Intracellular signaling in tumor and endothelial cells: The expected and, yet again, the unexpected.

In this issue of Cancer Cell, Phung and coworkers demonstrate that sustained endothelial activation of Akt by expression of constitutively activated Akt1 (myrAkt1) leads to blood vessels that essentially recapitulate the complex structural and functional abnormalities of tumor vessels. The authors provide evidence that rapamycin inhibition of PI3K/Akt/mTOR signaling in endothelial cells (ECs), by either reducing Akt activity or blocking mTOR, reverses the pathologic effects associated with excess VEGF signaling in the tumor vasculature. However, unexpected findings following mTOR inhibition in vivo highlight the seemingly paradoxical and complex effects of rapamycin on various cell types within the tumor microenvironment.

Implication of RICTOR in the mTOR inhibitor-mediated induction of insulin-like growth factor-I receptor (IGF-IR) and human epidermal growth factor receptor-2 (Her2) expression in gastrointestinal cancer cells.

Inhibition of mTORC1 with the mTOR inhibitor rapamycin may lead to an induction of Akt phosphorylation in cancer cells via mTORC2 activation. Using gastric and pancreatic cancer cells, we further investigated this paradoxical signaling response and found that rapamycin additionally up-regulates both IGF-IR and Her2 expression. Using RNAi for down-regulating RICTOR, this induction of receptor kinase expression was identified to be mediated via an mTORC2-induced Akt activation. Moreover, mTORC2 inhibition reduced the phosphorylation of GSK-3 and NF-kappaB, and significantly impaired cancer cell motility. In conclusion, inhibition of mTORC2 may abrogate unfavorable signaling effects of mTOR inhibitors, hence providing a novel rationale for therapy.

Overexpression of PDGF-BB decreases colorectal and pancreatic cancer growth by increasing tumor pericyte content.

We hypothesized that overexpression of PDGF-BB in colorectal cancer (CRC) and pancreatic cancer cells would result in increased pericyte coverage of ECs in vivo, rendering the tumor vasculature more resistant to antiangiogenic therapy. We stably transfected the cDNA for the PDGF-B into HT-29 human CRC and FG human pancreatic cancer cells. Surprisingly, when HT-29 or FG parental and transfected cells were injected into mice (subcutaneously and orthotopically), we observed marked inhibition of tumor growth in the PDGF-BB-overexpressing clones. In the PDGF-BB-overexpressing tumors, we observed an increase in pericyte coverage of ECs. Treatment of PDGF-BB-overexpressing tumors with imatinib mesylate (PDGFR inhibitor) resulted in increased growth and decreased total pericyte content compared with those in untreated PDGF-BB-overexpressing tumors. In vitro studies demonstrated the ability of VSMCs to inhibit EC proliferation by approximately 50%. These data show that increasing the pericyte content of the tumor microenvironment inhibits the growth of angiogenesis-dependent tumors. Single-agent therapy targeting PDGF receptor must be used with caution in tumors when PDGFR is not the target on the tumor cell itself.

GLUT1 expression is increased in hepatocellular carcinoma and promotes tumorigenesis.

Accelerated glycolysis is one of the biochemical characteristics of cancer cells. The glucose transporter isoform 1 (GLUT1) gene encodes a key rate-limiting factor in glucose transport into cancer cells. However, its expression level and functional significance in hepatocellular cancer (HCC) are still disputed. Therefore, we aimed to analyze the expression and function of the GLUT1 gene in cases of HCC. We found significantly higher GLUT1 mRNA expression levels in HCC tissues and cell lines compared with primary human hepatocytes and matched nontumor tissue. Immunohistochemical analysis of a tissue microarray of 152 HCC cases revealed a significant correlation between Glut1 protein expression levels and a higher Ki-67 labeling index, advanced tumor stages, and poor differentiation. Accordingly, suppression of GLUT1 expression by siRNA significantly impaired both the growth and migratory potential of HCC cells. Furthermore, inhibition of GLUT1 expression reduced both glucose uptake and lactate secretion. Hypoxic conditions further increased GLUT1 expression levels in HCC cells, and this induction was dependent on the activation of the transcription factor hypoxia-inducible factor-1alpha. In summary, our findings suggest that increased GLUT1 expression levels in HCC cells functionally affect tumorigenicity, and thus, we propose GLUT1 as an innovative therapeutic target for this highly aggressive tumor.

Targeting heat-shock protein 90 improves efficacy of rapamycin in a model of hepatocellular carcinoma in mice.

UNLABELLED: Hepatocellular carcinoma (HCC) remains associated with a poor prognosis, but novel targeted therapies in combination with anti-angiogenic substances may offer new perspectives. We hypothesized that simultaneous targeting of tumor cells, endothelial cells, and pericytes would reduce growth and angiogenesis of HCC, which represents a highly vascularized tumor entity. Recently, because of their anti-angiogenic properties, inhibitors of mammalian target of rapamycin (mTOR) have entered clinical trials for therapy of HCC. However, treatment with mTOR inhibitors may lead to paradoxical activation of Akt signaling in tumor cells via insulin-like growth factor-I receptor (IGF-IR)-dependent and IGF-IR-independent mechanisms. Because we have recently identified heat shock protein 90 (Hsp90) antagonists to impair both oncogenic and angiogenic signaling cascades in tumor cells, including Akt and IGF-IR, we sought to investigate whether Hsp90 blockade could improve growth-inhibitory and anti-angiogenic effects of the mTOR inhibitor rapamycin. Human HCC cells, a murine hepatoma cell line, endothelial cells (ECs), and vascular smooth muscle cells (VSMC) were employed in experiments. Results show that dual inhibition of mTOR and Hsp90 leads to effective disruption of oncogenic signaling cascades and substantially improves growth-inhibitory effects in vivo. Importantly, blocking Hsp90 abrogated the rapamycin-induced activation of Akt and of the downstream effector nuclear factor kappa-B (NF-kappaB) in HCC tumors. Furthermore, Hsp90 inhibition reduced the expression of platelet-derived growth factor-receptor-beta (PDGF-Rbeta) on VSMCs, and diminished vascular endothelial growth factor-receptor 2 (VEGFR-2) expression on ECs, which further improves the anti-angiogenic capacity of this regimen. CONCLUSION: Blocking Hsp90 disrupts rapamycin-induced activation of alternative signaling pathways in HCCs and substantially improves the growth-inhibitory effects of mTOR inhibition in vivo. Hence, the concept of targeting tumor cells, ECs, and VSMCs by blocking Hsp90/mTOR could prove valuable for treatment of HCC.

Activating transcription factor-3 (ATF3) functions as a tumor suppressor in colon cancer and is up-regulated upon heat-shock protein 90 (Hsp90) inhibition.

BACKGROUND: Activating transcription factor-3 (ATF3) is involved in the complex process of cellular stress response. However, its exact role in cancer is discussed controversially because both tumor suppressive and oncogenic effects have been described. Here we followed-up on our previous observation that inhibition of Hsp90 may increase ATF3 expression and sought to determine the role of ATF3 in colon cancer. METHODS: Regulation of ATF3 was determined in cancer cells using signaling inhibitors and a heat-shock protein-90 (Hsp90) antagonist. Human HCT116 cancer cells were stably transfected with an ATF3-shRNA or a luciferase-shRNA expression plasmid and alterations in cell motility were assessed in migration assays. The impact of ATF3 down-regulation on cancer growth and metastasis were investigated in a subcutaneous tumor model, a model of hepatic tumor growth and in a model of peritoneal carcinomatosis. Human colon cancer tissues were analyzed for ATF3 expression. RESULTS: The results show that therapeutic Hsp90 inhibition substantially up-regulates the expression of ATF3 in various cancer cells, including colon, gastric and pancreatic cancer. This effect was evident both in vitro and in vivo. RNAi mediated knock-down of ATF3 in HCT116 colon cancer cells significantly increased cancer cell migration in vitro. Moreover, in xenogenic mouse models, ATF3 knock-down promoted subcutaneous tumor growth and hepatic metastasis, as well as peritoneal carcinomatosis. Importantly, ATF3 expression was lower in human colon cancer specimens, as compared to corresponding normal surrounding tissues, suggesting that ATF3 may represent a down-regulated tumor suppressor in colon cancer. CONCLUSION: In conclusion, ATF3 down-regulation in colon cancer promotes tumor growth and metastasis. Considering that blocking Hsp90 induces ATF3 expression, Hsp90 inhibition may represent a valid strategy to treat metastatic colon cancer by up-regulating this anti-metastatic transcription factor.

Staged surgery with neoadjuvant 90Y-DOTATOC therapy for down-sizing synchronous bilobular hepatic metastases from a neuroendocrine pancreatic tumor.

PURPOSE: Treatment with DOTA-d-Phe(1)-Tyr(3)-octreotide (DOTATOC), labeled with beta-emitting radioisotope yttrium-90 ((90)Y-DOTATOC), has successfully been used for the palliative treatment of patients with advanced somatostatin receptor-expressing neuroendocrine tumors (NETs). However, controversy persists as to whether patients with metastatic NETs of the pancreas should undergo radical (salvage) surgery or receive palliative therapy. We proposed that (90)Y-DOTATOC could be used in a neoadjuvant intention for improving therapy of hepatic NET metastases. MATERIALS AND METHODS: We investigated a novel therapy concept in a 49-year-old patient presenting with a neuroendocrine tumor of the pancreatic tail and synchronous multiple bilobular hepatic metastases. After surgical removal of the large primary tumor by extended left en bloc resection of the pancreatic tail, the patient received neoadjuvant (90)Y-DOTATOC for therapy of primarily non-resectable bilobular hepatic metastases. RESULTS: The (90)Y-DOTATOC therapy resulted in an impressive regression of hepatic lesions, thus facilitating surgical removal of all remaining liver metastases in a second operation (staged surgery). In addition, one lesion was ablated using radiofrequency ablation (RFA). At 1-year of follow-up after hepatic R0 resection/RFA, there was no evidence of tumor recurrence or extrahepatic metastasis. CONCLUSIONS: The neoadjuvant use of (90)Y-DOTATOC therapy could prove valuable for treatment of advanced pancreatic NETs metastatic to the liver in terms of facilitating R0 resection by applying staged surgery concepts.

Heat-shock protein 90 (Hsp90) as a molecular target for therapy of gastrointestinal cancer.

Anticancer drug development strategies critically involve the identification of novel molecular targets which are crucial for tumorigenesis and metastasis. In this context, the molecular chaperone heat-shock protein 90 (Hsp90) has gained interest as a promising anticancer drug target, due to its importance in maintaining the stability, integrity, conformation and function of key oncogenic proteins. These Hsp90 "client proteins" have been demonstrated to play fundamental roles in the processes of signal transduction, cell proliferation and survival, cell cycle progression and apoptosis, as well as other features of malignant cells, such as invasion, tumor angiogenesis and metastasis. The cancer selectivity and antitumoral effects of Hsp90 inhibitors are mediated by simultaneous and combined actions, in terms of directly affecting multiple cancer targets and pathways. Several Hsp90 inhibitors, including the geldanamycin derivative 17-allylamino-17-demethoxygeldanamycin (17AAG), have displayed convincing antineoplastic efficacy and cancer selectivity in a variety of preclinical models, including gastrointestinal carcinomas. Importantly, some Hsp90 inhibitors have now progressed to phase I/II clinical testing. Against this background, the following review focuses on the current preclinical experience and value of targeting Hsp90 for the therapy of gastrointestinal carcinomas.

Dual targeting of Raf and VEGF receptor 2 reduces growth and metastasis of pancreatic cancer through direct effects on tumor cells, endothelial cells, and pericytes.

The Ras/Raf/MEK pathway represents an important oncogenic signaling pathway in gastrointestinal malignancies, including pancreatic cancer. Although activating B-Raf mutations are infrequent in pancreatic cancer, we hypothesized that targeting Raf could be valuable for therapy of this cancer entity. Moreover, as vascular endothelial growth factor receptor 2 (VEGFR2) is involved in tumor angiogenesis, we sought to investigate the effects of dual inhibition of Raf and VEGFR2 on pancreatic tumor growth, vascularization, and metastasis. Effects of a Raf/VEGFR2 inhibitor (NVP-AAL881) on pancreatic cancer cells, endothelial cells, and vascular smooth muscle cells were determined by Western blotting, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis, and migration assays, respectively. Changes in the expression of VEGF-A or survivin were investigated by ELISA and/or real-time PCR. The growth-inhibitory effects of Raf/VEGFR2 inhibition were additionally evaluated in orthotopic tumor models. Results showed that various Raf isoforms were activated in pancreatic cancer cells and NVP-AAL881 diminished the activation of MEK, Akt, Erk, and also STAT3. Moreover, dual inhibition of Raf/VEGFR2 significantly reduced VEGF expression and impaired cancer cell migration. Importantly, besides blocking VEGF-induced Erk and SAPK phosphorylation in endothelial cells, the Raf inhibitor diminished STAT3 phosphorylation, independent of a VEGFR2 blockade, and reduced the expression of survivin. In addition, cell proliferation and migration of both endothelial cells and vascular smooth muscle cells were significantly reduced. In vivo, blocking Raf/VEGFR2 significantly inhibited orthotopic tumor growth and vascularization and reduced cancer metastasis. In conclusion, blocking Raf exerts growth-inhibitory effects on pancreatic tumor cells, endothelial cells, and pericytes and elicits antiangiogenic properties. Dual targeting of Raf and VEGFR2 appears to be a valid strategy for therapy of pancreatic cancer.

Inhibition of insulin-like growth factor-I receptor (IGF-IR) using NVP-AEW541, a small molecule kinase inhibitor, reduces orthotopic pancreatic cancer growth and angiogenesis.

The insulin-like growth factor-I receptor (IGF-IR) is frequently overexpressed and constitutively activated in pancreatic cancer, thus representing a promising target for therapy. We investigated the impact of a novel inhibitor of IGF-IR (NVP-AEW541) on signalling and growth of pancreatic cancer. Human pancreatic cancer cells and endothelial cells were employed, and effects of NVP-AEW541 on signalling pathways investigated by Western blotting. NVP-AEW541 diminished the activation of IGF-IR, IRS-1, Erk, Akt and STAT3. Furthermore, NVP-AEW541 reduced cancer cell proliferation and abrogated migratory effects of IGF-I. NVP-AEW541 elicited a direct effect on endothelial cells in terms of reducing endothelial cell migration. In vivo, treatment of mice with NVP-AEW541 significantly reduced orthotopic pancreatic tumour growth, vascularisation, and VEGF expression. Interestingly, NVP-AEW541 lowered serum levels of IGF-binding-protein-3 (IGFBP-3). In conclusion, the IGF-IR inhibitor NVP-AEW541 effectively disrupts IGF-I signalling and reduces pancreatic tumour growth. Hence, blocking IGF-IR could prove valuable for targeted therapy of pancreatic cancer.

ENMD-1198, a novel tubulin-binding agent reduces HIF-1alpha and STAT3 activity in human hepatocellular carcinoma(HCC) cells, and inhibits growth and vascularization in vivo.

BACKGROUND: Hepatocellular carcinoma (HCC) represents a highly vascularized tumor entity and the process of angiogenesis is essential for the growth of HCC. Importantly, the pro-angiogenic transcription factors HIF-1alpha and STAT3 have been implicated in HCC progression, thus representing interesting targets for molecular targeted therapy. We hypothesized that therapeutic inhibition of HIF-1alpha could be achieved by using a novel tubulin-binding agent (ENMD-1198). ENMD-1198 is an analog of 2-methoxyestradiol (2ME2) with antiproliferative and antiangiogenic activity. METHODS: The human HCC cell lines HUH-7 and HepG2 were used for experiments. Effects of ENMD-1198 on constitutive and inducible (hypoxia, growth factors) activation of signaling cascades, including HIF-1alpha and STAT3, were investigated by Western blotting. Changes in VEGF expression were determined by real-time PCR. Effects of ENMD-1198 on cancer cell migration and invasion were evaluated in in vitro-assays. The growth-inhibitory effects of ENMD-1198 (200 mg/kg/day) were determined in a subcutaneous tumor model (HUH-7). RESULTS: ENMD-1198 inhibited the phosphorylation of MAPK/Erk, PI-3K/Akt and FAK. Moreover, activation of HIF-1alpha and STAT3 was dramatically reduced by ENMD-1198, which resulted in lower VEGF mRNA expression (P < 0.05). In addition, tumor cell migratory and invasive properties were significantly inhibited (P < 0.05, for both). In vivo, treatment with ENMD-1198 led to a significant reduction in tumor growth, tumor vascularization, and numbers of proliferating tumor cells (P < 0.05 for all). CONCLUSION: The novel microtubule destabilizing agent ENMD-1198 is suitable for inhibiting HIF-1alpha and STAT3 in human HCC cells and leads to reduced tumor growth and vascularization in vivo. Hence, inhibition of HIF-1alpha and STAT3 could prove valuable for therapy of hepatocellular carcinoma.

Targeting heat shock protein 90 in pancreatic cancer impairs insulin-like growth factor-I receptor signaling, disrupts an interleukin-6/signal-transducer and activator of transcription 3/hypoxia-inducible factor-1alpha autocrine loop, and reduces orthotopic tumor growth.

PURPOSE: Inhibitors of heat-shock protein 90 (Hsp90) may interfere with oncogenic signaling pathways, including Erk, Akt, and hypoxia-inducible factor-1alpha (HIF-1alpha). Because insulin-like growth factor-I receptor (IGF-IR) and signal transducer and activator of transcription 3 (STAT3) signaling pathways are implicated in the progression of pancreatic cancer, we hypothesized that blocking Hsp90 with geldanamycin derivates [17-allylamino-geldanamycin (17-AAG), 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG)] would impair IGF-I- and interleukin-6-mediated signaling and thus reduce pancreatic tumor growth and angiogenesis in vivo. EXPERIMENTAL DESIGN: Human pancreatic cancer cells (HPAF-II, L3.6pl) were used for experiments. Changes in signaling pathway activation upon Hsp90 blockade were investigated by Western blotting. Effects of Hsp90 inhibition (17-AAG) on vascular endothelial growth factor were determined by ELISA and real-time PCR. Effects of 17-DMAG (25 mg/kg; thrice a week; i.p.) on tumor growth and vascularization were investigated in a s.c. xenograft model and in an orthotopic model of pancreatic cancer. RESULTS: 17-AAG inhibited IGF-IR signaling by down-regulating IGF-IRbeta and directly impairing IGF-IR phosphorylation. Hypoxia- and IL-6-mediated activation of HIF-1alpha or STAT3/STAT5 were substantially inhibited by 17-AAG. Moreover, a novel IL-6/STAT3/HIF-1alpha autocrine loop was effectively disrupted by Hsp90 blockade. In vivo, 17-DMAG significantly reduced s.c. tumor growth and diminished STAT3 phosphorylation and IGF-IRbeta expression in tumor tissues. In an orthotopic model, pancreatic tumor growth and vascularization were both significantly reduced upon Hsp90 inhibition, as reflected by final tumor weights and CD31 staining, respectively. CONCLUSIONS: Blocking Hsp90 disrupts IGF-I and IL-6-induced proangiogenic signaling cascades by targeting IGF-IR and STAT3 in pancreatic cancer, leading to significant growth-inhibitory effects. Therefore, we suggest that Hsp90 inhibitors could prove to be valuable in the treatment of pancreatic cancer.

Dual inhibition of Raf and VEGFR2 reduces growth and vascularization of hepatocellular carcinoma in an experimental model.

BACKGROUND AND AIMS: Activation of the mitogen-activated protein kinase-extracellular-signal-regulated kinase (ERK) pathways plays an important role in the progression of hepatocellular carcinoma (HCC). Importantly, Raf kinases are principal effectors within this oncogenic signaling cascade. We hypothesized that concomitant inhibition of Raf and vascular endothelial growth factor receptor 2 (VEGFR2) will affect tumor growth and angiogenesis of HCC. MATERIALS AND METHODS: Human HCC cell lines, endothelial cells (EC), and vascular smooth muscle cells (VSMC) were used. For blocking Raf kinase and VEGFR2, the small molecule inhibitor NVP-AAL881 (Novartis, USA) was used. Activation of signaling intermediates was assessed by Western blotting, and changes in cell motility were evaluated in migration assays. Effects of NVP-AAL881 on HCC growth were determined in a subcutaneous tumor model. RESULTS: NVP-AAL881 disrupted activation of ERK and STAT3 in HCC cells and reduced cancer cell motility. In addition, the migration of ECs and VSMC was also significantly impaired. In ECs, HCC-conditioned media-induced activation of STAT3 was diminished by NVP-AAL881 treatment. In vivo, NVP-AAL881 significantly reduced tumor growth, CD31-vessel area, and numbers of BrdU-positive proliferating tumor cells. CONCLUSIONS: Combined inhibition of Raf and VEGFR2 disrupts oncogenic signaling and efficiently reduces tumor growth and vascularization of HCC. Hence, this strategy could prove valuable for therapy of HCC.

Sirolimus promotes tolerance for donor and recipient antigens after MHC class II disparate bone marrow transplantation in rats.

OBJECTIVE: Mixed chimerism after allogeneic bone marrow transplantation (BMT) promotes immunologic tolerance. Graft-vs-host disease (GvHD) can occur when immunosuppressive control of the graft fails. Here we evaluate the influence of concurrent immunosuppression after irradiation-based induction therapy on development of tolerance and GvHD. METHODS: Conditioning was performed by different doses of total body irradiation (TBI) in a major histocompatibility complex (MHC) class II disparate rat BMT model. Animals received subsequent immunosuppression with either cyclosporine A (CsA) or sirolimus. Nonresponsiveness toward donor and recipient antigens was demonstrated by development of mixed chimerism and/or GvHD. RESULTS: Administration of 10 Gy of TBI prior to BMT alone was associated with severe GvHD. Induction therapy with 8 Gy of TBI alone led to graft rejection in the long-term. Two weeks of immunosuppression with CsA after 8 Gy of TBI resulted in transient chimerism, but was finally associated with a combination of fatal GvHD and graft rejection. Six gray of TBI with CsA treatment for 14 or 28 days caused only mild GvHD, but did not lead to stable chimerism. In contrast, treatment with sirolimus was associated with stable chimerism after 8 Gy of TBI (14-day course) and 6 Gy of TBI (28-day course) accompanied by a low incidence of GvHD. CONCLUSIONS: In contrast to CsA, sirolimus facilitates development of tolerance after MHC class II disparate BMT and irradiation-based conditioning, with a low risk of GvHD. Therefore, sirolimus has promising characteristics for inclusion in immunosuppressive protocols.

Blocking heat shock protein-90 inhibits the invasive properties and hepatic growth of human colon cancer cells and improves the efficacy of oxaliplatin in p53-deficient colon cancer tumors in vivo.

We recently showed that inhibition of heat shock protein 90 (Hsp90) decreases tumor growth and angiogenesis in gastric cancer through interference with oncogenic signaling pathways. However, controversy still exists about the antimetastatic potential of Hsp90 inhibitors. Moreover, in vitro studies suggested that blocking Hsp90 could overcome p53-mediated resistance of cancer cells to oxaliplatin. We therefore hypothesized that blocking oncogenic signaling with a Hsp90 inhibitor would impair metastatic behavior of colon cancer cells and also improve the efficacy of oxaliplatin in vivo. Human colon cancer cells (HCT116, HT29, and SW620) and the Hsp90 inhibitor 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) were used for experiments. In vitro, 17-DMAG substantially inhibited phosphorylation of epidermal growth factor receptor, c-Met, and focal adhesion kinase, overall resulting in a significant decrease in cancer cell invasiveness. Importantly, 17-DMAG led to an up-regulation of the transcription factor activating transcription factor-3, a tumor suppressor and antimetastatic factor, on mRNA and protein levels. In a cell death ELISA, 17-DMAG markedly induced apoptosis in both p53-wt and p53-deficient cells. In vivo, 17-DMAG significantly reduced tumor growth and vascularization. Furthermore, blocking Hsp90 reduced hepatic tumor burden and metastatic nodules in an experimental model of hepatic colon cancer growth. Importantly, combining oxaliplatin with 17-DMAG in vivo significantly improved growth inhibitory and proapoptotic effects on p53-deficient cells, compared with either substance alone. In conclusion, inhibition of Hsp90 abrogates the invasive properties of colon cancer cells and modulates the expression of the antimetastatic factor activating transcription factor-3. Hence, targeting Hsp90 could prove valuable for treatment of advanced colorectal cancer by effectively inhibiting colon cancer growth and hepatic metastasis and improving the efficacy of oxaliplatin.

Inhibition of heat shock protein 90 impairs epidermal growth factor-mediated signaling in gastric cancer cells and reduces tumor growth and vascularization in vivo.

Oncogenic signaling through activation of epidermal growth factor receptor (EGFR), HER-2, and hypoxia inducible-factor-1alpha (HIF-1alpha) has been implicated in gastric cancer growth and angiogenesis through up-regulation of vascular endothelial growth factor (VEGF). Recently, heat shock protein 90 (Hsp90) has been identified as a critical regulator of oncogenic protein stability, including EGFR, HER-2, and HIF-1alpha. We hypothesized that inhibition of Hsp90 impairs EGF- and hypoxia-mediated angiogenic signaling in gastric cancer cells and consequently inhibits angiogenesis and tumor growth. In vitro, the geldanamycin derivate 17-allylamino-17-demethoxygeldanamycin (17-AAG) led to marked reduction in constitutive and inducible activation of extracellular signal-regulated kinase 1/2, Akt, and signal transducer and activator of transcription 3 and decreased nuclear HIF-1alpha protein. In addition, EGFR and HER-2 were down-regulated after Hsp90 inhibition. With respect to regulation of angiogenic molecules, 17-AAG significantly reduced EGF-mediated VEGF secretion. Phosphorylation of focal adhesion kinase and paxillin were both abrogated by 17-AAG, which resulted in significant impairment of cancer cell motility. Interestingly, cytotoxic effects of 17-AAG in vitro were higher on cancer cells and gastric fibroblasts than on pericytes. In vivo, the water-soluble compound 17-dimethylaminoethylamino-17-demethoxygeldanamycin (17-DMAG; 25 mg/kg, thrice per week) significantly reduced s.c. xenografted tumor growth. By immunohistochemistry, 17-DMAG significantly reduced vessel area and numbers of proliferating tumor cells in sections. Furthermore, similar significant growth-inhibitory effects of 17-DMAG were achieved when administered as low-dose therapy (5 mg/kg, thrice per week). In conclusion, blocking Hsp90 disrupts multiple proangiogenic signaling pathways in gastric cancer cells and inhibits xenografted tumor growth in vivo. Hence, gastric cancer harbors attractive molecular targets for therapy with Hsp90 inhibitors, which could lead to improved efficacy of antineoplastic therapy regimens.

Mycophenolate mofetil inhibits tumor growth and angiogenesis in vitro but has variable antitumor effects in vivo, possibly related to bioavailability.

BACKGROUND: Identifying immunosuppressive agents with antitumor effects could help address the problem of posttransplant malignancy. Here we tested for potential inhibitory effects of mycophenolate mofetil (MMF) on tumors in vitro and in vivo. METHODS: Mouse CT26 colon adenocarcinoma, B16 melanoma, and human TMK1 gastric adenocarcinoma cells were tested for in vitro growth in the presence of MMF. In vitro angiogenesis was tested with a rat aortic-ring assay. Tumor cells were implanted into dorsal skinfold chambers (DSFC) to assess in vivo angiogenesis. Subcutaneous tumor growth was determined in mice receiving MMF. RESULTS: MMF caused a dose-dependent reduction in tumor cell numbers in vitro, starting between 0.1 to 1 microM. Vessel sprouting from aortic rings was markedly blocked by similar concentrations of MMF. In vivo, however, DSFC results showed a marginal reduction in CT26 tumor angiogenesis with MMF doses of 40 or 80 mg/kg/day, although MMF did inhibit TMK1 vascularity. Moreover, 80 mg/kg/day MMF did not reduce subcutaneous CT26 tumor volumes, but did slightly inhibit B16 and TMK1 expansion. Interestingly, the mycophenolic acid (MPA) blood level 2 hr after 80 mg/kg/day MMF bolus dosing was near 14 mg/L, but decreased dramatically thereafter, suggesting a drug availability issue. Indeed, intermittent 2-hr MMF pulses in tumor-cell cultures substantially reduced the antiproliferative effect of MPA. CONCLUSION: MMF strongly inhibits tumor cell growth and angiogenesis in vitro, but only marginally inhibits tumors in vivo. These contrasting results may relate to drug availability, where intermittent exposure of tumor cells to immunosuppressive doses of MMF substantially reduce its potential antitumor effects.

Regulation of cyclooxygenase-2 (COX-2) expression in human pancreatic carcinoma cells by the insulin-like growth factor-I receptor (IGF-IR) system.

Both the insulin-like growth factor-I receptor (IGF-IR) and cyclooxygenase-2 (COX-2) are frequently overexpressed in pancreatic cancer. We hypothesized that IGF-IR is directly involved in induction of COX-2 and sought to investigate signaling pathways mediating this effect. Pancreatic cancer cells (L3.6pl) were stably transfected with a dominant-negative receptor (IGF-IR DN) construct or empty vector (pcDNA). Cells were stimulated with IGF-I to determine activated signaling intermediates and induction of COX-2. Signaling pathways mediating COX-2 induction were identified using signaling inhibitors. IGF-I up-regulated COX-2 selectively via the MAPK/(Erk-1/2) pathway. In addition, IGF-IR DN cells showed a marked decrease in constitutive COX-2 and a blunted response to IGF-I. Similarly, treatment with an anti-IGF-IR antibody effectively inhibited IGF-IR and MAPK/Erk activation and decreased COX-2 in parental cells. In conclusion, activation of IGF-IR mediates COX-2 expression in human pancreatic cancer cells.

The direct effects of anti-vascular endothelial growth factor therapy on tumor cells.

The vascular endothelial growth factor (VEGF) family members are essential mediators of tumor angiogenesis. The multiple functions of the VEGFs are mediated through complex interactions between their ligands, the high-affinity tyrosine kinase receptors, and co-receptors (neuropilins). Emerging evidence has shown that these receptors, formerly described as being exclusively expressed on endothelial cells, are also expressed on a number of nonendothelial cells, including tumor cells. Moreover, it has been shown that their receptors (VEGFRs) are functional in a number of nonendothelial systems, where they can serve as targets for anti-VEGF therapy. As the expression of VEGFRs on tumor cells contributes to the understanding of the complex roles of VEGF within the tumor microenvironment and elucidation of VEGF activity might further refine antineoplastic regimens, this article will review the main effects and selective interactions of the VEGFRs, the evidence for their expression and function on tumor cells, and the direct efforts of anti-VEGF therapy on tumor cells.