[Show abstract][Hide abstract] ABSTRACT: p21-activated kinases (Paks) have been shown to regulate cytoskeleton rearrangements, cell proliferation, attachment and migration in a variety of cellular contexts, including endothelial cells. However, the role of endothelial Pak in embryo development has not been reported, and currently there is no consensus on the endothelial function of individual Pak isoforms, in particular Pak2, the main Pak isoform expressed in endothelial cells. In this work, we employ genetic and molecular studies that show that Pak2, but not Pak1, is a critical mediator of development and maintenance of endothelial cell function. Endothelial depletion of Pak2 leads to early embryo lethality due to flawed blood vessel formation in the embryo body and the yolk sac. In adult endothelial cells, Pak2 depletion leads to severe apoptosis and acute angiogenesis defects, and in adult mice endothelial Pak2 deletion leads to increased vascular permeability. Furthermore, ubiquitous Pak2 deletion is lethal in adult mice. We show that many of these defects are mediated through a newly unveiled Pak2/Bmk1 pathway. Our results demonstrate that endothelial Pak2 is essential during embryogenesis and also for adult blood vessel maintenance, and also pinpoint the Bmk1/Erk5 pathway as a critical mediator of endothelial Pak2 signaling.
Molecular and Cellular Biology 09/2015; DOI:10.1128/MCB.00630-15 · 4.78 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Breast cancer, the most common cause of cancer-related deaths worldwide among women, is a molecularly and clinically heterogeneous disease. Extensive genetic and epigenetic profiling of breast tumors has recently revealed novel putative driver genes, including p21-activated kinase 1 (PAK1). PAK1 is a serine/threonine kinase downstream of small GTP-binding proteins, Rac1 and Cdc42, and is an integral component of growth factor signaling networks and cellular functions fundamental to tumorigenesis.
PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n = 980 and 1108). A novel small molecule inhibitor, FRAX1036, and RNA interference were used to examine PAK1 loss of function and combination with docetaxel in vitro. Mechanism of action for the therapeutic combination, both cellular and molecular, was assessed via time-lapse microscopy and immunoblotting.
We demonstrate that focal genomic amplification and over-expression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P = 1.29 x 10(-4) and P = 0.015, respectively). Given the role for PAK1 in regulating cytoskeletal organization, we hypothesized that combination of PAK1 inhibition with taxane treatment could be combined to further interfere with microtubule dynamics and cell survival. Consistent with this, administration of docetaxel with either a novel small molecule inhibitor of group I PAKs, FRAX1036, or PAK1 small interfering RNA oligonucleotides dramatically altered signaling to cytoskeletal-associated proteins, such as stathmin, and induced microtubule disorganization and cellular apoptosis. Live-cell imaging revealed that the duration of mitotic arrest mediated by docetaxel was significantly reduced in the presence of FRAX1036, and this was associated with increased kinetics of apoptosis.
Taken together, these findings further support PAK1 as a potential target in breast cancer and suggest combination with taxanes as a viable strategy to increase anti-tumor efficacy.
Breast cancer research: BCR 04/2015; 17(1):59. DOI:10.1186/s13058-015-0564-5 · 5.49 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Soft tissue sarcomas are a heterogeneous group of tumors with many different subtypes. In 2014 an estimated 12,020 newly diagnosed cases and 4,740 soft tissue sarcoma related deaths can be expected in the United States. Many soft tissue sarcomas are associated with poor prognosis and therapeutic options are often limited. The evolution of precision medicine has not yet fully reached the clinical treatment of sarcomas since therapeutically tractable genetic changes have not been comprehensively studied so far. We analyzed a total of 484 adult-type malignant mesenchymal tumors by MET fluorescence in situ hybridization and MET and hepatocyte growth factor immunohistochemistry. Eleven different entities were included, among them the most common and clinically relevant subtypes and tumors with specific translocations or complex genetic changes. MET protein expression was observed in 2.6% of the cases, all of which were either undifferentiated pleomorphic sarcomas or angiosarcomas, showing positivity rates of 14% and 17%, respectively. 6% of the tumors showed hepatocyte growth factor overexpression, mainly seen in undifferentiated pleomorphic sarcomas and angiosarcomas, but also in clear cell sarcomas, malignant peripheral nerve sheath tumors, leiomyosarcomas and gastrointestinal stromal tumors. MET and hepatocyte growth factor overexpression were significantly correlated and may suggest an autocrine activation in these tumors. MET FISH amplification and copy number gain were present in 4% of the tumors (15/413). Two samples, both undifferentiated pleomorphic sarcomas, fulfilled the criteria for high level amplification of MET, one undifferentiated pleomorphic sarcoma reached an intermediate level copy number gain, and 12 samples of different subtypes were categorized as low level copy number gains for MET. Our findings indicate that angiosarcomas and undifferentiated pleomorphic sarcomas rather than other.
PLoS ONE 04/2015; 10(4):e0120079. DOI:10.1371/journal.pone.0120079 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Hepatocyte growth factor (HGF) and vascular endothelial growth factor (VEGF) drive cancer through their respective receptors, MET and VEGF receptor 2 (VEGFR2). VEGFR2 inhibits MET by promoting MET dephosphorylation. However, whether MET conversely regulates VEGFR2 remains unknown. Here we show that MET suppresses VEGFR2 protein by inducing its endoplasmic-reticulum-associated degradation (ERAD), via intracrine VEGF action. HGF-MET signaling in epithelial cancer cells promoted VEGF biosynthesis through PI3-kinase. In turn, VEGF and VEGFR2 associated within the ER, activating inositol-requiring enzyme 1α, and thereby facilitating ERAD-mediated depletion of VEGFR2. MET disruption upregulated VEGFR2, inducing compensatory tumor growth via VEGFR2 and MEK. However, concurrent disruption of MET and either VEGF or MEK circumvented this, enabling more profound tumor inhibition. Our findings uncover unique cross-regulation between MET and VEGFR2—two RTKs that play important roles in tumor malignancy. Furthermore, these results suggest rational combinatorial strategies for targeting RTK signaling pathways more effectively, which has potentially important implications for cancer therapy.
[Show abstract][Hide abstract] ABSTRACT: Antibody drug conjugates (ADCs), in which cytotoxic drugs are linked to antibodies targeting antigens on tumor cells, represent promising novel agents for the treatment of malignant lymphomas. Pinatuzumab vedotin is an anti-CD22 ADC and polatuzumab vedotin an anti-CD79B ADC that are both linked to the microtubule-disrupting agent monomethyl auristatin E (MMAE). In the present study, we analyzed the activity of these agents in different molecular subtypes of diffuse large B-cell lymphoma (DLBCL) both in vitro and in early clinical trials. Both anti-CD22-MMAE and anti-CD79B-MMAE were highly active and induced cell death in the vast majority of activated B-cell-like (ABC) and germinal center B-cell-like (GCB) DLBCL cell lines. Similarly, both agents induced cytotoxicity in models with and without mutations in the signaling molecule CD79B. In line with these observations, relapsed and refractory DLBCL patients of both subtypes responded to these agents. Importantly, a strong correlation between CD22 and CD79B expression in vitro and in vivo was not detectable, indicating that patients should not be excluded from anti-CD22-MMAE or anti-CD79B-MMAE treatment due to low target expression. In summary, these studies suggest that pinatuzumab vedotin and polatuzumab vedotin are active agents for the treatment of patients with different subtypes of DLBCL.Leukemia accepted article preview online, 24 February 2015. doi:10.1038/leu.2015.48.
[Show abstract][Hide abstract] ABSTRACT: Pancreatic adenocarcinoma (PDAC) is a major unmet medical need and a deeper understanding of molecular drivers is needed to advance therapeutic options for patients. We report here that p21-activated kinase 1 (PAK1) is a central node in PDAC cells downstream of multiple growth factor signaling pathways, including hepatocyte growth factor (HGF) and MET receptor tyrosine kinase. PAK1 inhibition blocks signaling to cytoskeletal effectors and tumor cell motility driven by HGF/MET. MET antagonists, such as onartuzumab and crizotinib, are currently in clinical development. Given that even highly effective therapies have resistance mechanisms, we show that combination with PAK1 inhibition overcomes potential resistance mechanisms mediated either by activation of parallel growth factor pathways or direct amplification of PAK1. Inhibition of PAK1 attenuated in vivo tumor growth and metastasis in a model of pancreatic adenocarcinoma. In human tissues, PAK1 is highly expressed in a proportion of PDAC (33% IHC score 2 or 3; n = 304) and its expression is significantly associated with MET positivity (p < 0.0001) and linked to a widespread metastatic pattern in patients (p = 0.067). Taken together, our results provide evidence for a functional role of MET/PAK1 signaling in pancreatic adenocarcinoma and support further characterization of therapeutic inhibitors in this indication.
The Journal of Pathology 12/2014; 234(4). DOI:10.1002/path.4412 · 7.43 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The development of human cancer is a multistep process characterized by the accumulation of genetic and epigenetic alterations that drive or reflect tumour progression. These changes distinguish cancer cells from their normal counterparts, allowing tumours to be recognized as foreign by the immune system. However, tumours are rarely rejected spontaneously, reflecting their ability to maintain an immunosuppressive microenvironment. Programmed death-ligand 1 (PD-L1; also called B7-H1 or CD274), which is expressed on many cancer and immune cells, plays an important part in blocking the 'cancer immunity cycle' by binding programmed death-1 (PD-1) and B7.1 (CD80), both of which are negative regulators of T-lymphocyte activation. Binding of PD-L1 to its receptors suppresses T-cell migration, proliferation and secretion of cytotoxic mediators, and restricts tumour cell killing. The PD-L1-PD-1 axis protects the host from overactive T-effector cells not only in cancer but also during microbial infections. Blocking PD-L1 should therefore enhance anticancer immunity, but little is known about predictive factors of efficacy. This study was designed to evaluate the safety, activity and biomarkers of PD-L1 inhibition using the engineered humanized antibody MPDL3280A. Here we show that across multiple cancer types, responses (as evaluated by Response Evaluation Criteria in Solid Tumours, version 1.1) were observed in patients with tumours expressing high levels of PD-L1, especially when PD-L1 was expressed by tumour-infiltrating immune cells. Furthermore, responses were associated with T-helper type 1 (TH1) gene expression, CTLA4 expression and the absence of fractalkine (CX3CL1) in baseline tumour specimens. Together, these data suggest that MPDL3280A is most effective in patients in which pre-existing immunity is suppressed by PD-L1, and is re-invigorated on antibody treatment.
[Show abstract][Hide abstract] ABSTRACT: Breast cancers are categorized into three subtypes based on protein expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor-2 (HER2/ERBB2). Patients enroll onto experimental clinical trials based on ER, PR, and HER2 status and, as receptor status is prognostic and defines treatment regimens, central receptor confirmation is critical for interpreting results from these trials. Patients enrolling onto experimental clinical trials in the metastatic setting often have limited available archival tissue that might better be used for comprehensive molecular profiling rather than slide-intensive reconfirmation of receptor status. We developed a Random Forests-based algorithm using a training set of 158 samples with centrally confirmed IHC status, and subsequently validated this algorithm on multiple test sets with known, locally determined IHC status. We observed a strong correlation between target mRNA expression and IHC assays for HER2 and ER, achieving an overall accuracy of 97 and 96 %, respectively. For determining PR status, which had the highest discordance between central and local IHC, incorporation of expression of co-regulated genes in a multivariate approach added predictive value, outperforming the single, target gene approach by a 10 % margin in overall accuracy. Our results suggest that multiplexed qRT-PCR profiling of ESR1, PGR, and ERBB2 mRNA, along with several other subtype associated genes, can effectively confirm breast cancer subtype, thereby conserving tumor sections and enabling additional biomarker data to be obtained from patients enrolled onto experimental clinical trials.
Electronic supplementary material
The online version of this article (doi:10.1007/s10549-014-3163-8) contains supplementary material, which is available to authorized users.
Breast Cancer Research and Treatment 10/2014; 148(2). DOI:10.1007/s10549-014-3163-8 · 3.94 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Mesothelin (MSLN) is an attractive target for antibody-drug conjugate therapy because it is highly expressed in various epithelial cancers, with normal expression limited to non-dividing mesothelia. We generated novel anti-mesothelin antibodies and conjugated an internalizing one (7D9) to the microtubule-disrupting drugs monomethyl auristatin E (MMAE) and MMAF, finding the most effective to be MMAE with a lysosomal protease-cleavable valine-citrulline linker. The humanized (h7D9.v3) version, alphaMSLN-MMAE, specifically targeted mesothelin-expressing cells and inhibited their proliferation with an IC50 of 0.3nM. Because the anti-tumor activity of an anti-mesothelin immunotoxin (SS1P) in transfected mesothelin models did not translate to the clinic, we carefully selected in vivo efficacy models endogenously expressing clinically relevant levels of mesothelin, after scoring mesothelin levels in ovarian, pancreatic and mesothelioma tumors by immunohistochemistry. We found that endogenous mesothelin in cancer cells is upregulated in vivo and identified two suitable xenograft models for each of these three indications. A single dose of alphaMSLN-MMAE profoundly inhibited or regressed tumor growth in a dose-dependent manner in all six models, including two patient-derived tumor xenografts. The robust and durable efficacy of alphaMSLN-MMAE in preclinical models of ovarian, mesothelioma and pancreatic cancers justifies the ongoing phase I clinical trial.
Molecular Cancer Therapeutics 09/2014; 13(11). DOI:10.1158/1535-7163.MCT-14-0487-T · 5.68 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Molecularly-targeted drug therapies have revolutionized cancer treatment, however, resistance remains a major limitation to their overall efficacy. Epithelial to mesenchymal transition (EMT) has been linked to acquired resistance to tyrosine kinase inhibitors (TKIs), independent of mutational resistance mechanisms. AXL is a receptor tyrosine kinase (RTK) associated with EMT that has been implicated in drug resistance, and has emerged as a candidate therapeutic target. Across 643 human cancer cell lines that were analyzed, elevated AXL was strongly associated with a mesenchymal phenotype, particularly in triple negative breast cancer and non-small cell lung cancer. In an unbiased screen of small molecule inhibitors of cancer-relevant processes, we discovered that AXL inhibition was specifically synergistic with anti-mitotic agents in killing cancer cells that had undergone EMT and demonstrated associated TKI resistance. However, we did not find that AXL inhibition alone could overcome acquired resistance to EGFR TKIs in the EMT setting, as previously reported. These findings reveal a novel co-treatment strategy for tumors displaying mesenchymal features that otherwise render them treatment-refractory.
Cancer Research 08/2014; 74(20). DOI:10.1158/0008-5472.CAN-14-1009 · 9.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Aberrant activation of the HGF/MET signaling axis has been strongly implicated in the malignant transformation and progression of gastroesophageal cancer (GEC). MET receptor overexpression in tumor samples from GEC patients has been consistently correlated with an aggressive metastatic phenotype and poor prognosis. In preclinical GEC models, abrogation of HGF/MET signaling has been shown to induce tumor regression as well as inhibition of metastatic dissemination. Promising clinical results in patient subsets in which MET is overexpressed have spurned several randomized studies of HGF/MET-directed agents, including two pivotal global Phase III trials. Available data highlight the need for predictive biomarkers in order to select patients most likely to benefit from HGF/MET inhibition. In this review, we discuss the current knowledge of mechanisms of MET activation in GEC, the current status of the clinical evaluation of MET-targeted therapies in GEC, characteristics of ongoing randomized GEC trials and the associated efforts to identify and validate biomarkers. We also discuss the considerations and challenges for HGF/MET inhibitor drug development in the GEC setting.