Metastatic colorectal adenocarcinoma tumor purity assessment from whole exome sequencing data.

Tumors rich in stroma are associated with advanced stage and poor prognosis in colorectal adenocarcinoma (CRC). Abundance of stromal cells also has implications for genomic analysis of patient tumors as it may prevent detection of somatic mutations. As part of our efforts to interrogate stroma-cancer cell interactions and to identify actionable therapeutic targets in metastatic CRC, we aimed to determine the proportion of stroma embedded in hepatic CRC metastases by performing computational tumor purity analysis based on whole exome sequencing data (WES). Unlike previous studies focusing on histopathologically prescreened samples, we used an unbiased in-house collection of tumor specimens. WES from CRC liver metastasis samples were utilized to evaluate stromal content and to assess the performance of three in silico tumor purity tools, ABSOLUTE, Sequenza and PureCN. Matching tumor derived organoids were analyzed as a high purity control as they are enriched in cancer cells. Computational purity estimates were compared to those from a histopathological assessment conducted by a board-certified pathologist. According to all computational methods, metastatic specimens had a median tumor purity of 30% whereas the organoids were enriched for cancer cells with a median purity estimate of 94%. In line with this, variant allele frequencies (VAFs) of oncogenes and tumor suppressor genes were undetectable or low in most patient tumors, but higher in matching organoid cultures. Positive correlation was observed between VAFs and in silico tumor purity estimates. Sequenza and PureCN produced concordant results whereas ABSOLUTE yielded lower purity estimates for all samples. Our data shows that unbiased sample selection combined with molecular, computational, and histopathological tumor purity assessment is critical to determine the level of stroma embedded in metastatic colorectal adenocarcinoma.

xCT inhibition sensitizes tumors to γ-radiation via glutathione reduction.

About 3 million US cancer patients and 1.7 million EU cancer patients received multiple doses of radiation therapy (RT) in 2012, with treatment duration limited by normal adjacent tissue damage. Tumor-specific sensitization could allow treatment with lower radiation doses, reducing normal tissue damage. This is a longstanding, largely unrealized therapeutic goal. The cystine:glutamate exchanger xCT is expressed on poor prognosis subsets of most solid tumors, but not on most normal cells. xCT provides cells with environmental cystine for enhanced glutathione synthesis. Glutathione is used to control reactive oxygen species (ROS), which are therapeutic effectors of RT. We tested whether xCT inhibition would sensitize xCT+ tumor cells to ionizing radiation. We found that pretreatment with the xCT inhibitor erastin potently sensitized xCT+ but not xCT- cells, in vitro and in xenograft. Similarly, targeted gene inactivation also sensitized cells, and both modes of sensitization were overcome by glutathione supplementation. Sensitization prolongs DNA damage signaling, increases genome instability, and enhances cell death, revealing an unforeseen role for cysteine in genome integrity maintenance. We conclude that an xCT-specific therapeutic would provide tumor-specific sensitization to RT, allowing treatment with lower radiation doses, and producing far fewer side effects than other proposed sensitizers. Our data speaks to the need for the rapid development of such a drug.

Genome-independent hypoxic repression of estrogen receptor alpha in breast cancer cells.

BACKGROUND: About 75-80% of breast tumors express the estrogen receptor alpha (ER-α) and are treated with endocrine-target therapeutics, making this the premier therapeutic modality in the breast cancer clinic. However, acquired resistance is common and about 20% of resistant tumors loose ER-α expression via unknown mechanisms. Inhibition of ER-α loss could improve endocrine therapeutic efficacy, benefiting a significant number of patients. Here we test whether tumor hypoxia might commonly produce ER-α loss. METHODS: Using standard molecular and cellular biological assays and a work station/incubator with controllable oxygen levels, we analyze the effects of hypoxia on ER-α protein, mRNA, and transcriptional activity in a panel of independently-derived ER-α positive cell lines. These lines were chosen to represent the diverse genetic backgrounds and mutations commonly present in ER-α positive tumors. Using shRNA-mediated knockdown and overexpression studies we also elucidate the role of hypoxia-inducible factor 1-alpha (HIF-1α) in the hypoxia-induced decrease in ER-α abundance. RESULTS: We present the first comprehensive overview of the effects of bona fide low environmental oxygen (hypoxia) and HIF-1α activity on ER-α abundance and transcriptional activity. We find that stabilized HIF-1α induces rapid loss of ER-α protein in all members of our diverse panel of breast cancer cell lines, which involves proteolysis rather than transcriptional repression. Reduced ER-α severely attenuates ER-α directed transcription, and inhibits cell proliferation without overt signs of cell death in the cell lines tested, despite their varying genomic backgrounds. CONCLUSIONS: These studies reveal a common hypoxia response that produces reduced ER-α expression and cell cycle stalling, and demonstrate a common role for HIF-1α in ER-α loss. We hypothesize that inhibitors of HIF-1α or the proteasome might stabilize ER-α expression in breast tumors in vivo, and work in combination with endocrine therapies to reduce resistance. Our data also suggests that disease re-occurrence in patients with ER-α positive tumors may arise from tumor cells chronically resident in hypoxic environments. We hypothesize that these non-proliferating cells may survive undetected until conditions change to oxygenate the environment, or cells eventually switch to proliferation via other signaling pathways.

Glutamine sensitivity analysis identifies the xCT antiporter as a common triple-negative breast tumor therapeutic target.

A handful of tumor-derived cell lines form the mainstay of cancer therapeutic development, yielding drugs with an impact typically measured as months to disease progression. To develop more effective breast cancer therapeutics and more readily understand their clinical impact, we constructed a functional metabolic portrait of 46 independently derived breast cell lines. Our analysis of glutamine uptake and dependence identified a subset of triple-negative samples that are glutamine auxotrophs. Ambient glutamine indirectly supports environmental cystine acquisition via the xCT antiporter, which is expressed on one-third of triple-negative tumors in vivo. xCT inhibition with the clinically approved anti-inflammatory sulfasalazine decreases tumor growth, revealing a therapeutic target in breast tumors of poorest prognosis and a lead compound for rapid, effective drug development.

Lipoprotein lipase links dietary fat to solid tumor cell proliferation.

Many types of cancer cells require a supply of fatty acids (FA) for growth and survival, and interrupting de novo FA synthesis in model systems causes potent anticancer effects. We hypothesized that, in addition to synthesis, cancer cells may obtain preformed, diet-derived FA by uptake from the bloodstream. This would require hydrolytic release of FA from triglyceride in circulating lipoprotein particles by the secreted enzyme lipoprotein lipase (LPL), and the expression of CD36, the channel for cellular FA uptake. We find that selected breast cancer and sarcoma cells express and secrete active LPL, and all express CD36. We further show that LPL, in the presence of triglyceride-rich lipoproteins, accelerates the growth of these cells. Providing LPL to prostate cancer cells, which express low levels of the enzyme, did not augment growth, but did prevent the cytotoxic effect of FA synthesis inhibition. Moreover, LPL knockdown inhibited HeLa cell growth. In contrast to the cell lines, immunohistochemical analysis confirmed the presence of LPL and CD36 in the majority of breast, liposarcoma, and prostate tumor tissues examined (n = 181). These findings suggest that, in addition to de novo lipogenesis, cancer cells can use LPL and CD36 to acquire FA from the circulation by lipolysis, and this can fuel their growth. Interfering with dietary fat intake, lipolysis, and/or FA uptake will be necessary to target the requirement of cancer cells for FA.

PIK3CA cooperates with other phosphatidylinositol 3'-kinase pathway mutations to effect oncogenic transformation.

Mutations in genes functioning in different pathways frequently occur together in the same cancer, whereas mutations in the same pathway tend to be mutually exclusive. However, the majority of colon, breast, and endometrial cancers that possess mutations in PIK3CA, the catalytic subunit p110alpha of phosphatidylinositol 3'-kinase (PI3K), also possess mutations or alterations in genes upstream of PI3K such as Ras, ERBB2/ERBB3, or PTEN. PIK3CA mutations occur almost exclusively in invasive tumors, whereas upstream mutations occur as frequently in early-stage and late-stage tumors, suggesting that PIK3CA mutation is a late-stage event that may augment earlier activation of the PI3K pathway. Consistent with this, we find that levels of p-AKT (Ser(473)) induced by mutant Ras or knockdown of PTEN were dramatically increased by addition of mutant PIK3CA. Soft agar assays revealed that anchorage-independent growth induced by mutant Ras was greatly increased in the presence of mutant PIK3CA. In breast, colon, and endometrial cancers in which the PI3K pathway is activated by a combination of mutant PIK3CA and alterations in Ras, ERBB2/3, or PTEN, signaling to downstream elements such as Akt was mediated exclusively by the p110alpha isoform, rather than a combination of different PI3K isoforms. Our data therefore suggest that in tumors with co-occurring mutations in multiple components of the PI3K pathway, selective inhibition of the alpha isoform of p110 is an attractive therapeutic strategy, especially for late-stage tumors.

Notch and epithelial-mesenchyme transition in development and tumor progression: another turn of the screw.

Notch is an ancient cell signaling system that regulates cell fate specification, stem cell maintenance and initiation of differentiation in embryonic and postnatal tissues.(1) Alteration of these functions in the adult have been associated to various types of cancer in which Notch may act as an oncogene or as a tumor suppressor. As occurs during development, Notch cooperates with other signaling pathways in the transformation process. Notch has recently been shown to promote epithelial-to-mesenchymal transition (EMT) during cardiac valve formation, via snail induction and subsequent cadherin downregulation. One implication of this work is that Notch acting through a similar mechanism, may also be involved in the EMT process that occurs during tumor progression and converts polarized epithelial cells into motile, invasive cells.

Mechanisms of cell-cycle arrest in Spitz nevi with constitutive activation of the MAP-kinase pathway.

Spitz nevi are benign melanocytic nevi that overlap histopathologically with melanoma. We previously found copy number increases of chromosome 11p frequently paralleled by mutations in the HRAS oncogene mapping to this region. In this study, we explored mechanisms that inhibit proliferation in the presence of HRAS activation. We analyzed MAP-kinase activation using immunohistochemistry for phospho-ERK, cyclin D1, and microphthalmia transcription factor expression in 17 Spitz nevi with and 18 Spitz nevi without 11p copy number increase. We found relatively high levels of phospho-ERK and cyclin D1 expression suggesting MAP-kinase pathway activation in both groups of Spitz nevi. However, Spitz nevi with 11p copy number increases showed significantly higher levels of cyclin D1 expression and lower levels of microphthalmia transcription factor expression suggesting stronger MAP-kinase pathway activation in this group. Contrasting this apparent activation, the proliferation rate as assessed by Mib1 expression was low in both groups. An analysis of cell-cycle inhibitory proteins including p16, p21, and p27 showed that the majority of Spitz nevus cells expressed high levels of p16, with cells of the cases that had increased copy number of 11p expressing significantly higher levels than those of Spitz nevi with normal copy number of 11p. We propose that in benign nevi with constitutive activation of the MAP-kinase pathway, p16 functions as an essential mediator of oncogene-induced senescence preventing progression to melanoma.

Notch promotes epithelial-mesenchymal transition during cardiac development and oncogenic transformation.

Epithelial-to-mesenchymal transition (EMT) is fundamental to both embryogenesis and tumor metastasis. The Notch intercellular signaling pathway regulates cell fate determination throughout metazoan evolution, and overexpression of activating alleles is oncogenic in mammals. Here we demonstrate that Notch activity promotes EMT during both cardiac development and oncogenic transformation via transcriptional induction of the Snail repressor, a potent and evolutionarily conserved mediator of EMT in many tissues and tumor types. In the embryonic heart, Notch functions via lateral induction to promote a selective transforming growth factor-beta (TGFbeta)-mediated EMT that leads to cellularization of developing cardiac valvular primordia. Embryos that lack Notch signaling elements exhibit severely attenuated cardiac snail expression, abnormal maintenance of intercellular endocardial adhesion complexes, and abortive endocardial EMT in vivo and in vitro. Accordingly, transient ectopic expression of activated Notch1 (N1IC) in zebrafish embryos leads to hypercellular cardiac valves, whereas Notch inhibition prevents valve development. Overexpression of N1IC in immortalized endothelial cells in vitro induces EMT accompanied by oncogenic transformation, with corresponding induction of snail and repression of VE-cadherin expression. Notch is expressed in embryonic regions where EMT occurs, suggesting an intimate and fundamental role for Notch, which may be reactivated during tumor metastasis.