocyte PPARγ protects against breast tumorigenesis.

Peroxisome proliferator-activated receptor (PPAR)γ regulates the expression of genes essential for fat storage, primarily through its activity in adipocytes. It also has a role in carcinogenesis. PPARγ normally stops the in vivo progression of 7,12-dimethylbenz[a]anthracene (DMBA)-mediated breast tumours as revealed with PPARγ haploinsufficient mice. Since many cell types associated with the mammary gland express PPARγ, each with unique signal patterns, this study aimed to define which tissues are required for PPARγ-dependent antitumour effects. Accordingly, adipocyte-specific PPARγ knockout (PPARγ-A KO) mice and their wild-type (PPARγ-WT) controls were generated, and treated with DMBA for 6 weeks to initiate breast tumorigenesis. On week 7, mice were randomized to continue on normal chow diet or one supplemented with rosiglitazone (ROSI), and followed for 25 weeks for tumour outcomes. In PPARγ-A KO versus PPARγ-WT mice, malignant mammary tumour incidence was significantly higher and mammary tumour latency was decreased. DMBA + ROSI treatment reduced average mammary tumour volumes by 50%. Gene expression analyses of mammary glands by quantitative real-time polymerase chain reaction and immunofluorescence indicated that untreated PPARγ-A KOs had significantly decreased BRCA1 expression in mammary stromal adipocytes. Compared with PPARγ-WT mice, serum leptin levels in PPARγ-A KOs were also significantly higher throughout the study. Together, these data are the first to suggest that in vivo PPARγ expression in mammary stromal adipocytes attenuates breast tumorigenesis through BRCA1 upregulation and decreased leptin secretion. This study supports a protective effect of activating PPARγ as a novel chemopreventive therapy for breast cancer.