Small molecule inhibition of group I p21-activated kinases in breast cancer induces apoptosis and potentiates the activity of microtubule stabilizing agents.

INTRODUCTION: 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 (PAK)1. 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. METHODS: PAK1 dysregulation (copy number gain, mRNA and protein expression) was evaluated in two cohorts of breast cancer tissues (n=980 and 1,108). 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. RESULTS: We demonstrate that focal genomic amplification and overexpression of PAK1 are associated with poor clinical outcome in the luminal subtype of breast cancer (P=1.29×10(-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. CONCLUSIONS: 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.

Granulin-epithelin precursor interacts with heparan sulfate on liver cancer cells.

Granulin-epithelin precursor (GEP) is a pluripotent secretory growth factor which promotes cancer progression in a number of human cancers. However, how cancer cells interact with GEP remains unknown. In this study, we aimed to identify the cell surface-binding partner of GEP on liver cancer cells. Human recombinant GEP (rGEP) was expressed and purified to homogeneity. The rGEP was shown to trigger phosphorylation of AKT and ERK1/2 in liver cancer cells. We demonstrated cell surface attachment of rGEP, which was blocked by prebinding of platelet-derived growth factor-AA, platelet-derived growth factor-BB and fibroblast growth factor-2. Therefore, heparan sulfate (HS) had been reasoned as the binding partner of rGEP. Heparinase digestion validated the role of HS on supporting the attachment. The heparin-binding domain of GEP was mapped to RRH(555-557) in the C-terminal region. Suppression of the HS polymerase exostosin-1 reduced the rGEP binding and rGEP-mediated signaling transduction. Suppression of a specific HS proteoglycan, glypican-3, also showed a partial reduction of rGEP binding and an inhibition on rGEP-mediated activation of AKT. Furthermore, glypican-3 was shown to correlate with the expressions of GEP in clinical samples (Spearman's ρ = 0.363, P = 0.001). This study identified HS, partly through glypican-3, as a novel binding partner of GEP on the surface of liver cancer cells.

EGFR phosphorylates tumor-derived EGFRvIII driving STAT3/5 and progression in glioblastoma.

EGFRvIII, a frequently occurring mutation in primary glioblastoma, results in a protein product that cannot bind ligand, but signals constitutively. Deducing how EGFRvIII causes transformation has been difficult because of autocrine and paracrine loops triggered by EGFRvIII alone or in heterodimers with wild-type EGFR. Here, we document coexpression of EGFR and EGFRvIII in primary human glioblastoma that drives transformation and tumorigenesis in a cell-intrinsic manner. We demonstrate enhancement of downstream STAT signaling triggered by EGFR-catalyzed phosphorylation of EGFRvIII, implicating EGFRvIII as a substrate for EGFR. Subsequent phosphorylation of STAT3 requires nuclear entry of EGFRvIII and formation of an EGFRvIII-STAT3 nuclear complex. Our findings clarify specific oncogenic signaling relationships between EGFR and EGFRvIII in glioblastoma.

Dual blockade of lipid and cyclin-dependent kinases induces synthetic lethality in malignant glioma.

Malignant glioma, the most common primary brain tumor, is generally incurable. Although phosphatidylinositol-3-kinase (PI3K) signaling features prominently in glioma, inhibitors generally block proliferation rather than induce apoptosis. Starting with an inhibitor of both lipid and protein kinases that induced prominent apoptosis and that failed early clinical development because of its broad target profile and overall toxicity, we identified protein kinase targets, the blockade of which showed selective synthetic lethality when combined with PI3K inhibitors. Prioritizing protein kinase targets for which there are clinical inhibitors, we demonstrate that cyclin-dependent kinase (CDK)1/2 inhibitors, siRNAs against CDK1/2, and the clinical CDK1/2 inhibitor roscovitine all cooperated with the PI3K inhibitor PIK-90, blocking the antiapoptotic protein Survivin and driving cell death. In addition, overexpression of CDKs partially blocked some of the apoptosis caused by PIK-75. Roscovitine and PIK-90, in combination, were well tolerated in vivo and acted in a synthetic-lethal manner to induce apoptosis in human glioblastoma xenografts. We also tested clinical Akt and CDK inhibitors, demonstrating induction of apoptosis in vitro and providing a preclinical rationale to test this combination therapy in patients.

Identification and characterization of tropomyosin 3 associated with granulin-epithelin precursor in human hepatocellular carcinoma.

BACKGROUND AND AIM: Granulin-epithelin precursor (GEP) has previously been reported to control cancer growth, invasion, chemo-resistance, and served as novel therapeutic target for cancer treatment. However, the nature and characteristics of GEP interacting partner remain unclear. The present study aims to identify and characterize the novel predominant interacting partner of GEP using co-immunoprecipitation and mass spectrometry. METHODS AND RESULTS: Specific anti-GEP monoclonal antibody was used to capture GEP and its interacting partner from the protein extract of the liver cancer cells Hep3B. The precipitated proteins were analyzed by SDS-PAGE, followed by mass spectrometry and the protein identity was demonstrated to be tropomyosin 3 (TPM3). The interaction has been validated in additional cell models using anti-TPM3 antibody and immunoblot to confirm GEP as the interacting partner. GEP and TPM3 expressions were then examined by real-time quantitative RT-PCR in clinical samples, and their transcript levels were significantly correlated. Elevated TPM3 levels were observed in liver cancer compared with the adjacent non-tumorous liver, and patients with elevated TPM3 levels were shown to have poor recurrence-free survival. Protein expression of GEP and TPM3 was observed only in the cytoplasm of liver cancer cells by immunohistochemical staining. CONCLUSIONS: TPM3 is an interacting partner of GEP and may play an important role in hepatocarcinogenesis.

Granulin-epithelin precursor and ATP-dependent binding cassette (ABC)B5 regulate liver cancer cell chemoresistance.

BACKGROUND & AIMS: Chemotherapy is used to treat unresectable liver cancer with marginal efficacy; this might result from hepatic cancer cells with stem cell and chemoresistant features. Gene expression profiling studies have shown that hepatic cancer cells express granulin-epithelin precursor (GEP); we investigated its role in hepatic cancer stem cell functions and chemoresistance. METHODS: The effects of GEP and drug transporter signaling on chemoresistance were investigated in hepatic cancer stem cells. We analyzed the expression patterns of 142 clinical samples from liver tumors, adjacent nontumorous liver tissue, and liver tissue from patients who did not have cancer. RESULTS: GEP regulated the expression of the adenosine triphosphate-dependent binding cassette (ABC)B5 drug transporter in liver cancer cells. Chemoresistant cells that expressed GEP had increased levels of ABCB5; suppression of ABCB5 sensitized the cells to doxorubicin uptake and apoptosis. Most cells that expressed GEP and ABCB5 also expressed the hepatic cancer stem cell markers CD133 and EpCAM; blocking ABCB5 reduced their expression. Expression levels of GEP and ABCB5 were correlated in human liver tumor samples. ABCB5 levels were increased in liver cancer cells compared with nontumor liver tissue from patients with cirrhosis or hepatitis, or normal liver tissue. ABCB5 expression was associated with the recurrence of hepatocellular carcinoma after partial hepatectomy. CONCLUSIONS: Expression of GEP and ABCB5 in liver cancer stem cells is associated with chemoresistance and reduced survival times of patients with hepatocellular carcinoma. Reagents designed to target these proteins might be developed as therapeutics and given in combination with chemotherapy to patients with liver cancer.

PI3K signaling in glioma--animal models and therapeutic challenges.

The PI3 kinase (PI3K) family plays a complex role in cell biology and metabolism. Signaling through the PI3Ks is frequently activated in many human cancers, including glioblastoma, because of gain-of-function mutations in PIK3CA or loss of PTEN. Experiments involving genetic mouse models and small molecule inhibitors have helped to elucidate the roles of the regulatory and catalytic subunits of PI3K in metabolism and cancer. Downstream of PI3K is Akt, a critical effector of growth, proliferation and survival. The suggested dependence of glioblastoma tumors on PI3K signaling implies that PI3K inhibitors should lead to effective killing of these cancer cells, but that has been shown not to be the case. The engagement of other survival pathways in response to PI3K inhibition prompts the need to develop combination therapies that promote cytotoxicity in cancer cells.

A dual phosphoinositide-3-kinase alpha/mTOR inhibitor cooperates with blockade of epidermal growth factor receptor in PTEN-mutant glioma.

We have shown previously that blockade of epidermal growth factor receptor (EGFR) cooperates with a pan-selective inhibitor of phosphoinositide-3-kinase (PI3K) in EGFR-driven glioma. In this communication, we tested EGFR-driven glioma differing in PTEN status, treating with the EGFR inhibitor erlotinib and a novel dual inhibitor of PI3Kalpha and mTOR (PI-103). Erlotinib blocked proliferation only in PTEN(wt) cells expressing EGFR. Although erlotinib monotherapy showed little effect in PTEN(mt) glioma, PI-103 greatly augmented the antiproliferative efficacy of erlotinib in this setting. To address the importance of PI3K blockade, we showed in PTEN(mt) glioma that combining PI-103 and erlotinib was superior to either monotherapy or to therapy combining erlotinib with either rapamycin (an inhibitor of mTOR) or PIK-90 (an inhibitor of PI3Kalpha). These experiments show that a dual inhibitor of PI3Kalpha and mTOR augments the activity of EGFR blockade, offering a mechanistic rationale for targeting EGFR, PI3Kalpha, and mTOR in the treatment of EGFR-driven, PTEN-mutant glioma.