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bioRxiv2018

Electromagnetic Field and Temozolomide Increase Differentiation of Human Glioblastoma Cell Line; concise view of mechanism

Glioblastoma is a highly malignant brain tumor with an extremely dismal prognosis, with a median survival of 12 months. Promoting glioma stem cell (GSC) differentiation is a crucial therapeutic strategy in treating glioblastoma to improve survival. We combine standard chemotherapy drug Temozolomide (TMZ) with Electromagnetic Field to evaluate their differentiation effects on glioma U87 cell line.\n\nHuman glioma U87 cells exposed to electromagnetic field (EMF), Temozolomide (TMZ) alone and a combination of both were compared to control group. Nestin and CD133 were detected to identify stem-like cells (SLCs) changes during the experiment. The differentiation was found through detecting the expression of the glial fibrillary acidic protein (GFAP), and Notch4. We evaluated Ca+, SOD, and Notch as important chemical mediators in signal transduction to elucidate the mechanism of actions in the processes of differentiation.\n\nThe expression of cancer SLC markers CD133, and neural stem/progenitor Nestin decreased in EMF/ Drug combined group compared to control which shows depletion of stem-like cell pool. In contrast, the differentiation of SLCs was increased by detecting the expression of the glial fibrillary acidic protein (GFAP), but, Notch4 decreased in Electromagnetic field/Drug combined group compared to control which shows a benign process of treatment. The differentiation was also confirmed by light microscopy. Morphological changes such as an elongated shape with elongated neurites, intercellular connection, and neurite branching were observed. Superoxide dismutase (SOD), Ca+ and Notch were also increased as important chemical mediators in signal transduction.\n\nHere, we found the combination of pulsed electromagnetic fields (PEMFs) and TMZ significantly resulted in differentiation and glioma stem cells (GSCs) pool reduction that could have a profound therapeutic implication.

cancer biology↗

bioRxiv2018

Predicting clone genotypes from tumor bulk sequencing of multiple samples

MotivationAnalyses of data generated from bulk sequencing of tumors have revealed extensive genomic heterogeneity within patients. Many computational methods have been developed to enable the inference of genotypes of tumor cell populations (clones) from bulk sequencing data. However, the relative and absolute accuracy of available computational methods in estimating clone counts and clone genotypes is not yet known.\n\nResultsWe have assessed the performance of nine methods, including eight previously-published and one new method (CloneFinder), by analyzing computer simulated datasets. CloneFinder, LICHeE, CITUP, and cloneHD inferred clone genotypes with low error (<5% per clone) for a majority of datasets in which the tumor samples contained evolutionarily-related clones. Computational methods did not perform well for datasets in which tumor samples contained mixtures of clones from different clonal lineages. Generally, the number of clones was underestimated by cloneHD and overestimated by Phy-loWGS, and BayClone2, Canopy, and Clomial required prior information regarding the number of clones. AncesTree and Canopy did not produce results for a large number of datasets.\n\nConclusionsDeconvolution of clone genotypes from single nucleotide variant (SNV) frequency differences among tumor samples remains challenging, so there is a need to develop more accurate computational methods and robust software for clone genotype inference.\n\nAvailability and ImplementationCloneFinder is implemented in Python and is available from https://github.com/gstecher/CloneFinderAPI.\n\nContacts.kumar@temple.edu\n\nSupplementary informationSupplementary data are available at Bioinformatics online

cancer biology↗

bioRxiv2018

Influence of miR-520e-mediated MAPK signaling pathway on HBV replication and regulation of hepatocellular carcinoma cells via targeting EphA2

This paper aims to determine the role of miR-520e in the replication of hepatitis B virus (HBV) and the growth of hepatocellular carcinoma (HCC) cells. MiR-520e and EphA2 in HBV-positive HCC tissues and cells were detected. HepG2.2.15 and Huh7 cells transfected with pHBV1.2 were divided into Mock, NC, miR-520e mimic, miR-520e inhibitor, si-EphA2, and miR-520e inhibitor + si-EphA2 groups. MiR-520e, HBV DNA content, HBsAg and HBeAg levels, cell proliferation, apoptosis and protein expression of EphA2 and MAPK pathways were evaluated. Furthermore, rAAV81.3HBV infected-mouse model was established to detect HBV-DNA levels. MiR-520e was up-regulated and EphA2 was down-regulated in HBV-positive HCC tissues and cells (HepG2.2.15 and HepAD38). MiR-520e was decreased in Huh7-X and HepG2-X cells in which HBx was stably expressed, but miR-520e was dose-dependently elevated in Huh7-X, HepG2-X, and HepG2.2.15 cells after interfering HBx. Additionally, miR-520e mimic and si-EphA2 groups were apparently reduced in HBV DNA content, HBsAg and HBeAg levels, cell proliferation, and were enhanced in the expressions of EphA2, MAPK pathways and cell apoptosis. Furthermore, si-EphA2 can reverse the promotion effect of miR-520e inhibitor on the HBV replication and tumor cell growth Up-regulating miR-520e in rAAV81.3HBV infected-mouse resulted in the reduced EphA2 in liver tissues and HBV DNA content in serum. MiR-520e was found to be decreased in HBV-positive HCC tissues and cells, while over-expression of miR-520e blocked MAPK pathways via inhibiting EphA2, ultimately reducing HBV replication and inhibiting tumor cell growth.

cancer biology↗

bioRxiv2018

Novel significant stage-specific differentially expressed genes in liver hepatocellular carcinoma

Liver cancer is among the top deadly cancers worldwide with a very poor prognosis, and the liver is a particularly vulnerable site for metastasis of other cancers. In this study, we developed a novel computational framework for the stage-specific analysis of hepatocellular carcinoma initiation and progression. Using publicly available clinical and RNA-Seq data of cancer samples and controls, we annotated the gene expression matrix with sample stages. We performed a linear modelling analysis of gene expression across all stages and found significant genome-wide changes in gene expression in cancer samples relative to control. Using a contrast against the control, we were able to identify differentially expressed genes (log fold change >2) that were significant at an adjusted p-value < 10E-3. In order to identify genes that were specific to each stage without confounding differential expression in other stages, we developed a full set of pairwise stage contrasts and enforced a p-value threshold (<0.05) for each such contrast. Genes were specific for a stage if they passed all the significance filters for that stage. Our analysis yielded two stage-I specific genes (CA9, WNT7B), two stage-II specific genes (APOBEC3B, FAM186A), ten stage-III specific genes including DLG5, PARI and GNMT, and ten stage-IV specific genes including GABRD, PGAM2 and PECAM1. Of these, only APOBEC3B is an established cancer driver gene. DLG5 was found to be tumor-promoting contrary to the cancer literature on this gene. Further, GABRD, well studied in literature on other cancers, emerged as a stage-IV specific gene. Our findings could be validated using multiple sources of omics data as well as experimentally. The biomarkers identified herein could potentially underpin diagnosis as well as pinpoint drug targets.

cancer biology↗

bioRxiv2018

Microbial Dysbiosis and polyamine metabolism as predictive markers for early detection during progression of pancreatic adenocarcinoma in spontaneous mouse model

PurposeThe lack of tools for early detection of pancreatic ductal adenocarcinoma (PDAC) is directly correlated to the abysmal survival rate in patients. In addition to several potential detection tools under active investigation, we tested the gut microbiome and its metabolic complement as one of the earliest detection tools that could be useful in patients at high-risk for PDAC.\n\nExperimental DesignA combination of 16s pyrosequencing and whole-genome sequencing of gut microbiota was used in a spontaneous genetically engineered PDAC murine model (KRASG12DTP53R172HPdxCre or KPC). Metabolic reconstruction of microbiome was done using the HUmanN2 pipeline. Serum polyamine levels were measured from murine and patient samples using standard methods.\n\nResultsResults showed a progressive Proteobacterial and Firmicutes dominance in gut microbiota in early stages of PDAC development. Upon in silico reconstruction of active metabolic pathways within the altered microbial flora, polyamine and nucleotide biosynthetic pathways were significantly elevated. These metabolic products are known to be actively assimilated by the host and eventually utilized by rapidly dividing cells for proliferation validating their importance in the context of tumorigenesis. In KPC mice, as well as PDAC patients, we show significantly elevated serum polyamine concentration. Therefore, at the early stages of tumorigenesis, the gut microbial composition changes in a way to release metabolites that foster host tumorigenesis, thereby fulfilling the vicious cycle hypothesis of the role of the microbiome in health and disease states.\n\nConclusionsOur results provide a potential, precise, non-invasive tool for early detection of PDAC, which will result in improved outcomes.\n\nSynopsisGut microbiota changes during early stages of pancreatic ductal adenocarcinoma (PDAC) progression and contributes towards host polyamine pool. Both changes can be used as an early predictive marker for PDAC.\n\nTranslational RelevancePancreatic carcinogenesis progresses through pre-cancerous PanIN lesions to invasive cancer. Even though these morphological changes are histologically distinct, imaging techniques are not able to distinguish the pre-invasive PanINs from normal pancreas, making detection of a tumor at a precancerous stage impossible. Thus, majority of cases (85-90%) present with advanced pancreatic cancer at the time of diagnosis. This contributes to the dismal survival rate in this disease. Our study of gut microbiome analysis on KPC mice during tumor progression followed by metabolic reconstruction and experimental validation in human samples indicate that gut-microbiome analysis along with an analysis of the microbial metabolites can be developed as potential biomarkers for detection of PDAC at early stages when histological changes are not yet grossly apparent.

cancer biology↗

bioRxiv2018

Modulation of mutant KrasG12D-driven lung tumorigenesis in vivo by gain or loss of PCDH7 function

PROTOCADHERIN 7 (PCDH7), a transmembrane receptor and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinoma and is associated with poor clinical outcome. While PCDH7 was recently shown to promote transformation and facilitate brain metastasis in lung and breast cancers, decreased PCDH7 expression has also been documented in colorectal, gastric, and invasive bladder cancers. These data suggest context-dependent functions for PCDH7 in distinct tumor types. Given that PCDH7 is a potentially targetable molecule on the surface of cancer cells, further investigation of its role in tumorigenesis in vivo is needed to evaluate the therapeutic potential of its inhibition. Here we report the analysis of novel PCDH7 gain- and loss-of-function mouse models and provide compelling evidence that this cell-surface protein acts as a potent lung cancer driver. Employing a Cre-inducible transgenic allele, we demonstrated that enforced PCDH7 expression significantly accelerates KrasG12D-driven lung tumorigenesis and potentiates MAPK pathway activation. Furthermore, we performed in vivo somatic genome editing with CRISPR/Cas9 in KrasLSL-G12D; Tp53fl/fl (KP) mice to assess the consequences of PCDH7 loss of function. Inactivation of PCDH7 in KP mice significantly reduced lung tumor development, prolonged survival, and diminished phospho-activation of ERK1/2. Together, these findings establish a critical oncogenic function for PCDH7 in vivo and highlight the therapeutic potential of PCDH7 inhibition for lung cancer. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the new inducible transgenic model described here provides a robust experimental system for broadly elucidating the effects of PCDH7 overexpression in vivo.\n\nAUTHOR SUMMARYLung cancer is the leading cause of cancer-associated deaths worldwide. PROTOCADHERIN 7 (PCDH7), cell surface protein and member of the Cadherin superfamily, is frequently overexpressed in lung adenocarcinomas and is associated with poor clinical outcome. Nevertheless, it has yet to be shown in vivo whether PCDH7 plays a role in the initiation and progression of lung cancer, and whether it represents an actionable therapeutic target. Here we demonstrate, using a novel transgenic mouse model, that PCDH7 overexpression accelerates KrasG12D-driven lung tumorigenesis. Furthermore, we validate PCDH7 as a therapeutic target by knocking it out using in vivo somatic genome editing in the KrasLSL-G12D; Tp53fl/fl (KP) model. Our results provide new insight into the mechanisms that drive lung cancer pathogenesis and, because targeting oncogenic cell-surface proteins with antibodies has proven to be a highly effective anti-cancer therapeutic strategy, establish a new target for cancer treatment. Moreover, given recent reports of elevated or reduced PCDH7 in distinct tumor types, the transgenic PCDH7 model described here provides a robust experimental system for elucidating the effects of PCDH7 overexpression in different in vivo settings. This model will also provide an ideal system for future testing of therapeutics directed at PCDH7.

cancer biology↗

bioRxiv2018

The systemic activin response to pancreatic cancer: Implications for effective cancer cachexia therapy

Pancreatic ductal adenocarcinoma (PDAC) is a particularly lethal malignancy with high rates of cachexia. Serum activin correlates with PDAC cachexia and mortality, while activin administration causes cachexia in mice. We studied activin in human tumors and in mice with orthotopic or genetic PDAC. Cachexia severity correlated with activin expression in tumor lines. Activins were expressed in both cancer and tumor stromal cells, but also in organs in murine PDAC cachexia. Tumor cells expressed activin-{beta}A, or Inhba, while organs expressed both activin-{beta}A and activin-{beta}B, or Inhbb. PDAC elicits activin expression; PDAC conditioned medium induced activin and atrophy of myotubes. Treatment with the activin trap, ACVR2B/Fc, reduced cachexia and prolonged survival in mice with activin-low tumors, and reduced cachexia in activin-high tumors, without affecting activin expression in organs. Mice expressing dominant negative ACVR2B in muscle were protected for weight loss but not survival. Overall our results indicate that PDAC induces a systemic activin response, leading to cachexia, and that activin targets might include organs beyond muscle. Targeting of both tumor-derived and host-derived activins could improve cachexia therapy.

cancer biology↗

bioRxiv2018

Targeting BMI1 and MCL1 for Lung Adenocarcinoma Treatment

Lung cancer is the leading cause of cancer-associated death worldwide. Early metastasis and the recurrence remain major challenges for lung cancer treatment in clinic. Targeting the cancer stemness could be a potential strategy to restrain tumor progression. In the current study, we found that in lung adenocarcinoma (LAC), BMI1 and MCL1 play crucial roles in invasion, chemo-resistance, and tumor initiation. JNK signaling is a link between oncogenic pathway or environment stress to cancer stemness. The activation of JNK, either by EGFR or chemotherapy agent, stabilized BMI1 and MCL1 proteins through suppressing the expression of E3-ubiquitin ligase HUWE1. In lung cancer patient samples, high level of BMI1 is correlated with poor survival, and the expression of BMI1 is positively correlated with MCL1. A novel small-molecule BI-44 was synthesized, which effectively suppressed BMI1/MCL1 expression and inhibited tumor formation and progression in preclinical models. Targeting BMI1/MCL1 provides the basis for a new therapeutic approach in the treatment of LAC.

cancer biology↗

bioRxiv2018

Distinct oncogenes drive distinct genome and epigenome alterations in human mammary epithelial cells

Gene expression differences, combined with distinct patterns of genomic rearrangements and epigenetic modifications, have laid the bases of molecular classification of breast cancer. Different molecular subtypes are thought to originate from different cell lineages in the mammary gland, but the early activation of an oncogene could also play a role. It is, however, difficult to discriminate the respective inputs of oncogene activation or cell type of origin in the natural history of the tumor. In this work, we have designed an experimental strategy aiming at determining whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. We show that initial activation of CCNE1, WNT1 and RASv12, which activate distinct oncogenic pathways, in shp53 immortalized HMECs results in different and reproducible profiles of mRNA and miRNA expression, copy number alterations (CNA) and DNA methylation modifications. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs.\n\nGenes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes.\n\nAuthor summaryGenetic and epigenetic changes are at the center of cancer development. Breast cancer molecular subtypes are defined on differences in genetic and epigenetic profiles and it is generally assumed these subtypes originate from different cell lineages in the mammary gland. We propose that founding oncogenic mutations could also have an impact. To address this question, we designed an experimental model, based on the ectopic expression of different oncogenes in human mammary epithelial cells (HMEC), and monitored genetic and DNA methylation changes occurring at different stages of cell transformation. We show that transformation of HMEC by distinct oncogenes resulted in clearly different and reproducible patterns of genetic and DNA methylation changes. Genes whose expression was modified by either CNAs or CpG methylation were consistent with the dominant pathways activated and reflected the phenotypes in the respective models. We propose that DNA methylation and CNA changes correspond to adaptive responses to the activation of the oncogenic pathways. Our data strongly suggest that early activation of distinct oncogenic insults will not only impinge on the phenotypic characteristics of the resulting tumors, but also have a strong impact on their genomic and epigenetic landscapes.

cancer biology↗

bioRxiv2018

Selective inhibition of CA IX over CA XII using benzene sulfonamides: Disconnect between CA activity and growth inhibition in breast cancer cells

Carbonic anhydrases (CAs) have been linked to tumor progression, particularly membrane-bound CA isoform IX (CA IX). The role of CA IX in the context of breast cancer is to regulate the pH of the tumor microenvironment. In contrast to CA IX, expression of CA XII, specifically in breast cancer, is associated with better outcome despite performing the same catalytic function. In this study, we have structurally modeled the orientation of bound ureido-substituted benzene sulfonamides (USBs) within the active site of CA XII, in comparison to CA IX and cytosolic off-target CA II, to understand isoform specific inhibition. This has identified specific residues within the CA active site, which differ between isoforms that are important for inhibitor binding and isoform specificity. The ability of these sulfonamides to block CA IX activity in breast cancer cells is less effective than their ability to block activity of the recombinant protein (by one to two orders of magnitude depending on the inhibitor). The same is true for CA XII activity but now they are two to three orders of magnitude less effective. Thus, there is significantly greater specificity for CA IX activity over CA XII. While the inhibitors block cell growth, without inducing cell death, this again occurs at two orders of magnitude above the Ki values for inhibition of CA IX and CA XII activity in their respective cell types. Surprisingly, the USBs inhibited cell growth even in cells where CA IX and CA XII expression was ablated. Despite the potential for these sulfonamides as chemotherapeutic agents, these data suggest that we reconsider the role of CA activity on growth potentiation.

cancer biology↗

bioRxiv2018

Impaired hematopoiesis and leukemia development in mice with a \"knock-in\" allele of U2af1(S34F)

Mutations affecting the spliceosomal protein U2AF1 are commonly found in myelodysplastic syndromes (MDS) and secondary acute myeloid leukemia (sAML). We have generated mice that carry Cre-dependent \"knock-in\" alleles of U2af1(S34F), the murine version of the most common mutant allele of U2AF1 encountered in human cancers. Cre-mediated recombination in murine hematopoietic lineages caused changes in RNA splicing, as well as multilineage cytopenia, macrocytic anemia, decreased hematopoietic stem and progenitor cells, low-grade dysplasias, and impaired transplantability, but without lifespan shortening or leukemia development. In an attempt to identify U2af1(S34F)-cooperating changes that promote leukemogenesis, we combined U2af1(S34F) with Runx1 deficiency in mice and further treated the mice with a mutagen, N-Ethyl-N-Nitrosourea (ENU). Overall, three of sixteen ENU-treated compound transgenic mice developed AML. However, AML did not arise in mice with other genotypes or without ENU treatment. Sequencing DNA from the three AMLs revealed somatic mutations homologous to those considered to be drivers of human AML, including predicted loss-or gain-of-function mutations in Tet2, Gata2, Idh1, and Ikzfl. However, the engineered U2af1(S34F) missense mutation reverted to wild type (WT) in two of the three AML cases, implying that U2af1(S34F) is dispensable, or even selected against, once leukemia is established.\n\nSIGNIFICANCE STATEMENTSomatic mutations in four splicing factor genes (U2AF1, SRSF2, SF3B1, and ZRSR2) are found in MDS and MDS-related AML, blood cancers with few effective treatment options. However, the pathophysiological effects of these mutations remain poorly characterized, in part due to the paucity of disease-relevant models. Here, we report the establishment of mouse models to study the most common U2AF1 mutation, U2af1(S34F). Production of the mutant protein specifically in the murine hematopoietic compartment disrupts hematopoiesis in ways resembling human MDS. We further identified deletion of the Runx1 gene and other known oncogenic mutations as changes that might collaborate with U2af1(S34F) to give rise to frank AML in mice.

cancer biology↗

bioRxiv2018

Time dependent response of daunorubicin on cytotoxicity, cell cycle and DNA repair in acute lymphoblastic leukaemia

Daunorubicin is commonly used in the treatment of acute lymphoblastic leukaemia (ALL). Various mechanisms of action for daunorubicin have been proposed and its action is likely to be multi-modal. The aim of this study was to explore the kinetics of double strand break (DSB) formation of three ALL cell lines following exposure to daunorubicin and to investigate the effects of daunorubicin on the cell cycle and the protein kinases involved in specific checkpoints following DNA damage and recovery periods. Three ALL cell lines CCRF-CEM and MOLT-4 derived from T lymphocytes and SUP-B15 derived from B lymphocytes were examined following 4 hours treatment with daunorubicin chemotherapy and varying recovery periods. Daunorubicin induced different degrees of toxicity in all cell lines and consistently generated reactive oxygen species. Daunorubicin was more potent at inducing DSB in MOLT-4 and CCRF-CEM cell lines while SUP-B15 cells showed delays in DSB repair and significantly more resistance to daunorubicin compared to the other cell lines as measured by {gamma}H2AX assay. Daunorubicin also causes cell cycle arrest in all three cell lines at different checkpoints at different times. These effects were not due to mutations in Ataxia-telangiectasia mutated (ATM) as sequencing revealed none in any of the three cell lines. However, p53 was phosphorylated at serine 15 only in CCRF-CEM and MOLT-4 but not in SUP-B15 cells. The lack of active p53 may be correlated to the increase of SOD2 in SUP-B15 cells. The delay in DSB repair and lower sensitivity to daunorubicin seen in the B lymphocyte derived SUP-B15 cells could be due to loss of function of p53 thus causing variations in the DNA repair pathways.

cancer biology↗

bioRxiv2018

IQGAP3 Overexpression Correlates with Poor Prognosis and Radiation Therapy Resistance in Breast Cancer

Background: IQ motif-containing GTPase activating protein 3 (IQGAP3), the latest found protein of IQGAP family, may act as a crucial factor in the process of cancer development and progression; however, its clinical value in breast cancer remains unestablished so far. Our team explored the correlation between IQGAP3 expression profile and the clinicopathological features in breast cancer. Methods: IQGAP3 levels in breast cancer cell lines and tumor tissues were detected by real-time PCR and western blotting and compared to the normal control groups. Protein expression of IQGAP3 was evaluated immunohistochemically in specimens (archived paraffin embedded) of 257 breast cancer patients. We also analyze the association between IQGAP3 expression and the clinical characters and prognosis. The relationship between IQGAP3 expression and sensitivity to radiation therapy was determined by subgroup analysis. Results: There was significant upregulation of IQGAP3 in breast cancer cell lines and human tumor tissues at both the mRNA and protein level compared to the normal ones. In addition, 110/257 (42.8%) of archived paraffin embedded breast cancer specimens had high protein expression of IQGAP3. High expression of IQGAP3 was significantly related to clinical stage (P=0.001), T category (P=0.002), N category (P=0.001), locoregional recurrence(P=0.002), distant metastasis (P=0.001), and vital status (P=0.001). Univariate and multivariate statistical analysis showed that IQGAP3 was an independent prognostic factor of the whole cohort breast cancer patients (P=0.003, P=0.001). Subgroup analysis revealed IQGAP3 expression correlates with radiation therapy resistance and was also an independent predictor for radiation therapy outcome. Conclusions: Our findings suggest that high IQGAP3 expression predicts poor prognosis and radiation therapy resistance in breast cancer. In addition, IQGAP3 may be a reliable novel biomarker to provide personalized prognostication and identify patients who can profit from more aggressive RT regimen for improving the survival of breast cancer patients.

cancer biology↗

bioRxiv2018

Prognostic Relevance of CCDC88C (Daple) Transcripts in the Peripheral Blood of Patients with Malignant Melanoma

A loss of balance between G-protein activation and deactivation has been implicated in the initiation of melanomas, and non-canonical Wnt signaling via the Wnt5A/Frizzled (FZD) pathway has been shown to be critical for the switch to an invasive phenotype. Daple [CCDC88C gene], a cytosolic guanine nucleotide exchange factor (GEF) which enhances non-canonical Wnt5A/FZD signaling via activation of trimeric G protein, Gai has been shown to serve opposing roles-- as an inducer of EMT and invasiveness and a potent tumor suppressor -- via two isoforms, V1 (full-length) and V2, respectively. Here we report that the relative abundance of these isoforms in the peripheral circulation, presumably largely from circulating tumor cells (CTCs), is a prognostic marker of cutaneous melanomas. Expression of V1 is increased in both the early and late clinical stages (p<0.001, p=0.002, respectively); V2 is decreased exclusively in the late clinical stage (p=0.011). The two isoforms have opposing prognostic effects: high expression of V2 increases progression-free survival (PFS; p = 0.02), whereas high expression of V1 decreases PFS (p=0.013). Furthermore, these effects are additive, in that melanoma patients with a low V2-high V1 signature carry the highest risk of metastatic disease. We conclude that detection of Daple transcripts in the peripheral blood (i.e., liquid biopsies) of patients with melanoma may serve as a prognostic marker and an effective strategy for non-invasive long-term follow-up of patients with melanoma.

cancer biology↗

bioRxiv2018

G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma

Mutational inactivation of ATRX (-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

cancer biology↗

bioRxiv2018

Dynamics and predicted drug response of a gene network linking dedifferentiation with beta-catenin dysfunction in hepatocellular carcinoma

Alterations of individual genes variably affect development of hepatocellular carcinoma (HCC), prompting the need to characterize the function of tumor-promoting genes in the context of gene regulatory networks (GRN). Here, we identify a GRN which functionally links LIN28B-dependent dedifferentiation with dysfunction of CTNNB1 ({beta}-CATENIN). LIN28B and CTNNB1 form a functional GRN with SMARCA4 (BRG1), Let-7b, SOX9, TP53 and MYC. GRN activity is detected in HCC and gastrointestinal cancers; it negatively correlates with HCC prognosis and contributes to a transcriptomic profile typical of the proliferative class of HCC. Using data from The Cancer Genome Atlas and from transcriptomic, transfection and mouse transgenic experiments, we generated and validated a quantitative mathematical model of the GRN. The model predicts how the expression of GRN components changes when the expression of another GRN member varies or is inhibited by a pharmacological drug. The dynamics of GRN component expression reveal distinct cell states that can switch reversibly in normal condition, and irreversibly in HCC. We conclude that identification and modelling of the GRN provides insight into prognosis, mechanisms of tumor-promoting genes and response to pharmacological agents in HCC.

cancer biology↗

bioRxiv2018

E-cadherin represses anoikis resistance in sarcomas through both signaling and mechanical mechanisms

E-cadherin, an epithelial-specific cell-cell adhesion molecule, plays multiple roles in maintaining adherens junctions, regulating migration and invasion, and mediating intracellular signaling. Downregulation of E-cadherin is a hallmark of epithelial-mesenchymal transition (EMT) and correlates with poor prognosis in multiple carcinomas. Conversely, upregulation of E-cadherin is prognostic for improved survival in sarcomas. Yet, despite the prognostic benefit of E-cadherin expression in sarcoma, the mechanistic significance of E-cadherin in sarcomas remains poorly understood. Here, by combining mathematical models with wet-bench experiments, we identify the core regulatory networks mediated by E-cadherin in sarcomas, and decipher their functional consequences. Unlike in carcinomas, E-cadherin overexpression in sarcomas does not induce a mesenchymal-epithelial transition (MET). However, E-cadherin acts to reduce both anchorage-independent growth and spheroid formation of sarcoma cells. Ectopic E-cadherin expression acts to downregulate phosphorylated CREB (p-CREB) and the transcription factor, TBX2, to inhibit anoikis resistance. RNAi-mediated knockdown of TBX2 phenocopies the effect of E-cadherin on p-CREB levels and restores anoikis sensitivity to sarcoma cells. Beyond its signaling role, E-cadherin expression in sarcoma cells can also strengthen cell-cell adhesion and restricts spheroid growth through mechanical action. Together, our results demonstrate that E-cadherin inhibits sarcoma aggressiveness by inducing anoikis and restricting colony growth.

cancer biology↗

bioRxiv2018

A stochastic model of metastatic bottleneck predicts patient outcome and therapy response

Metastases are responsible for 90% of cancer-related deaths. Initiation of metastases, where newly seeded tumor cells expand into colonies, presents a tremendous bottleneck to metastasis formation. Despite its clinical importance, our understanding of this process is very limited. Here, we propose a simple stochastic model assuming that the initiating metastatic cells proliferate faster when surrounded by more of their kind. The model quantifies the severity of metastatic bottleneck as the probability that the seeded colony survives. Based on this model, we derive how metastasis occurrence depends on primary tumor size and affects patient outcome. Our predictions agree with epidemiological data for thirteen cancer types. The model predicts that impact of treatment decisions depends both on the primary tumor size and on the severity of the metastatic bottleneck, and that medical interventions that tighten the bottleneck would be much more efficient than therapies that decrease overall tumor burden, such as chemotherapy.

cancer biology↗