Humanized yeast genetic interaction mapping predicts synthetic lethal interactions of FBXW7 in breast cancer.

BACKGROUND: Synthetic lethal interactions (SLIs) that occur between gene pairs are exploited for cancer therapeutics. Studies in the model eukaryote yeast have identified ~ 550,000 negative genetic interactions that have been extensively studied, leading to characterization of novel pathways and gene functions. This resource can be used to predict SLIs that can be relevant to cancer therapeutics. METHODS: We used patient data to identify genes that are down-regulated in breast cancer. InParanoid orthology mapping was performed to identify yeast orthologs of the down-regulated genes and predict their corresponding SLIs in humans. The predicted network graphs were drawn with Cytoscape. CancerRXgene database was used to predict drug response. RESULTS: Harnessing the vast available knowledge of yeast genetics, we generated a Humanized Yeast Genetic Interaction Network (HYGIN) for 1009 human genes with 10,419 interactions. Through the addition of patient-data from The Cancer Genome Atlas (TCGA), we generated a breast cancer specific subnetwork. Specifically, by comparing 1009 genes in HYGIN to genes that were down-regulated in breast cancer, we identified 15 breast cancer genes with 130 potential SLIs. Interestingly, 32 of the 130 predicted SLIs occurred with FBXW7, a well-known tumor suppressor that functions as a substrate-recognition protein within a SKP/CUL1/F-Box ubiquitin ligase complex for proteasome degradation. Efforts to validate these SLIs using chemical genetic data predicted that patients with loss of FBXW7 may respond to treatment with drugs like Selumitinib or Cabozantinib. CONCLUSIONS: This study provides a patient-data driven interpretation of yeast SLI data. HYGIN represents a novel strategy to uncover therapeutically relevant cancer drug targets and the yeast SLI data offers a major opportunity to mine these interactions.

Molecular characterization of an MLL1 fusion and its role in chromosomal instability.

Chromosomal rearrangements involving the mixed-lineage leukemia (MLL1) gene are common in a unique group of acute leukemias, with more than 100 fusion partners in this malignancy alone. However, do these fusions occur or have a role in solid tumors? We performed extensive network analyses of MLL1-fusion partners in patient datasets, revealing that multiple MLL1-fusion partners exhibited significant interactions with the androgen-receptor signaling pathway. Further exploration of tumor sequence data from TCGA predicts the presence of MLL1 fusions with truncated SET domain in prostate tumors. To investigate the physiological relevance of MLL1 fusions in solid tumors, we engineered a truncated version of MLL1 by fusing it with one of its known fusion partners, ZC3H13, to use as a model system. Functional characterization with cell-based assays revealed that MLL1-ZC3H13 fusion induced chromosomal instability, affected mitotic progression, and enhanced tumorsphere formation. The MLL1-ZC3H13 chimera consistently increased the expression of a cancer stem cell marker (CD44); in addition, we detected potential collateral lethality between DOT1L and MLL1 fusions. Our work reveals that MLL1 fusions are likely prevalent in solid tumors and exhibit a potential pro-tumorigenic role.

Expression-based analyses indicate a central role for hypoxia in driving tumor plasticity through microenvironment remodeling and chromosomal instability.

Can transcriptomic alterations drive the evolution of tumors? We asked if changes in gene expression found in all patients arise earlier in tumor development and can be relevant to tumor progression. Our analyses of non-mutated genes from the non-amplified regions of the genome of 158 triple-negative breast cancer (TNBC) cases identified 219 exclusively expression-altered (EEA) genes that may play important role in TNBC. Phylogenetic analyses of these genes predict a "punctuated burst" of multiple gene upregulation events occurring at early stages of tumor development, followed by minimal subsequent changes later in tumor progression. Remarkably, this punctuated burst of expressional changes is instigated by hypoxia-related molecular events, predominantly in two groups of genes that control chromosomal instability (CIN) and those that remodel tumor microenvironment (TME). We conclude that alterations in the transcriptome are not stochastic and that early-stage hypoxia induces CIN and TME remodeling to permit further tumor evolution.

Repression of Fyn-related kinase in breast cancer cells is associated with promoter site-specific CpG methylation.

The triple-negative breast cancer subtype is highly aggressive and has no defined therapeutic target. Fyn-related kinase (FRK) is a non-receptor tyrosine kinase, reported to be downregulated in breast cancer and gliomas, where it is suggested to have tumor suppressor activity. We examined the expression profile of FRK in a panel of 40 breast cancer cells representing all the major subtypes, as well as in 4 non-malignant mammary epithelial cell lines. We found that FRK expression was significantly repressed in a proportion of basal B breast cancer cell lines. We then determined the mechanism of suppression of FRK in FRK-low or negative cell lines. In silico analyses of the FRK promoter region led to the identification of at least 17 CpG sites. Bisulphite sequencing of the promoter region revealed that two of these sites were consistently methylated in FRK-low/negative cell lines and especially in the basal B breast cancer subtype. We further show that treatment of these cells with histone deacetylase inhibitors, Entinostat and Mocetinostat' promoted re-expression of FRK mRNA and protein. Further, using luciferase reporter assays, we show that both GATA3-binding protein FOG1 and constitutively active STAT5A increased the activity of FRK promoter. Together, our results present the first evidence that site-specific promoter methylation contributes to the repression of FRK more so in basal B breast cancers. Our study also highlights the potential clinical significance of targeting FRK using epigenetic drugs specifically in basal B breast cancers which are usually triple negative and very aggressive.

FRK inhibits breast cancer cell migration and invasion by suppressing epithelial-mesenchymal transition.

The human fyn-related kinase (FRK) is a non-receptor tyrosine kinase known to have tumor suppressor activity in breast cancer cells. However, its mechanism of action has not been fully characterized. We generated FRK-stable MDA-MB-231 breast cancer cell lines and analyzed the effect on cell proliferation, migration, and invasiveness. We also used kinome analysis to identify potential FRK-regulated signaling pathways. We employed both immunoblotting and RT-PCR to identify/validate FRK-regulated targets (proteins and genes) in these cells. Finally, we interrogated the TCGA and GENT gene expression databases to determine the correlation between the expression of FRK and epithelial/mesenchymal markers. We observed that FRK overexpression suppressed cell proliferation, migration, and invasiveness, inhibited various JAK/STAT, MAPK and Akt signaling pathways, and suppressed the expression of some STAT3 target genes. Also, FRK overexpression increased the expression of epithelial markers including E-cadherin mRNA and down-regulated the transcript levels of vimentin, fibronectin, and slug. Finally, we observed an inverse correlation between FRK expression and mesenchymal markers in a large cohort of breast cancer cells. Our data, therefore, suggests that FRK represses cell proliferation, migration and invasiveness by suppressing epithelial to mesenchymal transition.

Therapeutic relevance of the protein phosphatase 2A in cancer.

Chromosomal Instability (CIN) is regarded as a unifying feature of heterogeneous tumor populations, driving intratumoral heterogeneity. Polo-Like Kinase 1 (PLK1), a serine-threonine kinase that is often overexpressed across multiple tumor types, is one of the key regulators of CIN and is considered as a potential therapeutic target. However, targeting PLK1 has remained a challenge due to the off-target effects caused by the inhibition of other members of the polo-like family. Here we use synthetic dosage lethality (SDL), where the overexpression of PLK1 is lethal only when another, normally non-lethal, mutation or deletion is present. Rather than directly inhibiting PLK1, we found that inhibition of PP2A causes selective lethality to PLK1-overexpressing breast, pancreatic, ovarian, glioblastoma, and prostate cancer cells. As PP2A is widely regarded as a tumor suppressor, we resorted to gene expression datasets from cancer patients to functionally dissect its therapeutic relevance. We identified two major classes of PP2A subunits that negatively correlated with each other. Interestingly, most mitotic regulators, including PLK1, exhibited SDL interactions with only one class of PP2A subunits (PPP2R1A, PPP2R2D, PPP2R3B, PPP2R5B and PPP2R5D). Validation studies and other functional cell-based assays showed that inhibition of PPP2R5D affects both levels of phospho-Rb as well as sister chromatid cohesion in PLK1-overexpressing cells. Finally, analysis of clinical data revealed that patients with high expression of mitotic regulators and low expression of Class I subunits of PP2A improved survival. Overall, these observations point to a context-dependent role of PP2A that warrants further exploration for therapeutic benefits.

BRK targets Dok1 for ubiquitin-mediated proteasomal degradation to promote cell proliferation and migration.

Breast tumor kinase (BRK), also known as protein tyrosine kinase 6 (PTK6), is a non-receptor tyrosine kinase overexpressed in more that 60% of human breast carcinomas. The overexpression of BRK has been shown to sensitize mammary epithelial cells to mitogenic signaling and to promote cell proliferation and tumor formation. The molecular mechanisms of BRK have been unveiled by the identification and characterization of BRK target proteins. Downstream of tyrosine kinases 1 or Dok1 is a scaffolding protein and a substrate of several tyrosine kinases. Herein we show that BRK interacts with and phosphorylates Dok1 specifically on Y362. We demonstrate that this phosphorylation by BRK significantly downregulates Dok1 in a ubiquitin-proteasome-mediated mechanism. Together, these results suggest a novel mechanism of action of BRK in the promotion of tumor formation, which involves the targeting of tumor suppressor Dok1 for degradation through the ubiquitin proteasomal pathway.

CREB3L1 is a metastasis suppressor that represses expression of genes regulating metastasis, invasion, and angiogenesis.

The unfolded protein response (UPR) is activated in response to hypoxia-induced stress such as in the tumor microenvironment. This study examined the role of CREB3L1 (cyclic AMP [cAMP]-responsive element-binding protein 3-like protein 1), a member of the UPR, in breast cancer development and metastasis. Initial experiments identified the loss of CREB3L1 expression in metastatic breast cancer cell lines compared to low-metastasis or nonmetastatic cell lines. When metastatic cells were transfected with CREB3L1, they demonstrated reduced invasion and migration in vitro, as well as a significantly decreased ability to survive under nonadherent or hypoxic conditions. Interestingly, in an in vivo rat mammary tumor model, not only did CREB3L1-expressing cells fail to form metastases compared to CREB3L1 null cells but regression of the primary tumors was seen in 70% of the animals as a result of impaired angiogenesis. Microarray and chromatin immunoprecipitation with microarray technology (ChIP on Chip) analyses identified changes in the expression of many genes involved in cancer development and metastasis, including a decrease in those involved in angiogenesis. These data suggest that CREB3L1 plays an important role in suppressing tumorigenesis and that loss of expression is required for the development of a metastatic phenotype.

Histone deacetylase inhibitors mediate post-transcriptional regulation of p21WAF1 through novel cis-acting elements in the 3' untranslated region.

Histone deacetylase inhibitors (HDACIs) are promising anti-tumor agents that selectively induce cell cycle arrest, differentiation and/or apoptosis of tumor cells. Fundamentally, HDACIs are proposed to function by activating the transcription of genes, including the potent cyclin dependent kinase inhibitor p21(WAF1). However, HDACIs primarily increase p21(WAF1) expression at the post-transcriptional level in HepG2 cells, implying that these anti-tumor agents regulate genes at multiple levels. Here, two novel cis-acting elements in the 3' untranslated region (UTR) of p21(WAF1) are identified that control the ability of HDACIs to induce p21(WAF1) mRNA stabilization. Collectively, these studies highlight the complexity of HDACIs in gene regulation.

Requirement of N-myristoyltransferase 1 in the development of monocytic lineage.

N-myristoyltransferase (NMT) exists in two isoforms, NMT1 and NMT2, that catalyze myristoylation of various proteins crucial in signal transduction, cellular transformation, and oncogenesis. We have recently demonstrated that NMT1 is essential for the early development of mouse embryo. In this report, we have demonstrated that an invariant consequence of NMT1 knock out is defective myelopoesis. Suppressed macrophage colony forming units were observed in M-CSF-stimulated bone marrow cells from heterozygous (+/-) Nmt1-deficient mice. Homozygous (-/-) Nmt1-deficient mouse embryonic stem cells resulted in drastic reduction of macrophages when stimulated to differentiate by M-CSF. Furthermore, to understand the requirement of NMT1 in the monocytic differentiation we investigated the role of NMT, pp60c-Src (NMT substrate) and heat shock cognate protein 70 (inhibitor of NMT), during PMA-induced differentiation of U937 cells. Src kinase activity and protein expression increased during the differentiation process along with regulation of NMT activity by hsc70. NMT1 knock down in PMA treated U937 cells showed defective monocytic differentiation. We report in this study novel observation that regulated total NMT activity and NMT1 is essential for proper monocytic differentiation of the mouse bone marrow cells.

Short nucleotide polymorphic insertions in the MCL-1 promoter affect gene expression.

We have recently reported novel short nucleotide (six and eighteen) polymorphic insertions, in the MCL-1 promoter and their association with higher mRNA and protein levels. The aim of the present study was to test the hypothesis that these insertions directly affect MCL-1 gene expression. Haematopoietic and epithelial human cell lines were transfected with +0, +6, or +18 MCL-1 promoter fragments positioned upstream of the Firefly luciferase reporter gene. The cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and granulocyte macrophage colony-stimulating factor (GM-CSF). Compared to +0, both polymorphic insertions (+6 and +18) were associated with increased promoter activity. Although chromatin immunoprecipitation assay showed that there are Sp1/Sp3 binding sites in the MCL-1 promoter, electrophoretic mobility shift assay showed that it is unlikely that these sites are in the region harboring these insertions. These results provide further evidence for the biological effect of MCL-1 promoter polymorphisms on gene expression.

The modification of Sp3 isoforms by SUMOylation has differential effects on the SRC1A promoter.

Previously, we had described a housekeeping like promoter that regulates expression of the SRC gene in many cell types. This promoter was found to be regulated by Sp1 and hnRNP-K. However, at that time we could find little evidence supporting a significant role for Sp3 in SRC activation. Interestingly, despite its first description some 12 years ago, a full length Sp3 clone has only recently been described. Previous mechanistic studies, including our own, employed a version of Sp3 that was significantly N-terminally truncated. In addition, several shorter Sp3 isoforms exist that result from internally initiated translation sites. To complicate matters further, all Sp3 isoforms can be modified by SUMO-1. Due to this newly emerging information few reports exist that systematically explore these various Sp3 isoforms (SUMOylated or not) and how they affect activity of specific mammalian promoters. We therefore undertook such a study to re-evaluate regulation of SRC by these various Sp3 isoforms. Using human and insect cells we found that the newly isolated full length version of Sp3 was only a weak to moderate activator of SRC. However, to our surprise, the more commonly used N-terminally truncated version of Sp3 was up to five times more active. We also found that mutations preventing SUMOylation of the shorter Sp3 isoforms were sufficient to convert them into potent transactivators of SRC. In contrast to other studies, however, we found that SUMOylation of full length Sp3 had little effect on its transcriptional properties. These results provide new insights into the complexity of Sp3 mediated transcription which appears to be highly dependent on the isoform bound, SUMOylation status and the promoter context.

N-myristoyltransferase 2 expression in human colon cancer: cross-talk between the calpain and caspase system.

A number of viral and eukaryotic proteins which undergo a lipophilic modification by the enzyme N-myristoyltransferase (NMT: NMT1 and NMT2) are required for signal transduction and regulatory functions. To investigate whether NMT2 contributes to the pathogenesis of colorectal carcinoma, we observed a higher expression of NMT2 in most of the cases of cancerous tissues compared to normal tissues (84.6% of cases; P < 0.05) by Western blot analysis. Furthermore, protein-protein interaction of NMTs revealed that m-calpain interacts with NMT1 while caspase-3 interacts with NMT2. Our findings provide the first evidence of higher expression of NMT2 in human colorectal adenocarcinomas and the interaction of both forms of NMT with various signaling molecules.

Src family kinase members have a common response to histone deacetylase inhibitors in human colon cancer cells.

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation and/or apoptosis in numerous cancer cell types and have shown promise in clinical trials. These agents are particularly novel, given their ability to selectively influence gene expression. Previously, we demonstrated that the HDIs butyrate and trichostatin A (TSA) directly repress c-Src proto-oncogene expression in many cancer cell lines. Activation and/or overexpression of c-Src have been frequently observed in numerous malignancies, especially of the colon. Therefore, our observation was particularly interesting since butyrate is a naturally abundant component of the large intestine and has been suggested to be a cancer-preventive agent. However, c-Src is not the only Src family kinase (SFK) member to be implicated in the development of human cancers, including those of the colon. Therefore, the relative expression levels of known SFKs were examined in a panel of human colon cancer cell lines. We found a surprisingly diverse expression pattern but noted that most cell lines expressed relatively high levels of at least 2 SFKs. When the effects of butyrate and TSA were examined in representative cell lines, the expression of all SFKs was repressed in a dose- and time-dependent manner. Further, detailed examination of Lck, Yes and Lyn demonstrated that this repression had a direct effect on transcription and was independent of new protein synthesis. These results mirror our earlier data obtained with c-Src and suggest that SFKs are a major target of HDIs and likely account in part for the anticancer effects of these promising new drugs.

Expression of methionine aminopeptidase 2, N-myristoyltransferase, and N-myristoyltransferase inhibitor protein 71 in HT29.

Protein myristoylation is a co-translational process, catalyzed by N-myristoyltransferase (NMT) that occurs after the initiating methionine is removed by methionine aminopeptidase (MetAP). The enzymes NMT and MetAP play a major role in the process of myristoylation of oncoproteins including the c-src family. In this study, we examined the levels of expression of MetAP2, NMT, and NMT inhibitor protein 71 (NIP71) in human colon cancer cell lines (HCCLs). We examined the influence of cell density on the expression of the above proteins in HT29 cells. Western blot analysis of MetAP2 and NMT demonstrated higher levels of protein expression in low density of HT29 while low expression in high density was observed. In addition, we observed that NIP71 and pp60(c-src) expressions were dependent on the cell density of HT29. This is the first study demonstrating the expression of MetAP2, NMT, pp60(c-src), and NIP71 in HCCLs.

Regulation of alternative SRC promoter usage in HepG2 hepatocellular carcinoma cells.

Alternative promoters allow for increased spatial and temporal diversity in expression patterns for a single gene. The human SRC gene, encoding the non-receptor c-Src tyrosine kinase, is regulated by two alternative promoters separated by approximately 1 kb. The distal SRC1alpha promoter is tissue-restricted, while expression of the proximal SRC1A promoter appears to be ubiquitous. A barrier to elucidating the mechanisms of SRC transcriptional regulation has been the finding that the individual strengths of the SRC promoters in isolation do not match their relative strength of use seen in vivo. For example, in HepG2 hepatocellular carcinoma cells, SRC1A is significantly stronger in isolation than SRC1alpha, despite SRC1alpha being the predominant promoter used in this cell line. Previously, we have shown that HepG2 cells, as well as various colon cancer cell lines, display activated SRC transcription, which is linked to the elevated c-Src expression and activity necessary for growth and survival of these cells. These findings thus highlight the importance of understanding the mechanisms of SRC transcriptional regulation in human cancer. We hypothesize the discrepancy between individual SRC promoter strength and relative usage in vivo stems from a lack of linked promoter context. Therefore, we have developed and validated a novel dual SRC promoter reporter strategy to allow the simultaneous mechanistic study of both SRC promoters in their natural linked context. This approach has yielded evidence that SRC activation proceeds through genomic element(s) outside the promoter region in HepG2 cells. Therefore, we performed a preliminary study of DNaseI hypersensitive (DH) site composition within the SRC locus. This approach identified a HepG2-specific DH site that displayed activating potential towards the SRC1alpha promoter. These results thus provide important insight to the mechanism of SRC transcriptional activation in liver cancer cells.

Histone deacetylase inhibitors regulate p21WAF1 gene expression at the post-transcriptional level in HepG2 cells.

Histone deacetylase inhibitors (HDIs) are thought to act primarily at the level of transcription inducing cell cycle arrest, differentiation and/or apoptosis in many cancer cell types. Induction of the potent cdk/cyclin inhibitor p21WAF1 is a key feature of this HDI mediated transcriptional re-programming phenomenon. However, in the current study we report that HDIs are also capable of inducing p21WAF1 through purely post-transcriptional events, namely increased mRNA stability. These studies highlight our growing appreciation for the complexities of HDI mediated effects and challenge our preconceptions regarding the action of these promising anti-neoplastics.

SRC gene expression in human cancer: the role of transcriptional activation.

Human pp60c-Src (or c-Src) is a 60 kDa nonreceptor tyrosine kinase encoded by the SRC gene and is the cellular homologue to the potent transforming v-Src viral oncogene. c-Src functions at the hub of a vast array of signal transduction cascades that influence cellular proliferation, differentiation, motility, and survival. c-Src activation has been documented in upwards of 50% of tumors derived from the colon, liver, lung, breast, and pancreas. Therefore, a major focus has been to understand the mechanisms of c-Src activation in human cancer. Early studies concentrated on post-translational mechanisms that lead to increased c-Src kinase activity, which often correlated with overexpression of c-Src protein. More recently, the discovery of an activating SRC mutation in a small subset of advanced colon tumors has been reported. In addition, elevated SRC transcription has been identified as yet another mechanism contributing significantly to c-Src activation in a subset of human colon cancer cell lines. Interestingly, histone deacetylase (HDAC) inhibitors, agents with well-documented anti-cancer activity, repress SRC transcription in a wide variety of human cancer cell lines. Analysis of the mechanisms behind HDAC inhibitor mediated repression could be utilized in the future to specifically inhibit SRC gene expression in human cancer.

SRC proximal and core promoter elements dictate TAF1 dependence and transcriptional repression by histone deacetylase inhibitors.

Histone deacetylase inhibitors (HDIs) induce cell cycle arrest, differentiation, or apoptosis in numerous cancer cell types both in vivo and in vitro. These dramatic effects are the result of a specific reprogramming of gene expression. However, the mechanism by which these agents activate the transcription of some genes, such as p21(WAF1), but repress others, such as cyclin D1, is currently unknown. We have been studying the human SRC gene as a model for HDI-mediated transcriptional repression. We found previously that both the tissue-specific and housekeeping SRC promoters were equally repressed by HDIs. Here we show that, despite an overt dissimilarity, both SRC promoters do share similar core promoter elements and transcription is TAF1 dependent. Detailed analysis of the SRC promoters suggested that both core and proximal promoter elements were responsible for HDI-mediated repression. This was confirmed in a series of promoter-swapping experiments with the HDI-inducible, TAF1-independent p21(WAF1) promoter. Remarkably, all the SRC-p21(WAF1) chimeric promoter constructs were not only repressed by HDIs but also dependent on TAF1. Together these experiments suggest that the overall promoter architecture, rather than discrete response elements, is responsible for HDI-mediated repression, and they implicate core promoter elements in particular as potential mediators of this response.

Identification of the SRC pyrimidine-binding protein (SPy) as hnRNP K: implications in the regulation of SRC1A transcription.

The human SRC gene encodes pp60(c-src), a non-receptor tyrosine kinase involved in numerous signaling pathways. Activation or overexpression of c-Src has also been linked to a number of important human cancers. Transcription of the SRC gene is complex and regulated by two closely linked but highly dissimilar promoters, each associated with its own distinct non-coding exon. In many tissues SRC expression is regulated by the housekeeping-like SRC1A promoter. In addition to other regulatory elements, three substantial polypurine:polypyrimidine (TC) tracts within this promoter are required for full transcriptional activity. Previously, we described an unusual factor called SRC pyrimidine-binding protein (SPy) that could bind to two of these TC tracts in their double-stranded form, but was also capable of interacting with higher affinity to all three pyrimidine tracts in their single-stranded form. Mutations in the TC tracts, which abolished the ability of SPy to interact with its double-stranded DNA target, significantly reduced SRC1A promoter activity, especially in concert with mutations in critical Sp1 binding sites. Here we expand upon our characterization of this interesting factor and describe the purification of SPy from human SW620 colon cancer cells using a DNA affinity-based approach. Subsequent in-gel tryptic digestion of purified SPy followed by MALDI-TOF mass spectrometric analysis identified SPy as heterogeneous nuclear ribonucleoprotein K (hnRNP K), a known nucleic-acid binding protein implicated in various aspects of gene expression including transcription. These data provide new insights into the double- and single-stranded DNA-binding specificity, as well as functional properties of hnRNP K, and suggest that hnRNP K is a critical component of SRC1A transcriptional processes.