l-2-Hydroxyglutarate remodeling of the epigenome and epitranscriptome creates a metabolic vulnerability in kidney cancer models.

Tumor cells are known to undergo considerable metabolic reprogramming to meet their unique demands and drive tumor growth. At the same time, this reprogramming may come at a cost with resultant metabolic vulnerabilities. The small molecule l-2-hydroxyglutarate (l-2HG) is elevated in the most common histology of renal cancer. Similarly to other oncometabolites, l-2HG has the potential to profoundly impact gene expression. Here, we demonstrate that l-2HG remodels amino acid metabolism in renal cancer cells through combined effects on histone methylation and RNA N6-methyladenosine. The combined effects of l-2HG result in a metabolic liability that renders tumors cells reliant on exogenous serine to support proliferation, redox homeostasis, and tumor growth. In concert with these data, high-l-2HG kidney cancers demonstrate reduced expression of multiple serine biosynthetic enzymes. Collectively, our data indicate that high-l-2HG renal tumors could be specifically targeted by strategies that limit serine availability to tumors.

Annual mpMRI surveillance: PI-RADS upgrading and increasing trend correlated with patients who harbor clinically significant disease.

INTRODUCTION: Prostate cancer screening has routinely identified men with very low- or low-risk disease, per the National Comprehensive Cancer Network guidelines. Current literature has demonstrated that the most appropriate management strategy for these patients is active surveillance (AS). The mainstay of AS includes periodic biopsies and biannual prostate-specific antigen tests. However, multiparametric magnetic resonance imaging (mpMRI) is uniquely posed to improve patient surveillance. This study aimed to evaluate the utility of an annual mpMRI in patients on AS, focusing on radiologic upgrading and Prostate Imaging-Reporting and Data System (PI-RADS) trends as indicators of clinically significant disease. METHODS: This prospective, single intuition, study enrolled 208 patients on AS who had at least two biopsies and 1 mpMRI with a median follow-up of 5.03 years. The main outcome variable was time to Gleason grade (GG) reclassification. RESULTS: After delineating patients on their initial PI-RADS score, men with score 3 and 5 lesions at first MRI had comparable GG reclassification-free survival to their counterparts. Conversely, men with initial PI-RADS 4 lesions showed a lower 5-year GG reclassification-free survival compared to those with PI-RADS score 1-2. The cohort was then subset to 70 patients who obtained ≥2 mpMRIs on protocol. Men experiencing uptrending mpMRI scores had an increased risk of GG reclassification, with a 35.4% difference in 5 year GG reclassification-free survival probability on the Kaplan-Meier curve analysis. CONCLUSION: In conclusion, this study demonstrates that for men on AS with stable recapitulated disease, an annual MRI may replace repeat biopsies after confirmatory sampling has been obtained. On the other hand, men who initiate AS with PI-RADS 4 and/or who display uptrending mpMRI scores require periodic biopsies along with repeat imaging. This study highlights the utility of integrating an annual MRI into AS protocols, thus promising a more effective approach to management.

Integration of magnetic resonance imaging into prostate cancer nomograms.

The decision whether to undergo prostate biopsy must be carefully weighed. Nomograms have widely been utilized as risk calculators to improve the identification of prostate cancer by weighing several clinical factors. The recent inclusion of multiparametric magnetic resonance imaging (mpMRI) findings into nomograms has drastically improved their nomogram's accuracy at identifying clinically significant prostate cancer. Several novel nomograms have incorporated mpMRI to aid in the decision-making process in proceeding with a prostate biopsy in patients who are biopsy-naïve, have a prior negative biopsy, or are on active surveillance. Furthermore, novel nomograms have incorporated mpMRI to aid in treatment planning of definitive therapy. This literature review highlights how the inclusion of mpMRI into prostate cancer nomograms has improved upon their performance, potentially reduce unnecessary procedures, and enhance the individual risk assessment by improving confidence in clinical decision-making by both patients and their care providers.

The TGF-ß/HDAC7 axis suppresses TCA cycle metabolism in renal cancer.

Mounting evidence points to alterations in mitochondrial metabolism in renal cell carcinoma (RCC). However, the mechanisms that regulate the TCA cycle in RCC remain uncharacterized. Here, we demonstrate that loss of TCA cycle enzyme expression is retained in RCC metastatic tissues. Moreover, proteomic analysis demonstrates that reduced TCA cycle enzyme expression is far more pronounced in RCC relative to other tumor types. Loss of TCA cycle enzyme expression is correlated with reduced expression of the transcription factor PGC-1α, which is also lost in RCC tissues. PGC-1α reexpression in RCC cells restores the expression of TCA cycle enzymes in vitro and in vivo and leads to enhanced glucose carbon incorporation into TCA cycle intermediates. Mechanistically, TGF-ß signaling, in concert with histone deacetylase 7 (HDAC7), suppresses TCA cycle enzyme expression. Our studies show that pharmacologic inhibition of TGF-ß restores the expression of TCA cycle enzymes and suppresses tumor growth in an orthotopic model of RCC. Taken together, this investigation reveals a potentially novel role for the TGF-ß/HDAC7 axis in global suppression of TCA cycle enzymes in RCC and provides insight into the molecular basis of altered mitochondrial metabolism in this malignancy.

The oncometabolite L-2-hydroxyglutarate is a common product of dipteran larval development.

The oncometabolite L-2-hydroxyglutarate (L-2HG) is considered an abnormal product of central carbon metabolism that is capable of disrupting chromatin architecture, mitochondrial metabolism, and cellular differentiation. Under most circumstances, mammalian tissues readily dispose of this compound, as aberrant L-2HG accumulation induces neurometabolic disorders and promotes renal cell carcinomas. Intriguingly, Drosophila melanogaster larvae were recently found to accumulate high L-2HG levels under normal growth conditions, raising the possibility that L-2HG plays a unique role in insect metabolism. Here we explore this hypothesis by analyzing L-2HG levels in 18 insect species. While L-2HG was present at low-to-moderate levels in most of these species (<100 pmol/mg; comparable to mouse liver), dipteran larvae exhibited a tendency to accumulate high L-2HG concentrations (>100 pmol/mg), with the mosquito Aedes aegypti, the blow fly Phormia regina, and three representative Drosophila species harboring concentrations that exceed 1 nmol/mg - levels comparable to those measured in mutant mice that are unable to degrade L-2HG. Overall, our findings suggest that one of the largest groups of animals on earth commonly generate high concentrations of an oncometabolite during juvenile growth, hint at a role for L-2HG in the evolution of dipteran development, and raise the possibility that L-2HG metabolism could be targeted to restrict the growth of key disease vectors and agricultural pests.

Teleological role of L-2-hydroxyglutarate dehydrogenase in the kidney.

L-2-hydroxyglutarate (L-2HG) is an oncometabolite found elevated in renal tumors. However, this molecule might have physiological roles that extend beyond its association with cancer, as L-2HG levels are elevated in response to hypoxia and during Drosophila larval development. L-2HG is known to be metabolized by L-2HG dehydrogenase (L2HGDH), and loss of L2HGDH leads to elevated L-2HG levels. Despite L2HGDH being highly expressed in the kidney, its role in renal metabolism has not been explored. Here, we report our findings utilizing a novel CRISPR/Cas9 murine knockout model, with a specific focus on the role of L2HGDH in the kidney. Histologically, L2hgdh knockout kidneys have no demonstrable histologic abnormalities. However, GC-MS metabolomics demonstrates significantly reduced levels of the TCA cycle intermediate succinate in multiple tissues. Isotope labeling studies with [U-13C] glucose demonstrate that restoration of L2HGDH in renal cancer cells (which lowers L-2HG) leads to enhanced incorporation of label into TCA cycle intermediates. Subsequent biochemical studies demonstrate that L-2HG can inhibit the TCA cycle enzyme α-ketoglutarate dehydrogenase. Bioinformatic analysis of mRNA expression data from renal tumors demonstrates that L2HGDH is co-expressed with genes encoding TCA cycle enzymes as well as the gene encoding the transcription factor PGC-1α, which is known to regulate mitochondrial metabolism. Restoration of PGC-1α in renal tumor cells results in increased L2HGDH expression with a concomitant reduction in L-2HG levels. Collectively, our analyses provide new insight into the physiological role of L2HGDH as well as mechanisms that promote L-2HG accumulation in disease states.

PRDM16 suppresses HIF-targeted gene expression in kidney cancer.

Analysis of transcriptomic data demonstrates extensive epigenetic gene silencing of the transcription factor PRDM16 in renal cancer. We show that restoration of PRDM16 in RCC cells suppresses in vivo tumor growth. RNaseq analysis reveals that PRDM16 imparts a predominantly repressive effect on the RCC transcriptome including suppression of the gene encoding semaphorin 5B (SEMA5B). SEMA5B is a HIF target gene highly expressed in RCC that promotes in vivo tumor growth. Functional studies demonstrate that PRDM16's repressive properties, mediated by physical interaction with the transcriptional corepressors C-terminal binding proteins (CtBP1/2), are required for suppression of both SEMA5B expression and in vivo tumor growth. Finally, we show that reconstitution of RCC cells with a PRDM16 mutant unable to bind CtBPs nullifies PRDM16's effects on both SEMA5B repression and tumor growth suppression. Collectively, our data uncover a novel epigenetic basis by which HIF target gene expression is amplified in kidney cancer and a new mechanism by which PRDM16 exerts its tumor suppressive effects.

PGC1α suppresses kidney cancer progression by inhibiting collagen-induced SNAIL expression.

The transcriptional events that promote invasive and metastatic phenotypes in renal cell carcinoma (RCC) remain poorly understood. Here we report that the decreased expression of peroxisome proliferator-activated receptor gamma, coactivator 1 alpha (PGC1α) and the increased expression of several genes encoding collagen family members are associated with RCC tumor progression. PGC1α restoration attenuates invasive phenotypes and suppresses tumor progression in vivo. In contrast, collagens produced by RCC cells promote invasive and migratory phenotypes. PGC1α restoration suppresses the expression of collagens and tumor phenotypes via the induction of miR-29a. Furthermore, decreased collagens via the PGC1α/miR-29a axis suppresses collagen-mediated activation of discoidin domain receptor 1 (DDR1)/ERK signaling. In turn, the suppression of collagen/DDR1 signaling by PGC1α leads to decreased levels of the known EMT regulators SNAIL1 and 2. Collectively, our results demonstrate a novel role for PGC1α in the regulation of proinvasive SNAIL proteins.

14-3-3 proteins protect AMPK-phosphorylated ten-eleven translocation-2 (TET2) from PP2A-mediated dephosphorylation.

Ten-eleven translocation-2 (TET2) is a member of the methylcytosine dioxygenase family of enzymes and has been implicated in cancer and aging because of its role as a global epigenetic modifier. TET2 has a large N-terminal domain and a catalytic C-terminal region. Previous reports have demonstrated that the TET2 catalytic domain remains active independently of the N-terminal domain. As such, the function of the N terminus of this large protein remains poorly characterized. Here, using yeast two-hybrid screening, co-immunoprecipitation, and several biochemical assays, we found that several isoforms of the 14-3-3 family of proteins bind TET2. 14-3-3 proteins bound TET2 when it was phosphorylated at Ser-99. In particular, we observed that AMP-activated protein kinase-mediated phosphorylation at Ser-99 promotes TET2 stability and increases global DNA 5-hydroxymethylcytosine levels. The interaction of 14-3-3 proteins with TET2 protected the Ser-99 phosphorylation, and disruption of this interaction both reduced TET2 phosphorylation and decreased TET2 stability. Furthermore, we noted that protein phosphatase 2A can interact with TET2 and dephosphorylate Ser-99. Collectively, these results provide detailed insights into the role of the TET2 N-terminal domain in TET2 regulation. Moreover, they reveal the dynamic nature of TET2 protein regulation that could have therapeutic implications for disease states resulting from reduced TET2 levels or activity.

Biochemical and Epigenetic Insights into L-2-Hydroxyglutarate, a Potential Therapeutic Target in Renal Cancer.

PURPOSE: Elevation of L-2-hydroxylgutarate (L-2-HG) in renal cell carcinoma (RCC) is due in part to reduced expression of L-2-HG dehydrogenase (L2HGDH). However, the contribution of L-2-HG to renal carcinogenesis and insight into the biochemistry and targets of this small molecule remains to be elucidated. EXPERIMENTAL DESIGN: Genetic and pharmacologic approaches to modulate L-2-HG levels were assessed for effects on in vitro and in vivo phenotypes. Metabolomics was used to dissect the biochemical mechanisms that promote L-2-HG accumulation in RCC cells. Transcriptomic analysis was utilized to identify relevant targets of L-2-HG. Finally, bioinformatic and metabolomic analyses were used to assess the L-2-HG/L2HGDH axis as a function of patient outcome and cancer progression. RESULTS: L2HGDH suppresses both in vitro cell migration and in vivo tumor growth and these effects are mediated by L2HGDH's catalytic activity. Biochemical studies indicate that glutamine is the predominant carbon source for L-2-HG via the activity of malate dehydrogenase 2 (MDH2). Inhibition of the glutamine-MDH2 axis suppresses in vitro phenotypes in an L-2-HG-dependent manner. Moreover, in vivo growth of RCC cells with basal elevation of L-2-HG is suppressed by glutaminase inhibition. Transcriptomic and functional analyses demonstrate that the histone demethylase KDM6A is a target of L-2-HG in RCC. Finally, increased L-2-HG levels, L2HGDH copy loss, and lower L2HGDH expression are associated with tumor progression and/or worsened prognosis in patients with RCC. CONCLUSIONS: Collectively, our studies provide biochemical and mechanistic insight into the biology of this small molecule and provide new opportunities for treating L-2-HG-driven kidney cancers.

Chemotherapy induces secretion of exosomes loaded with heparanase that degrades extracellular matrix and impacts tumor and host cell behavior.

The heparan sulfate-degrading enzyme heparanase promotes the progression of many cancers by driving tumor cell proliferation, metastasis and angiogenesis. Heparanase accomplishes this via multiple mechanisms including its recently described effect on enhancing biogenesis of tumor exosomes. Because we recently discovered that heparanase expression is upregulated in myeloma cells that survive chemotherapy, we were prompted to investigate the impact of anti-myeloma drugs on exosome biogenesis. When myeloma cells were exposed to the commonly utilized anti-myeloma drugs bortezomib, carfilzomib or melphalan, exosome secretion by the cells was dramatically enhanced. These chemotherapy-induced exosomes (chemoexosomes) have a proteome profile distinct from cells not exposed to drug including a dramatic elevation in the level of heparanase present as exosome cargo. The chemoexosome heparanase was not found inside the chemoexosome, but was present on the exosome surface where it was capable of degrading heparan sulfate embedded within an extracellular matrix. When exposed to myeloma cells, chemoexosomes transferred their heparanase cargo to those cells, enhancing their heparan sulfate degrading activity and leading to activation of ERK signaling and an increase in shedding of the syndecan-1 proteoglycan. Exposure of chemoexosomes to macrophages enhanced their secretion of TNF-α, an important myeloma growth factor. Moreover, chemoexosomes stimulated macrophage migration and this effect was blocked by H1023, a monoclonal antibody that inhibits heparanase enzymatic activity. These data suggest that anti-myeloma therapy ignites a burst of exosomes having a high level of heparanase that remodels extracellular matrix and alters tumor and host cell behaviors that likely contribute to chemoresistance and eventual patient relapse. SUMMARY: We find that anti-myeloma chemotherapy dramatically stimulates secretion of exosomes and alters exosome composition. Exosomes secreted during therapy contain high levels of heparanase on their surface that can degrade ECM and also can be transferred to both tumor and host cells, altering their behavior in ways that may enhance tumor survival and progression.

A novel culture system for adult porcine intestinal crypts.

Porcine models are useful for investigating therapeutic approaches to short bowel syndrome and potentially to intestinal stem cell (ISC) transplantation. Whereas techniques for the culture and genetic manipulation of ISCs from mice and humans are well established, similar methods for porcine stem cells have not been reported. Jejunal crypts were isolated from murine, human, and juvenile and adult porcine small intestine, suspended in Matrigel, and co-cultured with syngeneic intestinal subepithelial myofibroblasts (ISEMFs) or cultured without feeder cells in various culture media. Media containing epidermal growth factor, noggin, and R-spondin 1 (ENR medium) were supplemented with various combinations of Wnt3a- or ISEMF-conditioned medium (CM) and with glycogen synthase kinase 3 inhibitor (GSK3i), and their effects were studied on cultured crypts. Cell lineage differentiation was assessed by immunohistochemistry and quantitative polymerase chain reaction. Cultured porcine cells were serially passaged and transduced with a lentiviral vector. Whereas ENR medium supported murine enteroid growth, it did not sustain porcine crypts beyond 5 days. Supplementation of Wnt3a-CM and GSK3i resulted in the formation of complex porcine enteroids with budding extensions. These enteroids contained a mixture of stem and differentiated cells and were successfully passaged in the presence of GSK3i. Crypts grown in media supplemented with porcine ISEMF-CM formed spheroids that were less well differentiated than enteroids. Enteroids and spheroids were transfected with a lentivirus with high efficiency. Thus, our method maintains juvenile and adult porcine crypt cells long-term in culture. Porcine enteroids and spheroids can be successfully passaged and transduced by using lentiviral vectors.

Type I collagen as an extracellular matrix for the in vitro growth of human small intestinal epithelium.

BACKGROUND: We previously reported in vitro maintenance and proliferation of human small intestinal epithelium using Matrigel, a proprietary basement membrane product. There are concerns over the applicability of Matrigel-based methods for future human therapies. We investigated type I collagen as an alternative for the culture of human intestinal epithelial cells. METHODS: Human small intestine was procured from fresh surgical pathology specimens. Small intestinal crypts were isolated using EDTA chelation. Intestinal subepithelial myofibroblasts were isolated from a pediatric sample and expanded in vitro. After suspension in Matrigel or type I collagen gel, crypts were co-cultured above a confluent layer of myofibroblasts. Crypts were also grown in monoculture with exposure to myofibroblast conditioned media; these were subsequently sub-cultured in vitro and expanded with a 1∶2 split ratio. Cultures were assessed with light microscopy, RT-PCR, histology, and immunohistochemistry. RESULTS: Collagen supported viable human epithelium in vitro for at least one month in primary culture. Sub-cultured epithelium expanded through 12 passages over 60 days. Histologic sections revealed polarized columnar cells, with apical brush borders and basolaterally located nuclei. Collagen-based cultures gave rise to monolayer epithelial sheets at the gel-liquid interface, which were not observed with Matrigel. Immunohistochemical staining identified markers of differentiated intestinal epithelium and myofibroblasts. RT-PCR demonstrated expression of α-smooth muscle actin and vimentin in myofibroblasts and E-Cadherin, CDX2, villin 1, intestinal alkaline phosphatase, chromogranin A, lysozyme, and Lgr5 in epithelial cells. These markers were maintained through several passages. CONCLUSION: Type I collagen gel supports long-term in vitro maintenance and expansion of fully elaborated human intestinal epithelium. Collagen-based methods yield familiar enteroid structures as well as a new pattern of sheet-like growth, and they eliminate the need for Matrigel for in vitro human intestinal epithelial growth. Future research is required to further develop this cell culture system for tissue engineering applications.

Intestinal subepithelial myofibroblasts support the growth of intestinal epithelial stem cells.

Intestinal epithelial stem cells (ISCs) are the focus of recent intense study. Current in vitro models rely on supplementation with the Wnt agonist R-spondin1 to support robust growth, ISC self-renewal, and differentiation. Intestinal subepithelial myofibroblasts (ISEMFs) are important supportive cells within the ISC niche. We hypothesized that co-culture with ISEMF enhances the growth of ISCs in vitro and allows for their successful in vivo implantation and engraftment. ISC-containing small intestinal crypts, FACS-sorted single ISCs, and ISEMFs were procured from C57BL/6 mice. Crypts and single ISCs were grown in vitro into enteroids, in the presence or absence of ISEMFs. ISEMFs enhanced the growth of intestinal epithelium in vitro in a proximity-dependent fashion, with co-cultures giving rise to larger enteroids than monocultures. Co-culture of ISCs with supportive ISEMFs relinquished the requirement of exogenous R-spondin1 to sustain long-term growth and differentiation of ISCs. Mono- and co-cultures were implanted subcutaneously in syngeneic mice. Co-culture with ISEMFs proved necessary for successful in vivo engraftment and proliferation of enteroids; implants without ISEMFs did not survive. ISEMF whole transcriptome sequencing and qPCR demonstrated high expression of specific R-spondins, well-described Wnt agonists that supports ISC growth. Specific non-supportive ISEMF populations had reduced expression of R-spondins. The addition of ISEMFs in intestinal epithelial culture therefore recapitulates a critical element of the intestinal stem cell niche and allows for its experimental interrogation and biodesign-driven manipulation.

Use of collagen gel as an alternative extracellular matrix for the in vitro and in vivo growth of murine small intestinal epithelium.

Methods for the in vitro culture of primary small intestinal epithelium have improved greatly in recent years. A critical barrier for the translation of this methodology to the patient's bedside is the ability to grow intestinal stem cells using a well-defined extracellular matrix. Current methods rely on the use of Matrigel(™), a proprietary basement membrane-enriched extracellular matrix gel produced in mice that is not approved for clinical use. We demonstrate for the first time the capacity to support the long-term in vitro growth of murine intestinal epithelium in monoculture, using type I collagen. We further demonstrate successful in vivo engraftment of enteroids co-cultured with intestinal subepithelial myofibroblasts in collagen gel. Small intestinal crypts were isolated from 6 to 10 week old transgenic enhanced green fluorescent protein (eGFP+) mice and suspended within either Matrigel or collagen gel; cultures were supported using previously reported media and growth factors. After 1 week, cultures were either lysed for DNA or RNA extraction or were implanted subcutaneously in syngeneic host mice. Quantitative real-time polymerase chain reaction (qPCR) was performed to determine expansion of the transgenic eGFP-DNA and to determine the mRNA gene expression profile. Immunohistochemistry was performed on in vitro cultures and recovered in vivo explants. Small intestinal crypts reliably expanded to form enteroids in either Matrigel or collagen in both mono- and co-cultures as confirmed by microscopy and eGFP-DNA qPCR quantification. Collagen-based cultures yielded a distinct morphology with smooth enteroids and epithelial monolayer growth at the gel surface; both enteroid and monolayer cells demonstrated reactivity to Cdx2, E-cadherin, CD10, Periodic Acid-Schiff, and lysozyme. Collagen-based enteroids were successfully subcultured in vitro, whereas pure monolayer epithelial sheets did not survive passaging. Reverse transcriptase-polymerase chain reaction demonstrated evidence of Cdx2, villin 1, mucin 2, chromogranin A, lysozyme 1, and Lgr5 expression, suggesting a fully elaborated intestinal epithelium. Additionally, collagen-based enteroids co-cultured with myofibroblasts were successfully recovered after 5 weeks of in vivo implantation, with a preserved immunophenotype. These results indicate that collagen-based techniques have the capacity to eliminate the need for Matrigel in intestinal stem cell culture. This is a critical step towards producing neo-mucosa using good manufacturing practices for clinical applications in the future.