FABP5 Inhibition against PTEN-Mutant Therapy Resistant Prostate Cancer.

Resistance to standard of care taxane and androgen deprivation therapy (ADT) causes the vast majority of prostate cancer (PC) deaths worldwide. We have developed RapidCaP, an autochthonous genetically engineered mouse model of PC. It is driven by the loss of PTEN and p53, the most common driver events in PC patients with life-threatening diseases. As in human ADT, surgical castration of RapidCaP animals invariably results in disease relapse and death from the metastatic disease burden. Fatty Acid Binding Proteins (FABPs) are a large family of signaling lipid carriers. They have been suggested as drivers of multiple cancer types. Here we combine analysis of primary cancer cells from RapidCaP (RCaP cells) with large-scale patient datasets to show that among the 10 FABP paralogs, FABP5 is the PC-relevant target. Next, we show that RCaP cells are uniquely insensitive to both ADT and taxane treatment compared to a panel of human PC cell lines. Yet, they share an exquisite sensitivity to the small-molecule FABP5 inhibitor SBFI-103. We show that SBFI-103 is well tolerated and can strongly eliminate RCaP tumor cells in vivo. This provides a pre-clinical platform to fight incurable PC and suggests an important role for FABP5 in PTEN-deficient PC.

SOAT1 promotes mevalonate pathway dependency in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis, and new therapies are needed. Altered metabolism is a cancer vulnerability, and several metabolic pathways have been shown to promote PDAC. However, the changes in cholesterol metabolism and their role during PDAC progression remain largely unknown. Here we used organoid and mouse models to determine the drivers of altered cholesterol metabolism in PDAC and the consequences of its disruption on tumor progression. We identified sterol O-acyltransferase 1 (SOAT1) as a key player in sustaining the mevalonate pathway by converting cholesterol to inert cholesterol esters, thereby preventing the negative feedback elicited by unesterified cholesterol. Genetic targeting of Soat1 impairs cell proliferation in vitro and tumor progression in vivo and reveals a mevalonate pathway dependency in p53 mutant PDAC cells that have undergone p53 loss of heterozygosity (LOH). In contrast, pancreatic organoids lacking p53 mutation and p53 LOH are insensitive to SOAT1 loss, indicating a potential therapeutic window for inhibiting SOAT1 in PDAC.

Novel insights into breast cancer copy number genetic heterogeneity revealed by single-cell genome sequencing.

Copy number alterations (CNAs) play an important role in molding the genomes of breast cancers and have been shown to be clinically useful for prognostic and therapeutic purposes. However, our knowledge of intra-tumoral genetic heterogeneity of this important class of somatic alterations is limited. Here, using single-cell sequencing, we comprehensively map out the facets of copy number alteration heterogeneity in a cohort of breast cancer tumors. Ou/var/www/html/elife/12-05-2020/backup/r analyses reveal: genetic heterogeneity of non-tumor cells (i.e. stroma) within the tumor mass; the extent to which copy number heterogeneity impacts breast cancer genomes and the importance of both the genomic location and dosage of sub-clonal events; the pervasive nature of genetic heterogeneity of chromosomal amplifications; and the association of copy number heterogeneity with clinical and biological parameters such as polyploidy and estrogen receptor negative status. Our data highlight the power of single-cell genomics in dissecting, in its many forms, intra-tumoral genetic heterogeneity of CNAs, the magnitude with which CNA heterogeneity affects the genomes of breast cancers, and the potential importance of CNA heterogeneity in phenomena such as therapeutic resistance and disease relapse.

Cells in the body remain healthy by tightly preventing and repairing random changes, or mutations, in their genetic material. In cancer cells, however, these mechanisms can break down. When these cells grow and multiply, they can then go on to accumulate many mutations. As a result, cancer cells in the same tumor can each contain a unique combination of genetic changes. This genetic heterogeneity has the potential to affect how cancer responds to treatment, and is increasingly becoming appreciated clinically. For example, if a drug only works against cancer cells carrying a specific mutation, any cells lacking this genetic change will keep growing and cause a relapse. However, it is still difficult to quantify and understand genetic heterogeneity in cancer. Copy number alterations (or CNAs) are a class of mutation where large and small sections of genetic material are gained or lost. This can result in cells that have an abnormal number of copies of the genes in these sections. Here, Baslan et al. set out to explore how CNAs might vary between individual cancer cells within the same tumor. To do so, thousands of individual cancer cells were isolated from human breast tumors, and a technique called single-cell genome sequencing used to screen the genetic information of each of them. These experiments confirmed that CNAs did differ ­ sometimes dramatically ­ between patients and among cells taken from the same tumor. For example, many of the cells carried extra copies of well-known cancer genes important for treatment, but the exact number of copies varied between cells. This heterogeneity existed for individual genes as well as larger stretches of DNA: this was the case, for instance, for an entire section of chromosome 8, a region often affected in breast and other tumors. The work by Baslan et al. captures the sheer extent of genetic heterogeneity in cancer and in doing so, highlights the power of single-cell genome sequencing. In the future, a finer understanding of the genetic changes present at the level of an individual cancer cell may help clinicians to manage the disease more effectively.

Utility of Single-Cell Genomics in Diagnostic Evaluation of Prostate Cancer.

A distinction between indolent and aggressive disease is a major challenge in diagnostics of prostate cancer. As genetic heterogeneity and complexity may influence clinical outcome, we have initiated studies on single tumor cell genomics. In this study, we demonstrate that sparse DNA sequencing of single-cell nuclei from prostate core biopsies is a rich source of quantitative parameters for evaluating neoplastic growth and aggressiveness. These include the presence of clonal populations, the phylogenetic structure of those populations, the degree of the complexity of copy-number changes in those populations, and measures of the proportion of cells with clonal copy-number signatures. The parameters all showed good correlation to the measure of prostatic malignancy, the Gleason score, derived from individual prostate biopsy tissue cores. Remarkably, a more accurate histopathologic measure of malignancy, the surgical Gleason score, agrees better with these genomic parameters of diagnostic biopsy than it does with the diagnostic Gleason score and related measures of diagnostic histopathology. This is highly relevant because primary treatment decisions are dependent upon the biopsy and not the surgical specimen. Thus, single-cell analysis has the potential to augment traditional core histopathology, improving both the objectivity and accuracy of risk assessment and inform treatment decisions.Significance: Genomic analysis of multiple individual cells harvested from prostate biopsies provides an indepth view of cell populations comprising a prostate neoplasm, yielding novel genomic measures with the potential to improve the accuracy of diagnosis and prognosis in prostate cancer. Cancer Res; 78(2); 348-58. ©2017 AACR.

Whole-genome single-cell copy number profiling from formalin-fixed paraffin-embedded samples.

A substantial proportion of tumors consist of genotypically distinct subpopulations of cancer cells. This intratumor genetic heterogeneity poses a substantial challenge for the implementation of precision medicine. Single-cell genomics constitutes a powerful approach to resolve complex mixtures of cancer cells by tracing cell lineages and discovering cryptic genetic variations that would otherwise be obscured in tumor bulk analyses. Because of the chemical alterations that result from formalin fixation, single-cell genomic approaches have largely remained limited to fresh or rapidly frozen specimens. Here we describe the development and validation of a robust and accurate methodology to perform whole-genome copy-number profiling of single nuclei obtained from formalin-fixed paraffin-embedded clinical tumor samples. We applied the single-cell sequencing approach described here to study the progression from in situ to invasive breast cancer, which revealed that ductal carcinomas in situ show intratumor genetic heterogeneity at diagnosis and that these lesions may progress to invasive breast cancer through a variety of evolutionary processes.

TGF-ß reduces DNA ds-break repair mechanisms to heighten genetic diversity and adaptability of CD44+/CD24- cancer cells.

Many lines of evidence have indicated that both genetic and non-genetic determinants can contribute to intra-tumor heterogeneity and influence cancer outcomes. Among the best described sub-population of cancer cells generated by non-genetic mechanisms are cells characterized by a CD44+/CD24- cell surface marker profile. Here, we report that human CD44+/CD24- cancer cells are genetically highly unstable because of intrinsic defects in their DNA-repair capabilities. In fact, in CD44+/CD24- cells, constitutive activation of the TGF-beta axis was both necessary and sufficient to reduce the expression of genes that are crucial in coordinating DNA damage repair mechanisms. Consequently, we observed that cancer cells that reside in a CD44+/CD24- state are characterized by increased accumulation of DNA copy number alterations, greater genetic diversity and improved adaptability to drug treatment. Together, these data suggest that the transition into a CD44+/CD24- cell state can promote intra-tumor genetic heterogeneity, spur tumor evolution and increase tumor fitness.

Optimizing sparse sequencing of single cells for highly multiplex copy number profiling.

Genome-wide analysis at the level of single cells has recently emerged as a powerful tool to dissect genome heterogeneity in cancer, neurobiology, and development. To be truly transformative, single-cell approaches must affordably accommodate large numbers of single cells. This is feasible in the case of copy number variation (CNV), because CNV determination requires only sparse sequence coverage. We have used a combination of bioinformatic and molecular approaches to optimize single-cell DNA amplification and library preparation for highly multiplexed sequencing, yielding a method that can produce genome-wide CNV profiles of up to a hundred individual cells on a single lane of an Illumina HiSeq instrument. We apply the method to human cancer cell lines and biopsied cancer tissue, thereby illustrating its efficiency, reproducibility, and power to reveal underlying genetic heterogeneity and clonal phylogeny. The capacity of the method to facilitate the rapid profiling of hundreds to thousands of single-cell genomes represents a key step in making single-cell profiling an easily accessible tool for studying cell lineage.

Genome-wide copy number analysis of single cells.

Copy number variation (CNV) is increasingly recognized as an important contributor to phenotypic variation in health and disease. Most methods for determining CNV rely on admixtures of cells in which information regarding genetic heterogeneity is lost. Here we present a protocol that allows for the genome-wide copy number analysis of single nuclei isolated from mixed populations of cells. Single-nucleus sequencing (SNS), combines flow sorting of single nuclei on the basis of DNA content and whole-genome amplification (WGA); this is followed by next-generation sequencing to quantize genomic intervals in a genome-wide manner. Multiplexing of single cells is discussed. In addition, we outline informatic approaches that correct for biases inherent in the WGA procedure and allow for accurate determination of copy number profiles. All together, the protocol takes ∼3 d from flow cytometry to sequence-ready DNA libraries.

Fragile x mental retardation 1 and filamin a interact genetically in Drosophila long-term memory.

The last decade has witnessed the identification of single-gene defects associated with an impressive number of mental retardation syndromes. Fragile X syndrome, the most common cause of mental retardation for instance, results from disruption of the FMR1 gene. Similarly, Periventricular Nodular Heterotopia, which includes cerebral malformation, epilepsy and cognitive disabilities, derives from disruption of the Filamin A gene. While it remains unclear whether defects in common molecular pathways may underlie the cognitive dysfunction of these various syndromes, defects in cytoskeletal structure nonetheless appear to be common to several mental retardation syndromes. FMR1 is known to interact with Rac, profilin, PAK and Ras, which are associated with dendritic spine defects. In Drosophila, disruptions of the dFmr1 gene impair long-term memory (LTM), and the Filamin A homolog (cheerio) was identified in a behavioral screen for LTM mutants. Thus, we investigated the possible interaction between cheerio and dFmr1 during LTM formation in Drosophila. We show that LTM specifically is defective in dFmr1/cheerio double heterozygotes, while it is normal in single heterozygotes for either dFmr1 or cheerio. In dFmr1 mutants, Filamin (Cheerio) levels are lower than normal after spaced training. These observations support the notion that decreased actin cross-linking may underlie the persistence of long and thin dendritic spines in Fragile X patients and animal models. More generally, our results represent the first demonstration of a genetic interaction between mental retardation genes in an in vivo model system of memory formation.

Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory.

We used Drosophila olfactory memory as a model to study the molecular basis of cognitive defects in Fragile X syndrome in vivo. We observed that fragile X protein was acutely required and interacted with argonaute1 and staufen in the formation of long-term memory. Occlusion of long-term memory formation in Fragile X mutants could be rescued by protein synthesis inhibitors, suggesting that excess baseline protein synthesis could negatively affect cognition.

Genetic deletion of ghrelin does not decrease food intake but influences metabolic fuel preference.

Ghrelin is a recently identified growth hormone (GH) secretogogue whose administration not only induces GH release but also stimulates food intake, increases adiposity, and reduces fat utilization in mice. The effect on food intake appears to be independent of GH release and instead due to direct activation of orexigenic neurons in the arcuate nucleus of the hypothalamus. The effects of ghrelin administration on food intake have led to the suggestion that inhibitors of endogenous ghrelin could be useful in curbing appetite and combating obesity. To further study the role of endogenous ghrelin in appetite and body weight regulation, we generated ghrelin-deficient (ghrl(-/-)) mice, in which the ghrelin gene was precisely replaced with a lacZ reporter gene. ghrl(-/-) mice were viable and exhibited normal growth rates as well as normal spontaneous food intake patterns, normal basal levels of hypothalamic orexigenic and anorexigenic neuropeptides, and no impairment of reflexive hyperphagia after fasting. These results indicate that endogenous ghrelin is not an essential regulator of food intake and has, at most, a redundant role in the regulation of appetite. However, analyses of ghrl(-/-) mice demonstrate that endogenous ghrelin plays a prominent role in determining the type of metabolic substrate (i.e., fat vs. carbohydrate) that is used for maintenance of energy balance, particularly under conditions of high fat intake.