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High-level amplification of the protein phosphatase PPM1D (WIP1) is present in a subset of medulloblastomas (MBs) that have an expression profile consistent with active Sonic Hedgehog (SHH) signaling. We found that WIP1 overexpression increased expression of Shh target genes and cell proliferation in response to Shh stimulation in NIH3T3 and cerebellar granule neuron precursor cells in a p53-independent manner. Thus, we developed a mouse in which WIP1 is expressed in the developing brain under control of the Neurod2 promoter (ND2:WIP1). The external granule layer (EGL) in early postnatal ND2:WIP1 mice exhibited increased proliferation and expression of Shh downstream targets. MB incidence increased and survival decreased when ND2:WIP1 mice were crossed with an Shh-activated MB mouse model. Conversely, Wip1 knockout significantly suppressed MB formation in two independent mouse models of Shh-activated MB. Furthermore, Wip1 knockdown or treatment with a WIP1 inhibitor suppressed the effects of Shh stimulation and potentiated the growth inhibitory effects of SHH pathway-inhibiting drugs in Shh-activated MB cells in vitro. This suggests an important cross-talk between SHH and WIP1 pathways that accelerates tumorigenesis and supports WIP1 inhibition as a potential treatment strategy for MB.

Recent studies suggest that medulloblastoma, the most common malignant brain tumor of childhood, is comprised of four disease variants. The WIP1 oncogene is overexpressed in Group 3 and 4 tumors, which contain medulloblastomas with the most aggressive clinical behavior. Our data demonstrate increased WIP1 expression in metastatic medulloblastomas, and inferior progression-free and overall survival of patients with WIP1 high-expressing medulloblastoma. Microarray analysis identified upregulation of genes involved in tumor metastasis, including the G protein-coupled receptor CXCR4, in medulloblastoma cells with high WIP1 expression. Stimulation with the CXCR4 ligand SDF1α activated PI-3 kinase signaling, and promoted growth and invasion of WIP1 high-expressing medulloblastoma cells in a p53-dependent manner. When xenografted into the cerebellum of immunodeficient mice, medulloblastoma cells with stable or endogenous high WIP1 expression exhibited strong expression of CXCR4 and activated AKT in primary and invasive tumor cells. WIP1 or CXCR4 knockdown inhibited medulloblastoma growth and invasion. WIP1 knockdown also improved the survival of mice xenografted with WIP1 high-expressing medulloblastoma cells. WIP1 knockdown inhibited cell surface localization of CXCR4 by suppressing expression of the G protein receptor kinase 5, GRK5. Restoration of wild-type GRK5 promoted Ser339 phosphorylation of CXCR4 and inhibited the growth of WIP1-stable medulloblastoma cells. Conversely, GRK5 knockdown inhibited Ser339 phosphorylation of CXCR4, increased cell surface localization of CXCR4 and promoted the growth of medulloblastoma cells with low WIP1 expression. These results demonstrate crosstalk among WIP1, CXCR4 and GRK5, which may be important for the aggressive phenotype of a subclass of medulloblastomas in children.

Resistance to the human epidermal growth factor receptor (HER2)-targeted antibody trastuzumab is a major clinical concern in the treatment of HER2-positive metastatic breast cancer. Increased expression or signaling from the insulin-like growth factor-1 receptor (IGF-1R) has been reported to be associated with trastuzumab resistance. However, the specific molecular and biologic mechanisms through which IGF-1R promotes resistance or disease progression remain poorly defined. In this study, we found that the major biologic effect promoted by IGF-1R was invasion, which was mediated by both Src-focal adhesion kinase (FAK) signaling and Forkhead box protein M1 (FoxM1). Cotargeting IGF-1R and HER2 using either IGF-1R antibodies or IGF-1R short hairpin RNA in combination with trastuzumab resulted in significant but modest growth inhibition. Reduced invasion was the most significant biologic effect achieved by cotargeting IGF-1R and HER2 in trastuzumab-resistant cells. Constitutively active Src blocked the anti-invasive effect of IGF-1R/HER2 cotargeted therapy. Furthermore, knockdown of FoxM1 blocked IGF-1-mediated invasion, and dual targeting of IGF-1R and HER2 reduced expression of FoxM1. Re-expression of FoxM1 restored the invasive potential of IGF-1R knockdown cells treated with trastuzumab. Overall, our results strongly indicate that therapeutic combinations that cotarget IGF-1R and HER2 may reduce the invasive potential of cancer cells that are resistant to trastuzumab through mechanisms that depend in part on Src and FoxM1., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The standard targeted therapy for HER2-overexpressing breast cancer is the HER2 monoclonal antibody, trastuzumab. Although effective, many patients eventually develop trastuzumab resistance. The dual EGFR/HER2 small molecule tyrosine kinase inhibitor lapatinib is approved for use in trastuzumab-refractory metastatic HER2-positive breast cancer. However, lapatinib resistance is a problem as most patients with trastuzumab-refractory disease do not benefit from lapatinib. Understanding the mechanisms underlying lapatinib resistance may ultimately facilitate development of new therapeutic strategies for HER2-overexpressing breast cancer. Our current results indicate that MEK inhibition increases lapatinib-mediated cytotoxicity in resistant HER2-overexpressing breast cancer cells. We genetically and pharmacologically blocked MEK/ERK signaling and evaluated lapatinib response by trypan blue exclusion, anchorage-independent growth assays, flow cytometric cell cycle and apoptosis analysis, and in tumor xenografts. Combined MEK inhibition and lapatinib treatment reduced phosphorylated ERK more than single agent treatment. In addition, Western blots, immunofluorescence, and immunohistochemistry demonstrated that the combination of MEK inhibitor plus lapatinib reduced nuclear expression of the MEK/ERK downstream proto-oncogene FOXM1. Genetic knockdown of MEK was tested for the ability to increase lapatinib-mediated cell cycle arrest or apoptosis in JIMT-1 and MDA361 cells. Finally, xenograft studies demonstrated that combined pharmacological inhibition of MEK plus lapatinib suppressed tumor growth and reduced expression of FOXM1 in HER2-overexpressing breast cancers that are resistant to trastuzumab and lapatinib. Our results suggest that FoxM1 contributes to lapatinib resistance downstream of MEK signaling, and supports further study of pharmacological MEK inhibition to improve response to lapatinib in HER2-overexpressing trastuzumab-resistant breast cancer.

Medulloblastoma is the most common malignant childhood brain tumor. The protein phosphatase and oncogene WIP1 is over-expressed or amplified in a significant number of primary human medulloblastomas and cell lines. In the present study, we examine an important mechanism by which WIP1 promotes medulloblastoma growth using in vitro and in vivo models. Human cell lines and intracerebellar xenografted animal models were used to study the role of WIP1 and the major TP53 regulator, HDM2, in medulloblastoma growth. Stable expression of WIP1 enhances growth of TP53 wild-type medulloblastoma cells, compared with cells with stable expression of an empty-vector or mutant WIP1. In an animal model, WIP1 enhances proliferation and reduces the survival of immunodeficient mice bearing intracerebellar xenografted human medulloblastoma cells. Cells with increased WIP1 expression also exhibit increased expression of HDM2. HDM2 knockdown or treatment with the HDM2 inhibitor Nutlin-3a, the active enantomer of Nutlin-3, specifically inhibits the growth of medulloblastoma cells with increased WIP1 expression. Nutlin-3a does not affect growth of medulloblastoma cells with stable expression of an empty vector or of mutant WIP1. Knockdown of WIP1 or treatment with the WIP1 inhibitor CCT007093 results in increased phosphorylation of known WIP1 targets, reduced HDM2 expression, and reduced growth specifically in WIP1 wild-type and high-expressing medulloblastoma cells. Combined WIP1 and HDM2 inhibition is more effective than WIP1 inhibition alone in blocking growth of WIP1 high-expressing medulloblastoma cells. Our preclinical study supports a role for therapies that target WIP1 and HDM2 in the treatment of medulloblastoma.

Background: Recent publications have described an important role for cross talk between PI-3 kinase and sonic hedgehog signaling pathways in the pathogenesis of medulloblastoma., Methodology/principal Findings: We crossed mice with constitutive activation of Smoothened, SmoA1, with Pten deficient mice. Both constitutive and conditional Pten deficiency doubled the incidence of mice with symptoms of medulloblastoma and resulted in decreased survival. Analysis revealed a clear separation of gene signatures, with up-regulation of genes in the PI-3 kinase signaling pathway, including downstream activation of angiogenesis in SmoA1+/-; Pten +/- medulloblastomas. Western blotting and immunohistochemistry confirmed reduced or absent Pten, Akt activation, and increased angiogenesis in Pten deficient tumors. Down-regulated genes included genes in the sonic hedgehog pathway and tumor suppressor genes. SmoA1+/-; Pten +/+ medulloblastomas appeared classic in histology with increased proliferation and diffuse staining for apoptosis. In contrast, Pten deficient tumors exhibited extensive nodularity with neuronal differentiation separated by focal areas of intense staining for proliferation and virtually absent apoptosis. Examination of human medulloblastomas revealed low to absent PTEN expression in over half of the tumors. Kaplan-Meier analysis confirmed worse overall survival in patients whose tumor exhibited low to absent PTEN expression., Conclusions/significance: This suggests that PTEN expression is a marker of favorable prognosis and mouse models with activation of PI-3 kinase pathways may be important tools for preclinical evaluation of promising agents for the treatment of medulloblastoma.