Niranjan Awasthi

The efficacy of a novel, dual PI3K/mTOR inhibitor NVP-BEZ235 to enhance chemotherapy and antiangiogenic response in pancreatic cancer.

Gemcitabine has limited clinical benefits for pancreatic ductal adenocarcinoma (PDAC). The phosphatidylinositol‐3‐kinase (PI3K)/AKT and mammalian target of rapamycin (mTOR) signaling pathways are frequently dysregulated in PDAC. We investigated the effects of NVP‐BEZ235, a novel dual PI3K/mTOR inhibitor, in combination with gemcitabine and endothelial monocyte activating polypeptide II (EMAP) in experimental PDAC. Cell proliferation and protein expression were analyzed by WST‐1 assay and Western blotting. Animal survival experiments were performed in murine xenografts. BEZ235 caused a decrease in phospho‐AKT and phospho‐mTOR expression in PDAC (AsPC‐1), endothelial (HUVECs), and fibroblast (WI‐38) cells. BEZ235 inhibited in vitro proliferation of all four PDAC cell lines tested. Additive effects on proliferation inhibition were observed in the BEZ235‐gemcitabine combination in PDAC cells and in combination of BEZ235 or EMAP with gemcitabine in HUVECs and WI‐38 cells. BEZ235, alone or in combination with gemcitabine and EMAP, induced apoptosis in AsPC‐1, HUVECs, and WI‐38 cells as observed by increased expression of cleaved poly (ADP‐ribose) polymerase‐1 (PARP‐1) and caspase‐3 proteins. Compared to controls (median survival: 16 days), animal survival increased after BEZ235 and EMAP therapy alone (both 21 days) and gemcitabine monotherapy (28 days). Further increases in survival occurred in combination therapy groups BEZ235 + gemcitabine (30 days, P = 0.007), BEZ235 + EMAP (27 days, P = 0.02), gemcitabine + EMAP (31 days, P = 0.001), and BEZ235 + gemcitabine + EMAP (33 days, P = 0.004). BEZ235 has experimental PDAC antitumor activity in vitro and in vivo that is further enhanced by combination of gemcitabine and EMAP. These findings demonstrate advantages of combination therapy strategies targeting multiple pathways in pancreatic cancer treatment. J. Cell. Biochem. 113: 784–791, 2012.

Endothelial monocyte activating polypeptide II interferes with VEGF-induced proangiogenic signaling

Endothelial monocyte activating polypeptide II (EMAP II) is a proinflammatory cytokine with antiangiogenic properties. EMAP II functions as a potent inhibitor of primary and metastatic tumor growth, has strong inhibitory effects on endothelial cells (ECs), and can reduce intratumoral expression of the angiogenesis inducer vascular endothelial growth factor (VEGF). VEGF influences EC functions such as proliferation, migration, survival and tube formation. Therapeutic strategies that target VEGF have been demonstrated to reduce the tumor growth. We investigated the effects of EMAP II on VEGF-induced angiogenesis signaling. Primary human fetal lung ECs (HFLECs) and human umbilical vein ECs (HUVECs) were grown in E-Stim medium. Protein binding was analyzed using enzyme-linked immunosorbent assay (ELISA). Protein expression was determined by western blot analysis. EC proliferation and migration was determined using WST-1 reagent and transwell membrane, respectively. EMAP II efficiently and dose dependently binds to VEGF receptor 1 (VEGFR1) and VEGF receptor 2 (VEGFR2) as observed by ELISA. Bmax values for VEGFR1 and VEGFR2 were 0.45 and 0.17, respectively. In addition, EMAP II inhibited binding of VEGF to VEGFR1 and VEGFR2. EMAP II significantly reduced VEGF-induced expression of phosphorylated VEGFR1 (in HFLEC and HUVEC) by >50%, and of phosphorylated VEGFR2 (in HUVEC) by 66%. EMAP II also inhibited downstream VEGF signaling. Although VEGF-induced phosphorylation of Akt, Erk1/2, p38 and Raf 2.8-, 1.5-, 2.2- and 3.6-fold, respectively, EMAP II preincubation blocked this induction in phosphorylation to control levels. VEGF-induced EC proliferation 2.5-fold, and EMAP II pretreatment abrogated this effect. Similarly, VEGF-induced EC migration (2.5-fold) was significantly inhibited by EMAP II. These finding suggest that inhibition of VEGF signaling is one possible antiangiogenic mechanism of EMAP II, which may explain its in vivo antitumor activity and delineate therapeutic strategies to enhance anti-VEGF therapy to inhibit tumor growth.

Comparative benefits of Nab-paclitaxel over gemcitabine or polysorbate-based docetaxel in experimental pancreatic cancer

Gemcitabine has limited clinical benefits in pancreatic ductal adenocarcinoma. The solvent-based traditional taxanes docetaxel and paclitaxel have not shown clinical results superior to gemcitabine. Nab-paclitaxel, a water-soluble albumin-bound paclitaxel, may carry superior distribution properties into the tumor microenvironment and has shown efficacy in multiple tumor types. We evaluated nab-paclitaxel effects compared with gemcitabine or docetaxel. For pancreatic ductal adenocarcinoma cells AsPC-1, BxPC-3, MIA PaCa-2 and Panc-1, gemcitabine IC50 ranged from 494nM to 23.9 μM; docetaxel IC50 range was from 5 to 34nM; nab-paclitaxel IC50 range was from 243nM to 4.9 μM. Addition of IC25 dose of docetaxel or nab-paclitaxel decreased gemcitabine IC50. Net tumor growth inhibition after gemcitabine, docetaxel or nab-paclitaxel was 67, 31 and 72%, which corresponded with intratumoral proliferative and apoptotic indices. Tumor stromal density was decreased by nab-paclitaxel and to a lesser extent by docetaxel as measured through reduction in α-smooth muscle actin, S100A4 and collagen 1 expression. Animal survival was prolonged after nab-paclitaxel treatment (41 days, P < 0.002) compared with gemcitabine (32 days, P = 0.005), docetaxel (32 days, P = 0.005) and controls (20 days). Survival in nab-paclitaxel/gemcitabine and docetaxel/gemcitabine sequential treatment groups was not superior to nab-paclitaxel alone. Low-dose combination of gemcitabine with nab-paclitaxel or docetaxel was more effective compared with controls or gemcitabine alone but not superior to regular dose nab-paclitaxel alone. Combination treatment of gemcitabine+nab-paclitaxel or gemcitabine+docetaxel increased gemcitabine concentration in plasma and tumor. The superior antitumor activity of nab-paclitaxel provides a strong rationale for considering nab-paclitaxel as first-line monotherapy in pancreatic ductal adenocarcinoma.

BMS-754807, a Small Molecule Inhibitor of Insulin-like Growth Factor-1 Receptor / Insulin Receptor, Enhances Gemcitabine Response in Pancreatic Cancer

Gemcitabine has limited clinical benefits in pancreatic ductal adenocarcinoma (PDAC). Insulin-like growth factor (IGF) signaling proteins are frequently overexpressed in PDAC. The therapeutic potential of BMS-754807, a small-molecule inhibitor of IGF-type 1 receptor (IGF-1R) and insulin receptor (IR), and gemcitabine was evaluated in experimental PDAC. Cell proliferation and protein expression were measured by WST-1 assay and immunoblotting. Tumor growth and survival studies were conducted in murine xenografts. PDAC cells expressed phospho-IGF-1R protein. BMS-754807 and gemcitabine inhibited cell proliferation of PDAC cells; the combination of BMS-754807 with gemcitabine had additive effects. Addition of BMS-754807 decreased gemcitabine IC50 from 9.7 μmol/L to 75 nmol/L for AsPC-1, from 3 μmol/L to 70 nmol/L for Panc-1, from 72 to 16 nmol/L for MIA PaCa-2, and from 28 to 16 nmol/L for BxPC-3 cells. BMS-754807 caused a decrease in phospho-IGF-1R and phospho-AKT proteins in AsPC-1 and Panc-1 cells. BMS-754807 and gemcitabine caused an increase in PARP-1 and caspase-3 cleavage. Net tumor growth inhibition in BMS-754807, gemcitabine, and BMS-754807+gemcitabine groups was 59%, 35%, and 94% as compared with controls. Effects of therapy on intratumoral proliferation and apoptosis corresponded with tumor growth inhibition data. BMS-754807 also caused a decrease in phospho-IGF-1R and phospho-AKT in tumor tissue lysates. Median animal survival (controls: 21 days) with BMS-754807 was 27 days (P = 0.03), with gemcitabine 28 days (P = 0.05), and in the BMS-754807+gemcitabine combination group, 41 days (P = 0.007). The strong antitumor activity of BMS-754807 in experimental PDAC supports the potential of BMS-754807-induced mechanisms for clinical PDAC therapy. Mol Cancer Ther; 11(12); 2644–53. ©2012 AACR.

CXCL1 promotes tumor growth through VEGF pathway activation and is associated with inferior survival in gastric cancer.

The chemokine (C-X-C motif) ligand 1 (CXCL1) regulates tumor-stromal interactions and tumor invasion. However, the precise role of CXCL1 on gastric tumor growth and patient survival remains unclear. In the current study, protein expressions of CXCL1, vascular endothelial growth factor (VEGF) and phospho-signal transducer and activator of transcription 3 (p-STAT3) in primary tumor tissues from 98 gastric cancer patients were measured by immunohistochemistry (IHC). CXCL1 overexpressed cell lines were constructed using Lipofectamine 2000 reagent or lentiviral vectors. Effects of CXCL1 on VEGF expression and local tumor growth were evaluated in vitro and in vivo. CXCL1 was positively expressed in 41.4% of patients and correlated with VEGF and p-STAT3 expression. Higher CXCL1 expression was associated with advanced tumor stage and poorer prognosis. In vitro studies in AGS and SGC-7901 cells revealed that CXCL1 increased cell migration but had little effect on cell proliferation. CXCL1 activated VEGF signaling in gastric cancer (GC) cells, which was inhibited by STAT3 or chemokine (C-X-C motif) receptor 2 (CXCR2) blockade. CXCL1 also increased p-STAT3 expression in GC cells. In vivo, CXCL1 increased xenograft local tumor growth, phospho-Janus kinase 2 (p-JAK2), p-STAT3 levels, VEGF expression and microvessel density. These results suggested that CXCL1 increased local tumor growth through activation of VEGF signaling which may have mechanistic implications for the observed inferior GC survival. The CXCL1/CXCR2 pathway might be potent to improve anti-angiogenic therapy for gastric cancer.

PG545, an Angiogenesis and Heparanase Inhibitor, Reduces Primary Tumor Growth and Metastasis in Experimental Pancreatic Cancer

Aggressive tumor progression, metastasis, and resistance to conventional therapies lead to an extremely poor prognosis for pancreatic ductal adenocarcinoma (PDAC). Heparanase, an enzyme expressed by multiple cell types, including tumor cells in the tumor microenvironment, has been implicated in angiogenesis and metastasis, and its expression correlates with decreased overall survival in PDAC. We evaluated the therapeutic potential of PG545, an angiogenesis and heparanase inhibitor, in experimental PDAC. PG545 inhibited the proliferation, migration, and colony formation of pancreatic cancer cells in vitro at pharmacologically relevant concentrations. Heparanase inhibition also reduced the proliferation of fibroblasts but had only modest effects on endothelial cells in vitro. Furthermore, PG545 significantly prolonged animal survival in intraperitoneal and genetic models (mPDAC: LSL-KrasG12D; Cdkn2alox/lox; p48Cre) of PDAC. PG545 also inhibited primary tumor growth and metastasis in orthotopic and genetic endpoint studies. Analysis of tumor tissue revealed that PG545 significantly decreased cell proliferation, increased apoptosis, reduced microvessel density, disrupted vascular function, and elevated intratumoral hypoxia. Elevated hypoxia is a known driver of collagen deposition and tumor progression; however, tumors from PG545-treated animals displayed reduced collagen deposition and a greater degree of differentiation compared with control or gemcitabine-treated tumors. These results highlight the potent antitumor activity of PG545 and support the further exploration of heparanase inhibitors as a potential clinical strategy for the treatment of PDAC. Mol Cancer Ther; 12(7); 1190–201. ©2013 AACR.

Antitumor effects of EMAP II against pancreatic cancer through inhibition of fibronectin-dependent proliferation.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to conventional chemotherapy. The presence of both cellular and stromal fibronectin (FN) and its interaction with integrins is necessary for PDAC progression. We tested the efficacy of endothelial monocyte-activating polypeptide II (EMAP II) to inhibit PDAC progression and its ability to interfere with FN-integrin angiogenesis signaling. In heterotopic PDAC tumors EMAP II caused a significant reduction (>65%) in tumor growth, accompanied by a >50% and 44% decrease in microvessel density and proliferative activity, respectively. EMAP II therapy caused a 62% and 56% reduction in host and tumor cell FN expression. Cultured PDAC cells expressed aVb3 and a5b1 integrins. In vitro EMAP II had limited antiproliferative effects on ASPC-1, but a pronounced antiproliferative effect on endothellial cells. 3D FN matrices increased ASPC-1 cell proliferation by > 50%, and this induction was significantly blocked by a3, a5, a6 and aV integrin funtional blocking antibodies, while a1, a2 and a4 antibodies had no effect. EMAP II also inhibited 3D FN-matrix induced ASPC-1 proliferation by >43% at 20 mM. These findings suggest that EMAP II demonstrates significant antitumor activity against PDAC cells, and that this effect may be in part mediated through targeted interference with stromal FN-integrin dependent PDAC cell proliferation.

Profile of nintedanib in the treatment of solid tumors: the evidence to date

Angiogenesis is an essential process for tumor growth and metastasis, and remains a promising therapeutic target process in cancer treatment for several cancer types. Bevacizumab, a monoclonal antibody that targets vascular endothelial growth factor (VEGF), was the first antiangiogenic agent approved for cancer therapy. Novel antiangiogenic agents, such as sunitinib, sorafenib, pazopanib, or vandetanib that target additional proangiogenic signaling pathways beyond VEGF, have also been approved for the treatment of various malignant diseases. While most of these agents are approved in combination with cytotoxic chemotherapy for indications including metastatic colorectal cancer, non-small-cell lung cancer, breast cancer, renal cell carcinoma (RCC), and gastric cancer, some are used as approved monotherapy for advanced RCC, hepatocellular carcinoma and medullary thyroid cancer. Major challenges to the success of antiangiogenic therapy include associated toxicity risks, limitation of efficacy through the possible development of resistance and induction or promotion of metastatic progression. Nintedanib (formally known as BIBF 1120) is a triple angiokinase inhibitor of VEGF, fibroblast growth factor, platelet-derived growth factor signaling with lesser activity against RET, Flt-3, and Src. Through this unique targeting profile nintedanib has demonstrated significant antitumor activity in several tumor types in preclinical studies. Nintedanib has also shown promising clinical efficacy in combination with docetaxel and has been approved for treating patients with locally advanced and metastatic non-small-cell lung cancer in Europe. Nintedanib has also been found to be clinically promising in terms of efficacy and safety in several other solid tumors including ovarian cancer (Phase III), RCC (Phase II), and prostate cancer (Phase II). This review article provides a comprehensive summary of the preclinical and clinical efficacy of nintedanib in the treatment of solid tumors.

Evaluation of poly-mechanistic antiangiogenic combinations to enhance cytotoxic therapy response in pancreatic cancer

Gemcitabine (Gem) has limited clinical benefits in pancreatic ductal adenocarcinoma (PDAC). The present study investigated combinations of gemcitabine with antiangiogenic agents of various mechanisms for PDAC, including bevacizumab (Bev), sunitinib (Su) and EMAP II. Cell proliferation and protein expression were analyzed by WST-1 assay and Western blotting. In vivo experiments were performed via murine xenografts. Inhibition of in vitro proliferation of AsPC-1 PDAC cells by gemcitabine (10 µM), bevacizumab (1 mg/ml), sunitinib (10 µM) and EMAP (10 µM) was 35, 22, 81 and 6 percent; combination of gemcitabine with bevacizumab, sunitinib or EMAP had no additive effects. In endothelial HUVECs, gemcitabine, bevacizumab, sunitinib and EMAP caused 70, 41, 86 and 67 percent inhibition, while combination of gemcitabine with bevacizumab, sunitinib or EMAP had additive effects. In WI-38 fibroblasts, gemcitabine, bevacizumab, sunitinib and EMAP caused 79, 58, 80 and 29 percent inhibition, with additive effects in combination as well. Net in vivo tumor growth inhibition in gemcitabine, bevacizumab, sunitinib and EMAP monotherapy was 43, 38, 94 and 46 percent; dual combinations of Gem+Bev, Gem+Su and Gem+EMAP led to 69, 99 and 64 percent inhibition. Combinations of more than one antiangiogenic agent with gemcitabine were generally more effective but not superior to Gem+Su. Intratumoral proliferation, apoptosis and microvessel density findings correlated with tumor growth inhibition data. Median animal survival was increased by gemcitabine (26 days) but not by bevacizumab, sunitinib or EMAP monotherapy compared to controls (19 days). Gemcitabine combinations with bevacizumab, sunitinib or EMAP improved survival to similar extent (36 or 37 days). Combinations of gemcitabine with Bev+EMAP (43 days) or with Bev+Su+EMAP (46 days) led to the maximum survival benefit observed. Combination of antiangiogenic agents improves gemcitabine response, with sunitinib inducing the strongest effect. These findings demonstrate advantages of combining multi-targeting agents with standard gemcitabine therapy for PDAC.

Superior Antitumor Activity of Nanoparticle Albumin-Bound Paclitaxel in Experimental Gastric Cancer

Gastric cancer is the second common cause of cancer related death worldwide and lacks highly effective treatment for advanced disease. Nab-paclitaxel is a novel microtubule-inhibitory cytotoxic agent that has not been tested in gastric cancer as of yet. In this study, human gastric cancer cell lines AGS, NCI-N87 and SNU16 were studied. Nab-paclitaxel inhibited cell proliferation with an IC50 of 5 nM in SNU16, 23 nM in AGS and 49 nM in NCI-N87 cells after 72-hour treatment, which was lower than that of oxaliplatin (1.05 μM to 1.51 μM) and epirubicin (0.12 μM to 0.25 μM). Nab-paclitaxel treatment increased expression of the mitotic-spindle associated phospho-stathmin irrespective of the baseline total or phosphorylated stathmin level, and induced mitotic cell death as confirmed through increased expression of cleaved-PARP and caspase-3. After a two-week nab-paclitaxel, oxaliplatin or epirubicin treatment, the average in vivo local tumor growth inhibition rate was 77, 17.2 and 21.4 percent, respectively (p = 0.002). Effects of therapy on tumoral proliferative and apoptotic indices corresponded with tumor growth inhibition data, while expression of phospho-stathmin also increased in tissues. There was an increase in median animal survival after nab-paclitaxel treatment (93 days) compared to controls (31 days, p = 0.0007), oxaliplatin (40 days, p = 0.0007) or to docetaxel therapy (81 days, p = 0.0416). The strong antitumor activity of nab-paclitaxel in experimental gastric cancer supports such microtubule-inhibitory strategy for clinical application. Nab-paclitaxel benefits were observed independent from phosphorylated stathmin expression at baseline, putting into question the consideration of nab-paclitaxel use in gastric cancer based on this putative biomarker.