Targeting DNA Damage Response and Replication Stress in Pancreatic Cancer.

BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.

Precision Oncology in Surgery: Patient Selection for Operable Pancreatic Cancer.

OBJECTIVE: We aimed to define preoperative clinical and molecular characteristics that would allow better patient selection for operative resection. BACKGROUND: Although we use molecular selection methods for systemic targeted therapies, these principles are not applied to surgical oncology. Improving patient selection is of vital importance for the operative treatment of pancreatic cancer (pancreatic ductal adenocarcinoma). Although surgery is the only chance of long-term survival, 80% still succumb to the disease and approximately 30% die within 1 year, often sooner than those that have unresected local disease. METHOD: In 3 independent pancreatic ductal adenocarcinoma cohorts (total participants = 1184) the relationship between aberrant expression of prometastatic proteins S100A2 and S100A4 and survival was assessed. A preoperative nomogram based on clinical variables available before surgery and expression of these proteins was constructed and compared to traditional measures, and a postoperative nomogram. RESULTS: High expression of either S100A2 or S100A4 was independent poor prognostic factors in a training cohort of 518 participants. These results were validated in 2 independent patient cohorts (Glasgow, n = 198; Germany, n = 468). Aberrant biomarker expression stratified the cohorts into 3 distinct prognostic groups. A preoperative nomogram incorporating S100A2 and S100A4 expression predicted survival and nomograms derived using postoperative clinicopathological variables. CONCLUSIONS: Of those patients with a poor preoperative nomogram score, approximately 50% of patients died within a year of resection. Nomograms have the potential to improve selection for surgery and neoadjuvant therapy, avoiding surgery in aggressive disease, and justifying more extensive resections in biologically favorable disease.

ZNF300P1 encodes a lincRNA that regulates cell polarity and is epigenetically silenced in type II epithelial ovarian cancer.

BACKGROUND: We previously identified that the CpG island-associated promoter of the novel lincRNA ZNF300P1 (also known as LOC134466) is frequently hypermethylated and silenced in ovarian cancer tissues. However, the function of ZNF300P1 was unknown. In this report we demonstrate that ZNF300P1 is involved in the regulation of key cell cycle and cell motility networks in human ovarian surface epithelial cells, and may play a role in promoting metastasis in ovarian cancer cells. METHODS: We applied methylated DNA immunoprecipitation on whole genome promoter tiling arrays and Sequenom assays to examine methylation status of ZNF300P1 in multiple ovarian cancer cell lines, as well as in normal ovarian and ovarian tumor tissues. Transcript profiling was used to investigate the effects of ZNF300P1 suppression in ovarian cancer cells. We utilized siRNA knockdown in normal ovarian surface epithelial cells and performed cellular proliferation, migration and adhesion assays to validate and explore the profiling results. RESULTS: We demonstrate that ZNF300P1 is methylated in multiple ovarian cancer cell lines. Loss of ZNF300P1 results in decreased cell proliferation and colony formation. In addition, knockdown of the ZNF300P1 transcript results in aberrant and less persistent migration in wound healing assays due to a loss of cellular polarity. Using an ex vivo peritoneal adhesion assay, we also reveal a role for ZNF300P1 in the attachment of ovarian cancer cells to peritoneal membranes, indicating a potential function of ZNF300P1 expression in metastasis of ovarian cancer cells to sites within the peritoneal cavity. CONCLUSION: Our findings further support ZNF300P1 as frequently methylated in ovarian cancer and reveal a novel function for ZNF300P1 lincRNA expression in regulating cell polarity, motility, and adhesion and loss of expression may contribute to the metastatic potential of ovarian cancer cells.

Pharmacologic Targeting of TFIIH Suppresses KRAS-Mutant Pancreatic Ductal Adenocarcinoma and Synergizes with TRAIL.

Pancreatic ductal adenocarcinoma (PDAC) typically presents as metastatic disease at diagnosis and remains refractory to treatment. Next-generation sequencing efforts have described the genomic landscape, classified molecular subtypes, and confirmed frequent alterations in major driver genes, with coexistent alterations in KRAS and TP53 correlating with the highest metastatic burden and poorest outcomes. However, translating this information to guide therapy remains a challenge. By integrating genomic analysis with an arrayed RNAi druggable genome screen and drug profiling of a KRAS/TP53 mutant PDAC cell line derived from a patient-derived xenograft (PDCL), we identified numerous targetable vulnerabilities that reveal both known and novel functional aspects of pancreatic cancer biology. A dependence on the general transcription and DNA repair factor TFIIH complex, particularly the XPB subunit and the CAK complex (CDK7/CyclinH/MAT1), was identified and further validated utilizing a panel of genomically subtyped KRAS mutant PDCLs. TFIIH function was inhibited with a covalent inhibitor of CDK7/12/13 (THZ1), a CDK7/CDK9 kinase inhibitor (SNS-032), and a covalent inhibitor of XPB (triptolide), which led to disruption of the protein stability of the RNA polymerase II subunit RPB1. Loss of RPB1 following TFIIH inhibition led to downregulation of key transcriptional effectors of KRAS-mutant signaling and negative regulators of apoptosis, including MCL1, XIAP, and CFLAR, initiating caspase-8 dependent apoptosis. All three drugs exhibited synergy in combination with a multivalent TRAIL, effectively reinforcing mitochondrial-mediated apoptosis. These findings present a novel combination therapy, with direct translational implications for current clinical trials on metastatic pancreatic cancer patients. Significance: This study utilizes functional genetic and pharmacological profiling of KRAS-mutant pancreatic adenocarcinoma to identify therapeutic strategies and finds that TFIIH inhibition synergizes with TRAIL to induce apoptosis in KRAS-driven pancreatic cancer.

VEGFR1 (Flt1) regulates Rab4 recycling to control fibronectin polymerization and endothelial vessel branching.

The cell's main receptor for VEGF, VEGFR2 (Kdr) is one of the most important positive regulators of new blood vessel growth and its downstream signalling is well characterized. By contrast, VEGFR1 (Flt1) and the mechanisms by which this VEGF receptor promotes branching morphogenesis in angiogenesis remain relatively unclear.Here we report that engagement of VEGFR1 activates a Rab4A-dependent pathway that transports alphavbeta3 Integrin from early endosomes to the plasma membrane, and that this is required for VEGF-driven fibronectin polymerization in endothelial cells. Furthermore, VEGFR1 acts to promote endothelial tubule branching in an organotypic model of angiogenesis via a mechanism that requires Rab4A and alphavbeta3 Integrin. We conclude that a recycling pathway regulated by Rab4A is a critical effector of VEGFR1 during branching morphogenesis of the vasculature.

HNF4A and GATA6 Loss Reveals Therapeutically Actionable Subtypes in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) can be divided into transcriptomic subtypes with two broad lineages referred to as classical (pancreatic) and squamous. We find that these two subtypes are driven by distinct metabolic phenotypes. Loss of genes that drive endodermal lineage specification, HNF4A and GATA6, switch metabolic profiles from classical (pancreatic) to predominantly squamous, with glycogen synthase kinase 3 beta (GSK3ß) a key regulator of glycolysis. Pharmacological inhibition of GSK3ß results in selective sensitivity in the squamous subtype; however, a subset of these squamous patient-derived cell lines (PDCLs) acquires rapid drug tolerance. Using chromatin accessibility maps, we demonstrate that the squamous subtype can be further classified using chromatin accessibility to predict responsiveness and tolerance to GSK3ß inhibitors. Our findings demonstrate that distinct patterns of chromatin accessibility can be used to identify patient subgroups that are indistinguishable by gene expression profiles, highlighting the utility of chromatin-based biomarkers for patient selection in the treatment of PDAC.

hnRNP A2, a potential ssDNA/RNA molecular adapter at the telomere.

The heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multi-tasking protein that acts in the cytoplasm and nucleus. We have explored the possibility that this protein is associated with telomeres and participates in their maintenance. Rat brain hnRNP A2 was shown to have two nucleic acid binding sites. In the presence of heparin one site binds single-stranded oligodeoxyribonucleotides irrespective of sequence but not the corresponding oligoribonucleotides. Both the hnRNP A2-binding cis-acting element for the cytoplasmic RNA trafficking element, A2RE, and the ssDNA telomere repeat match a consensus sequence for binding to a second sequence-specific site identified by mutational analysis. hnRNP A2 protected the telomeric repeat sequence, but not the complementary sequence, against DNase digestion: the glycine-rich domain was found to be necessary, but not sufficient, for protection. The N-terminal RRM (RNA recognition motif) and tandem RRMs of hnRNP A2 also bind the single-stranded, template-containing segment of telomerase RNA. hnRNP A2 colocalizes with telomeric chromatin in the subset of PML bodies that are a hallmark of ALT cells, reinforcing the evidence for hnRNPs having a role in telomere maintenance. Our results support a model in which hnRNP A2 acts as a molecular adapter between single-stranded telomeric repeats, or telomerase RNA, and another segment of ssDNA.

The Therapeutic Potential of Targeting the HGF/cMET Axis in Ovarian Cancer.

Survival rates for ovarian cancer have remained relatively stable for the past 2 decades despite advances in surgical techniques and cytotoxic chemotherapeutics, indicating a requirement for better therapies. One pathway currently proposed for targeting is the HGF/cMET pathway. Upregulated in a number of tumour types, cMET is a tyrosine kinase receptor expressed on epithelial cells. In ovarian cancer, it has been identified as highly expressed in the four major subtypes, with expression estimates ranging from 11 to 68 % of cases. HGF, the only known ligand for cMET, is found at high levels in both serum and ascites in women with ovarian cancer, and is proposed to induce both migration and metastasis. However, clinically validated biomarkers are not yet available for either HGF or cMET, preventing a clear understanding of the true rate of overexpression, or its correlation with prognosis. Despite this, a number of agents against HGF and cMET are currently being investigated in clinical trials for multiple tumour types, including ovarian. However, a lack of patient selection, biomarker usage, and post hoc analysis correlating response with expression has resulted in the majority of these trials showing little beneficial effect from these agents, indicating that additional research is required to determine their usefulness in patients with ovarian cancer.

INC280, an orally available small molecule inhibitor of c-MET, reduces migration and adhesion in ovarian cancer cell models.

5-year survival rates for ovarian cancer are approximately 40%, and for women diagnosed at late stage (the majority), just 27%. This indicates a dire need for new treatments to improve survival rates. Recent molecular characterization has greatly improved our understanding of the disease and allowed the identification of potential new targets. One such pathway of interest is the HGF/c-MET axis. Activation of the HGF/c-MET axis has been demonstrated in certain ovarian tumours, and been found to be associated with decreased overall survival, suggesting its potential as a therapeutic target. The objective of this study was to determine the efficacy of a novel, highly potent, orally-bioavailable c-MET inhibitor, INC280, in blocking cell phenotypes important in ovarian cancer metastasis. Using in vitro and ex vivo models, we demonstrate that INC280 inhibits HGF-induced c-MET, and reduces downstream signalling. HGF-stimulated chemotactic and random migration are decreased by INC280 treatment, to levels seen in non-stimulated cells. Additionally, HGF-induced adhesion of cancer cells to peritoneal tissue is significantly decreased by INC280 treatment. Overall, these data indicate that INC280 inhibits many cell behaviours that promote ovarian cancer metastasis, and merits further investigation as a therapeutic candidate in the treatment of patients with ovarian cancer.

Connective tissue growth factor as a novel therapeutic target in high grade serous ovarian cancer.

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.

ß1 integrin deletion enhances progression of prostate cancer in the TRAMP mouse model.

ß1 integrin regulates the response of both normal and cancer cells to their local environment. Although mis-localised in prostate cancer, the role ß1 integrin plays in prostate development and carcinogenesis remains unknown. To assess the role of ß1 integrin in vivo, we conditionally deleted ß1 integrin from prostate epithelium and subsequently crossed these mice to the TRAMP prostate carcinogenesis model. Deletion of ß1 integrin following castration and subsequent androgen supplementation resulted in an expansion of the p63-positive basal cell population and decreased differentiation. Consistent with these findings, deletion of ß1 integrin in TRAMP mice decreased animal survival, decreased retention of normal prostate morphology, increased the percentage of tissue with poorly differentiated carcinoma, and increased cell proliferation. This study demonstrates that ß1 integrin regulates several aspects of normal prostate development and in contrast to its role in several other tissues, its loss is associated with increased rates of prostate tumour progression.

c-Myc regulates RNA splicing of the A-Raf kinase and its activation of the ERK pathway.

A-Raf kinase can inhibit apoptosis by binding to the proapoptotic mammalian sterile 20-like kinase (MST2). This function relies on expression of hnRNP H, which ensures the correct splicing of a-raf mRNA needed to produce full-length A-Raf protein. Here, we showed that expression of hnRNP H and production of full-length A-Raf is positively controlled by c-Myc. Low c-Myc reduces hnRNP H expression and switches a-raf splicing to produce A-Raf(short), a truncated protein. Importantly, A-Raf(short) fails to regulate MST2 but retains the Ras-binding domain such that it functions as a dominant negative mutant suppressing Ras activation and transformation. Human colon and head and neck cancers exhibit high hnRNP H and high c-Myc levels resulting in enhanced A-Raf expression and reduced expression of A-Raf(short). Conversely, in normal cells and tissues in which c-Myc and hnRNP H are low, A-Raf(short) suppresses extracellular signal regulated kinase activation such that it may act as a safeguard against oncogenic transformation. Our findings offered a new paradigm to understand how c-Myc coordinates diverse cell functions by directly affecting alternate splicing of key signaling components.

FAK deletion promotes p53-mediated induction of p21, DNA-damage responses and radio-resistance in advanced squamous cancer cells.

Focal adhesion kinase (FAK) is a cytoplasmic tyrosine kinase that is elevated in a variety of human cancers. While FAK is implicated in many cellular processes that are perturbed in cancer, including proliferation, actin and adhesion dynamics, polarisation and invasion, there is only some limited information regarding the role of FAK in radiation survival. We have evaluated whether FAK is a general radio-sensitising target, as has been suggested by previous reports. We used a clean genetic system in which FAK was deleted from mouse squamous cell carcinoma (SCC) cells (FAK -/-), and reconstituted with exogenous FAK wild type (wt). Surprisingly, the absence of FAK was associated with increased radio-resistance in advanced SCC cells. FAK re-expression inhibited p53-mediated transcriptional up-regulation of p21, and a sub-set of other p53 target genes involved in DNA repair, after treatment with ionizing radiation. Moreover, p21 depletion promoted radio-sensitisation, implying that FAK-mediated inhibition of p21 induction is responsible for the relative radio-sensitivity of FAK-proficient SCC cells. Our work adds to a growing body of evidence that there is a close functional relationship between integrin/FAK signalling and the p53/p21 pathway, but demonstrates that FAK's role in survival after stress is context-dependent, at least in cancer cells. We suggest that there should be caution when considering inhibiting FAK in combination with radiation, as this may not always be clinically advantageous.

hnRNP A2 regulates alternative mRNA splicing of TP53INP2 to control invasive cell migration.

Largely owing to widespread deployment of microarray analysis, many of the transcriptional events associated with invasive cell migration are becoming clear. However, the transcriptional drives to invasive migration are likely modified by alternative splicing of pre-mRNAs to produce functionally distinct patterns of protein expression. Heterogenous nuclear ribonucleoprotein (hnRNP A2) is a known regulator of alternative splicing that is upregulated in a number of invasive cancer types. Here, we report that although siRNA of hnRNP A2 had little influence on the ability of cells to migrate on plastic surfaces, the splicing regulator was clearly required for cells to move effectively on three-dimensional matrices and to invade into plugs of extracellular matrix proteins. We used exon-tiling microarrays to determine that hnRNP A2 controlled approximately six individual splicing events in a three-dimensional matrix-dependent fashion, one of which influenced invasive migration. Here, we show that alternative splicing of an exon in the 5' untranslated region of a gene termed TP53INP2 is a key event downstream of hnRNP A2 that is necessary for cells to invade the extracellular matrix. Furthermore, we report that the consequences of altered TP53INP2 splicing on invasion are likely mediated via alterations in Golgi complex integrity during migration on three-dimensional matrices.

Molecular recognition of an RNA trafficking element by heterogeneous nuclear ribonucleoprotein A2.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 is a multitasking protein involved in RNA packaging, alternative splicing of pre-mRNA, telomere maintenance, cytoplasmic RNA trafficking, and translation. It binds short segments of single-stranded nucleic acids, including the A2RE11 RNA element that is necessary and sufficient for cytoplasmic transport of a subset of mRNAs in oligodendrocytes and neurons. We have explored the structures of hnRNP A2, its RNA recognition motifs (RRMs) and Gly-rich module, and the RRM complexes with A2RE11. Circular dichroism spectroscopy showed that the secondary structure of the first 189 residues of hnRNP A2 parallels that of the tandem betaalpha betabeta alphabeta RRMs of its paralogue, hnRNP A1, previously deduced from X-ray diffraction studies. The unusual GRD was shown to have substantial beta-sheet and beta-turn structure. Sedimentation equilibrium and circular dichroism results were consistent with the tandem RRM region being monomeric and supported earlier evidence for the binding of two A2RE11 oligoribonucleotides to this domain, in contrast to the protein dimer formed by the complex of hnRNP A1 with the telomeric ssDNA repeat. A three-dimensional structure for the N-terminal, two-RRM-containing segment of hnRNP A2 was derived by homology modeling. This structure was used to derive a model for the complex with A2RE11 using the previously described interaction of pairs of stacked nucleotides with aromatic residues on the RRM beta-sheet platforms, conserved in other RRM-RNA complexes, together with biochemical data and molecular dynamics-based observations of inter-RRM mobility.

Beta IV tubulin is selectively expressed by oligodendrocytes in the central nervous system.

Oligodendrocyte differentiation and myelination involve dramatic changes in cell signaling pathways, gene expression patterns, cell shape, and cytoskeletal organization. In a pilot study investigating CNS angiogenesis, oligodendrocytes were intensely labeled by antisera directed against the C-terminal of Tie-2, a 140-kDa transmembrane receptor for angiopoietin. Immunoprecipitation of rat brain proteins with Tie-2 C-terminal antisera, however, produced a single spot of approximately 55-kDa pI approximately 5 by two-dimensional (2D) electrophoresis, which was identified as beta-tubulin by mass spectrometry. Isotype-specific antibodies for beta(IV) tubulin selectively labeled oligodendrocytes. First detected in premyelinating oligodendrocytes, beta(IV) tubulin was abundant in myelinating oligodendrocyte perinuclear cytoplasm and processes extending to and along developing myelin internodes. Beta(IV) tubulin-positive MTs were diffusely distributed in oligodendrocyte perinuclear cytoplasm and not organized around the centrosome. Beta(IV) tubulin may play a role in establishing the oligodendrocyte MT network, which is essential for the transport of myelin proteins, lipids, and RNA during myelination.

Heterogeneous nuclear ribonucleoprotein A3, a novel RNA trafficking response element-binding protein.

The cis-acting response element, A2RE, which is sufficient for cytoplasmic mRNA trafficking in oligodendrocytes, binds a small group of rat brain proteins. Predominant among these is heterogeneous nuclear ribonucleoprotein (hnRNP) A2, a trans-acting factor for cytoplasmic trafficking of RNAs bearing A2RE-like sequences. We have now identified the other A2RE-binding proteins as hnRNP A1/A1(B), hnRNP B1, and four isoforms of hnRNP A3. The rat and human hnRNP A3 cDNAs have been sequenced, revealing the existence of alternatively spliced mRNAs. In Western blotting, 38-, 39-, 41-, and 41.5-kDa components were all recognized by antibodies against a peptide in the glycine-rich region of hnRNP A3, but only the 41- and 41.5-kDa bands bound antibodies to a 15-residue N-terminal peptide encoded by an alternatively spliced part of exon 1. The identities of these four proteins were verified by Edman sequencing and mass spectral analysis of tryptic fragments generated from electrophoretically separated bands. Sequence-specific binding of bacterially expressed hnRNP A3 to A2RE has been demonstrated by biosensor and UV cross-linking electrophoretic mobility shift assays. Mutational analysis and confocal microscopy data support the hypothesis that the hnRNP A3 isoforms have a role in cytoplasmic trafficking of RNA.