Rohan Bareja

Oncogenic BRAF disrupts thyroid morphogenesis and function via twist expression

Thyroid cancer is common, yet the sequence of alterations that promote tumor formation are incompletely understood. Here, we describe a novel model of thyroid carcinoma in zebrafish that reveals temporal changes due to BRAFV600E. Through the use of real-time in vivo imaging, we observe disruption in thyroid follicle structure that occurs early in thyroid development. Combinatorial treatment using BRAF and MEK inhibitors reversed the developmental effects induced by BRAFV600E. Adult zebrafish expressing BRAFV600E in thyrocytes developed invasive carcinoma. We identified a gene expression signature from zebrafish thyroid cancer that is predictive of disease-free survival in patients with papillary thyroid cancer. Gene expression studies nominated TWIST2 as a key effector downstream of BRAF. Using CRISPR/Cas9 to genetically inactivate a TWIST2 orthologue, we suppressed the effects of BRAFV600E and restored thyroid morphology and hormone synthesis. These data suggest that expression of TWIST2 plays a role in an early step of BRAFV600E-mediated transformation. DOI: http://dx.doi.org/10.7554/eLife.20728.001

Suppression of insulin feedback enhances the efficacy of PI3K inhibitors

Mutations in PIK3CA, which encodes the p110α subunit of the insulin-activated phosphatidylinositol-3 kinase (PI3K), and loss of function mutations in PTEN, which encodes a phosphatase that degrades the phosphoinositide lipids generated by PI3K, are among the most frequent events in human cancers1,2. However, pharmacological inhibition of PI3K has resulted in variable clinical responses, raising the possibility of an inherent mechanism of resistance to treatment. As p110α mediates virtually all cellular responses to insulin, targeted inhibition of this enzyme disrupts glucose metabolism in multiple tissues. For example, blocking insulin signalling promotes glycogen breakdown in the liver and prevents glucose uptake in the skeletal muscle and adipose tissue, resulting in transient hyperglycaemia within a few hours of PI3K inhibition. The effect is usually transient because compensatory insulin release from the pancreas (insulin feedback) restores normal glucose homeostasis3. However, the hyperglycaemia may be exacerbated or prolonged in patients with any degree of insulin resistance and, in these cases, necessitates discontinuation of therapy3–6. We hypothesized that insulin feedback induced by PI3K inhibitors may reactivate the PI3K–mTOR signalling axis in tumours, thereby compromising treatment effectiveness7,8. Here we show, in several model tumours in mice, that systemic glucose–insulin feedback caused by targeted inhibition of this pathway is sufficient to activate PI3K signalling, even in the presence of PI3K inhibitors. This insulin feedback can be prevented using dietary or pharmaceutical approaches, which greatly enhance the efficacy/toxicity ratios of PI3K inhibitors. These findings have direct clinical implications for the multiple p110α inhibitors that are in clinical trials and provide a way to increase treatment efficacy for patients with many types of tumour.Glucose–insulin feedback can reactivate PI3K in tumours treated with PI3K inhibitors, reducing therapeutic efficacy, but this effect can be reduced by using drugs or diet to suppress the insulin response.

Concerted regulation of retinal pigment epithelium basement membrane and barrier function by angiocrine factors

The outer blood-retina barrier is established through the coordinated terminal maturation of the retinal pigment epithelium (RPE), fenestrated choroid endothelial cells (ECs) and Bruchs membrane, a highly organized basement membrane that lies between both cell types. Here we study the contribution of choroid ECs to this process by comparing their gene expression profile before (P5) and after (P30) the critical postnatal period when mice acquire mature visual function. Transcriptome analyses show that expression of extracellular matrix-related genes changes dramatically over this period. Co-culture experiments support the existence of a novel regulatory pathway: ECs secrete factors that remodel RPE basement membrane, and integrin receptors sense these changes triggering Rho GTPase signals that modulate RPE tight junctions and enhance RPE barrier function. We anticipate our results will spawn a search for additional roles of choroid ECs in RPE physiology and disease.

Patient derived organoids to model rare prostate cancer phenotypes

A major hurdle in the study of rare tumors is a lack of existing preclinical models. Neuroendocrine prostate cancer is an uncommon and aggressive histologic variant of prostate cancer that may arise de novo or as a mechanism of treatment resistance in patients with pre-existing castration-resistant prostate cancer. There are few available models to study neuroendocrine prostate cancer. Here, we report the generation and characterization of tumor organoids derived from needle biopsies of metastatic lesions from four patients. We demonstrate genomic, transcriptomic, and epigenomic concordance between organoids and their corresponding patient tumors. We utilize these organoids to understand the biologic role of the epigenetic modifier EZH2 in driving molecular programs associated with neuroendocrine prostate cancer progression. High-throughput organoid drug screening nominated single agents and drug combinations suggesting repurposing opportunities. This proof of principle study represents a strategy for the study of rare cancer phenotypes.There are few available models to study neuroendocrine prostate cancer. Here they develop and characterize patient derived organoids from metastatic lesions, use these models to show the role of EZH2 in driving neuroendocrine phenotype, and perform high throughput organoid screening to identify therapeutic drug combinations.

Loss of an Androgen-Inactivating and Isoform-Specific HSD17B4 Splice Form Enables Emergence of Castration-Resistant Prostate Cancer

SUMMARY Castration-resistant prostate cancer (CRPC) requires tumors to engage metabolic mechanisms that allow sustained testosterone and/or dihydrotestosterone to stimulate progression. 17β-Hydroxyste-roid dehydrogenase type 4 (17βHSD4), encoded by HSD17B4, is thought to inactivate testosterone and dihydrotestosterone by converting them to their respective inert 17-keto steroids. Counterintuitively, HSD17B4 expression increases in CRPC and predicts poor prognosis. Here, we show that, of five alternative splice forms, only isoform 2 encodes an enzyme capable of testosterone and dihydrotestosterone inactivation. In contrast with other transcripts, functional expression of isoform 2 is specifically suppressed in development of CRPC in patients. Genetically silencing isoform 2 shifts the metabolic balance toward 17β-OH androgens (testosterone and dihydrotestosterone), stimulating androgen receptor (AR) and CRPC development. Our studies specifically implicate HSD17B4 isoform 2 loss in lethal prostate cancer.

Rapid identification of BCR/ABL1-like acute lymphoblastic leukaemia patients using a predictive statistical model based on quantitative real time-polymerase chain reaction: clinical, prognostic and therapeutic implications

BCR/ABL1‐like acute lymphoblastic leukaemia (ALL) is a subgroup of B‐lineage acute lymphoblastic leukaemia that occurs within cases without recurrent molecular rearrangements. Gene expression profiling (GEP) can identify these cases but it is expensive and not widely available. Using GEP, we identified 10 genes specifically overexpressed by BCR/ABL1‐like ALL cases and used their expression values – assessed by quantitative real time‐polymerase chain reaction (Q‐RT‐PCR) in 26 BCR/ABL1‐like and 26 non‐BCR/ABL1‐like cases to build a statistical “BCR/ABL1‐like predictor”, for the identification of BCR/ABL1‐like cases. By screening 142 B‐lineage ALL patients with the “BCR/ABL1‐like predictor”, we identified 28/142 BCR/ABL1‐like patients (19·7%). Overall, BCR/ABL1‐like cases were enriched in JAK/STAT mutations (P < 0·001), IKZF1 deletions (P < 0·001) and rearrangements involving cytokine receptors and tyrosine kinases (P = 0·001), thus corroborating the validity of the prediction. Clinically, the BCR/ABL1‐like cases identified by the BCR/ABL1‐like predictor achieved a lower rate of complete remission (P = 0·014) and a worse event‐free survival (P = 0·0009) compared to non‐BCR/ABL1‐like ALL. Consistently, primary cells from BCR/ABL1‐like cases responded in vitro to ponatinib. We propose a simple tool based on Q‐RT‐PCR and a statistical model that is capable of easily, quickly and reliably identifying BCR/ABL1‐like ALL cases at diagnosis.

Bone biopsy protocol for advanced prostate cancer in the era of precision medicine.

Metastatic biopsies are increasingly being performed in patients with advanced prostate cancer to search for actionable targets and/or to identify emerging resistance mechanisms. Due to a predominance of bone metastases and their sclerotic nature, obtaining sufficient tissue for clinical and genomic studies is challenging.

Publisher Correction: Suppression of insulin feedback enhances the efficacy of PI3K inhibitors

In this Letter, author Xing Du was incorrectly listed as Du Xing; this has been corrected online.

CD38 is methylated in prostate cancer and regulates extracellular NAD

BackgroundCancer cell metabolism requires sustained pools of intracellular nicotinamide adenine dinucleotide (NAD+) which is maintained by a balance of NAD+ hydrolase activity and NAD+ salvage activity. We recently reported that human prostate cancer can be initiated following oncogene expression in progenitor-like luminal cells marked by low expression of the NAD+-consuming enzyme CD38. CD38 expression is reduced in prostate cancer compared to benign prostate, suggesting that tumor cells may reduce CD38 expression in order to enhance pools of NAD+. However, little is known about how CD38 expression is repressed in advanced prostate cancer and whether CD38 plays a role in regulating NAD+ levels in prostate epithelial cells.MethodsCD38 expression, its association with recurrence after prostatectomy for clinically localized prostate cancer, and DNA methylation of the CD38 promoter were evaluated in human prostate tissues representing various stages of disease progression. CD38 was inducibly over-expressed in benign and malignant human prostate cell lines in order to determine the effects on cell proliferation and levels of NAD+ and NADH. NAD+ and NADH were also measured in urogenital tissues from wild-type and CD38 knockout mice.ResultsCD38 mRNA expression was reduced in metastatic castration-resistant prostate cancer compared to localized prostate cancer. In a large cohort of men undergoing radical prostatectomy, CD38 protein expression was inversely correlated with recurrence. We identified methylation of the CD38 promoter in primary and metastatic prostate cancer. Over-expression of wild-type CD38, but not an NAD+ hydrolase-deficient mutant, depleted extracellular NAD+ levels in benign and malignant prostate cell lines. However, expression of CD38 did not significantly alter intracellular NAD+ levels in human prostate cell lines grown in vitro and in urogenital tissues isolated from wild-type and CD38 knockout mice.ConclusionsCD38 protein expression in prostate cancer is associated with risk of recurrence. Methylation results suggest that CD38 is epigenetically regulated in localized and metastatic prostate cancer tissues. Our study provides support for CD38 as a regulator of extracellular, but not intracellular, NAD+ in epithelial cells. These findings suggest that repression of CD38 by methylation may serve to increase the availability of extracellular NAD+ in prostate cancer tissues.

Abstract IA19: Phenotype plasticity—a novel mechanism of targeted therapy resistance

Prostate cancer (PCa) is the most commonly diagnosed cancer and the third highest cause of cancer-related death in men in Europe, where it is responsible for over 90,000 deaths a year. The mainstay of treatment for metastatic PCa is androgen-deprivation therapy (ADT). Although initially effective, the treatment ultimately fails and progression to castration-resistant prostate cancer (CRPC) occurs. Given that CRPC is still driven by hormonal signaling through aberrant activation of the androgen receptor (AR), improved, more potent AR-targeting therapies have been developed for CRPC patients. However, resistance to these therapies ultimately occurs as well. One form of resistance identified by my group results in a phenotypic switch leading to androgen receptor indifference and progression to neuroendocrine prostate cancer (NEPC), which shows a distinct histomorphology and expresses neural-like markers. Unlike more commonly recognized mechanisms of ADT resistance due to AR mutations or amplification, NEPC no longer responds to AR-targeting therapy and has a mean survival of 7 months. There is mounting evidence supporting the role of epigenetic events as a mechanism for transdifferentiation of PCa to an androgen signaling-indifferent state under specific genomic conditions, involving but not limited to TP53, RB1, and PTEN loss. However, the epigenetic regulators at work and the specific changes in the epigenetic landscape are unknown. The SWI/SNF complex is a major epigenetic player, both in regulating normal cell differentiation and in cancer biology. This presentation will focus on novel data supporting the role of alterations in this complex that could contribute to PCa phenotype plasticity. Citation Format: Joanna Cyrta, David Wilkes, Sung Suk Chae, Matteo Benelli, Rohan Bareja, Davide Prandi, Paola Maria Giovanna Cavaliere, Himisha Beltran, Andrea Sboner, Francesca Demichelis, Mark A. Rubin. Phenotype plasticity—a novel mechanism of targeted therapy resistance [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr IA19.