Mitogenic Pathways Research Articles

Abstract 3208: Identification and characterization of a small molecule involved in the downregulation of RET transcription in MTC

Abstract The gain of function mutations in the RET proto-oncogene, which results in the constitutive activation of the mitogenic pathways, were found mainly in Medullary Thyroid Carcinoma (MTC). In our study, a new therapeutic strategy has been explored to repress the expression of the RET gene by targeting the transcriptional activation of this gene with small molecules. The transcriptional inhibitor of the RET gene was initially screened from the NCI chemical libraries using the cell-based luciferase reporter gene assay, which has a promoter sequence identical to the RET promoter. As a result of our screening effort, a small molecule, nonactin, has been identified as a potential lead compound, downregulating the transcriptional activity of the RET gene promoter. The effect of this molecule in the downregulation of the RET mRNA levels and the protein levels was confirmed by RT-PCR and western blotting respectively. We also observed that Egr1 expression is downergulated in a dose dependent manner by this small molecule, which is in correlation with the RET downregulation. The RET promoter has a highly conserved GC rich region, which acts as a binding site for Egr-1, which is involved in the transcription of many growth factor genes. Thus, we conclude that Egr1 would be one of important transcriptional factors involved in the activation of the RET and nonactin inhibits the expression of Egr1, which in turn downregulates the transcription of RET oncogene. Citation Format: Vishnu Muthuraj Kumarasamy, Yoon-Joo Shin, Daekyu Sun. Identification and characterization of a small molecule involved in the downregulation of RET transcription in MTC. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3208. doi:10.1158/1538-7445.AM2014-3208

Abstract 1820: Dynamics of HER-2 loss in mammary carcinoma of human HER-2 trangenic mice

Abstract Progression of HER-2+ breast cancer can result in the emergence of HER-2-negative tumor variants that activate alternative mitogenic pathways, either spontaneously or after therapy. We found that HER-2 loss occurs even in transgenic mouse models in which the oncogene is driven by viral promoters, thus mammary carcinoma of human HER-2 transgenic mice (huHER-2 mice) can be used to study not only the early phases of HER-2-driven mammary carcinogenesis, but also tumor progression beyond HER-2 addiction. Primary mammary carcinomas of huHER-2 mice express high levels of the oncogene, with a marked intratumoral heterogeneity. Cell lines grown from HER-2+ mammary carcinomas frequently undergo a progressive loss of expression. We have established a model system consisting of cell lines, clones and variants that exhibit one of three phenotypes: a) high and stable HER-2 expression in vitro and in vivo, b) high but labile HER-2 expression which is lost either during in vitro culture, after tumor growth in mice or after in vitro treatment with trastuzumab, and c) complete loss of HER-2 expression After HER-2 loss, most variants displayed a transition to an elongated, motile phenotype (epithelial to mesenchymal transition), an increased ability to generate mammospheres, a reduced expression of CD24 and an increased expression of CD44 (denoting mammary cancer stem cells). Tumorigenic and metastatic ability of HER-2-negative cells was increased in comparison to HER-2+ cells. As expected, HER-2 loss was accompanied by resistance to HER-2 targeted monoclonal antibodies and small molecule inhibitors. The study of therapeutic agents directed against downstream targets showed that HER-2-loss was accompanied by a loss of sensitvity to a Src inhibitor, whereas a PI3K inhibitor was highly effective regardless of HER-2 expression. Our results indicate that human HER-2 transgenic mice are a useful model to study the dynamics of HER-2 loss in advanced HER-2+ mammary carcinoma, and to analyze alternative therapeutic strategies. Supported by grants from the Italian Association for Cancer Research (AIRC). Citation Format: Patrizia Nanni, Arianna Palladini, Lorena Landuzzi, Massimiliano Dall'Ora, Marianna Ianzano, Valentina Grosso, Dario Ranieri, Giordano Nicoletti, Roberta Laranga, Carla De Giovanni, Manuela Iezzi, Pier-Luigi Lollini. Dynamics of HER-2 loss in mammary carcinoma of human HER-2 trangenic mice. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1820. doi:10.1158/1538-7445.AM2014-1820

Distinct signaling mechanisms of mTORC1 and mTORC2 in glioblastoma multiforme: a tale of two complexes.

Mechanistic target of rapamycin (mTOR) is a serine-threonine kinase that functions via two multiprotein complexes, namely mTORC1 and mTORC2, each characterized by different binding partners that confer separate functions. mTORC1 function is tightly regulated by PI3-K/Akt and is sensitive to rapamycin. mTORC2 is sensitive to growth factors, not nutrients, and is associated with rapamycin-insensitivity. mTORC1 regulates protein synthesis and cell growth through downstream molecules: 4E-BP1 (also called EIF4E-BP1) and S6K. Also, mTORC2 is thought to modulate growth factor signaling by phosphorylating the C-terminal hydrophobic motif of some AGC kinases such as Akt and SGK. Recent evidence has suggested that mTORC2 may play an important role in maintenance of normal as well as cancer cells by virtue of its association with ribosomes, which may be involved in metabolic regulation of the cell. Rapamycin (sirolimus) and its analogs known as rapalogues, such as RAD001 (everolimus) and CCI-779 (temsirolimus), suppress mTOR activity through an allosteric mechanism that acts at a distance from the ATP-catalytic binding site, and are considered incomplete inhibitors. Moreover, these compounds suppress mTORC1-mediated S6K activation, thereby blocking a negative feedback loop, leading to activation of mitogenic pathways promoting cell survival and growth. Consequently, mTOR is a suitable target of therapy in cancer treatments. However, neither of these complexes is fully inhibited by the allosteric inhibitor rapamycin or its analogs. In recent years, new pharmacologic agents have been developed which can inhibit these complexes via ATP-binding mechanism, or dual inhibition of the canonical PI3-K/Akt/mTOR signaling pathway. These compounds include WYE-354, KU-003679, PI-103, Torin1, and Torin2, which can target both complexes or serve as a dual inhibitor for PI3-K/mTOR. This investigation describes the mechanism of action of pharmacological agents that effectively target mTORC1 and mTORC2 resulting in suppression of growth, proliferation, and migration of tumor and cancer stem cells.

Myocyte growth, repair, and oxidative stress following pediatric heart transplantation.

Cardiac remodeling is associated with plasma biomarkers of fibrinogenesis, inflammation, and oxidative stress, and upregulation of mitogenic, pro-fibrotic, and apoptotic signaling pathways. Our primary objective was to evaluate biomarker and subcellular myocardial changes in pediatric heart transplant recipients. Fifty-two-week prospective, randomized (tacrolimus, Tac, vs. cyclosporine, CsA), open-label, parallel group study. Serial myocardial biopsies were probed for mitogenic and pro-inflammatory proteins. Plasma biomarkers of oxidative stress (F2α isoprostanes, nitrotyrosine), and inflammation and oxidation (hsCRP and cystatin-C) were measured. Nine of 11 randomized patients completed the study (four Tac, five CsA). Mean levels of F2α isoprostanes, hsCRP, and cystatin-C were maximal at Week 2. Peak activation of all MAP kinases in myocardial tissue was maximal at Week 10; no association was seen with rejection. Cardiac Bax/Bcl-2 levels (index of apoptosis) correlated negatively with F2α isoprostanes at Week 2 (r=-0.88) and with hsCRP at Week 52 (r=-0.67). At Week 52, hsCRP levels correlated positively with molecular indices of cardiac cell growth. We found evidence of systemic and myocardial oxidative damage and inflammation early posttransplant, which may be related to the remodeling process. Further study is needed to better understand the cardiac and systemic repair processes following pediatric heart transplantation.

Broussonetia kazinoki modulates the expression of VEGFR-2 and MMP-2 through the inhibition of ERK, Akt and p70S6K‑dependent signaling pathways: Its implication in endothelial cell proliferation, migration and tubular formation.

Broussonetia kazinoki (BK) has been used as a traditional medicine to improve vision, as well as for inflammatory and infectious diseases. In the present study, we investigated the effects and molecular mechanism of the ethanolic extract of BK on cell proliferation, migration and tubular formation in vascular endothelial growth factor-A (VEGF-A)-treated human umbilical vein endothelial cells. BK treatment inhibited VEGF-A-stimulated endothelial cell proliferation through the downregulation of cell cycle-related proteins including cyclin-dependent kinases and cyclins. Moreover, BK treatment suppressed cell migration and tubular formation in response to VEGF-A. These anti-angiogenic activities of BK were associated with the inactivation of mitogenic signaling pathways including extracellular signal-regulated kinase, Akt and p70S6K, and the subsequent downregulation of VEGFR-2 and matrix metalloproteinase-2. Taken together, these findings suggest further evaluation and development of BK as a potential therapeutic agent for the treatment and prevention of angiogenesis-related diseases including cancer.

One Hippo and many masters: differential regulation of the Hippo pathway in cancer.

The Hippo/MST2 (mammalian sterile 20-like kinase 2) pathway is a signalling cascade evolutionarily conserved in its structure. Originally described in Drosophila melanogaster as a regulator of organ size, this pathway has greater functions in mammals. Disturbance of mammalian MST2 pathway is associated with tumorigenesis by affecting apoptosis, cell cycle and polarity. In addition, this pathway has been shown to cross-talk with mitogenic pathways at multiple levels. In the present mini-review, we discuss our contribution highlighting the regulation of MST2 signalling by frequently observed oncogenic perturbations affecting mitogenic pathways. In particular, we review the role of RAS isoforms and PI3K (phosphoinositide 3-kinase)/Akt in the regulation of MST2 activity by phosphorylation. We also put the emphasis on RAF-induced control of MST2 signalling by protein-protein interactions. Finally, we recapitulate some of the direct mechanisms, such as ubiquitin-dependent degradation or gene silencing by promoter hypermethylation, involved in MST2 pathway component down-regulation in cancers.

Time-dependent effect of orchidectomy on vascular nitric oxide and thromboxane A2 release. Functional implications to control cell proliferation through activation of the epidermal growth factor receptor.

This study analyzes whether the release of nitric oxide (NO) and thromboxane A2 (TXA2) depends on the time lapsed since gonadal function is lost, and their correlation with the proliferation of vascular smooth muscle cells (VSMC) mediated by the epidermal growth factor receptor (EGFR). For this purpose, aortic and mesenteric artery segments from control and 6-weeks or 5-months orchidectomized rats were used to measure NO and TXA2 release. The results showed that the basal and acetylcholine (ACh)-induced NO release were decreased 6 weeks post-orchidectomy both in aorta and mesenteric artery, but were recovered 5 months thereafter up to levels similar to those found in arteries from control rats. The basal and ACh-induced TXA2 release increased in aorta and mesenteric artery 6 weeks post-orchidectomy, and was maintained at high levels 5 months thereafter. Since we previously observed that orchidectomy, which decreased testosterone level, enlarged the muscular layer of mesenteric arteries, the effect of testosterone on VSMC proliferation was analyzed. The results showed that treatment of cultured VSMC with testosterone downregulated mitogenic signaling pathways initiated by the ligand-dependent activation of the EGFR. In contrast, the EGFR pathways were constitutively active in mesenteric arteries of long-term orchidectomized rats. Thus, the exposure of mesenteric arteries from control rats to epidermal growth factor (EGF) induced the activation of EGFR signaling pathways. However, the addition of EGF to arteries from orchidectomized rats failed to induce a further activation of these pathways. In conclusion, this study shows that the release of NO depends on the time lapsed since the gonadal function is lost, while the release of TXA2 is already increased after short periods post-orchidectomy. The alterations in these signaling molecules could contribute to the constitutive activation of the EGFR and its downstream signaling pathways after long period post-orchidectomy enhancing the proliferation of the vascular muscular layer.

Growth hormone-releasing hormone antagonists abolish the transactivation of human epidermal growth factor receptors in advanced prostate cancer models.

Growth hormone-releasing hormone (GHRH) and its receptors have been implicated in a variety of cellular phenotypes related with tumorigenesis process. Human epidermal growth factor receptor family members (HER) such as EGFR and HER2 are involved in mitogenic signaling pathways implicated in the progression of prostate cancer. We analyzed the cross-talk between GHRH and EGF receptors in prostate cancer. The effects of GHRH in HER signaling were evaluated on human androgen-independent PC3 prostate cancer cells in vitro and GHRH antagonist in vitro and in nude mice xenografts of PC3 prostate cancer. Time-course studies indicated that GHRH had a stimulatory activity on both the expression of EGFR and HER2. GHRH analogues, JMR-132 and JV-1-38, endowed with antagonistic activity for GHRH receptors, abrogated the response to GHRH in PC3 cells. GHRH stimulated a rapid ligand-independent activation of EGFR and HER2 involving at least cAMP/PKA and Src family signaling pathways. GHRH also stimulated a slow ligand-dependent activation of EGFR and HER2 involving an extracellular pathway with an important role for ADAM. Preliminary results also revealed an increase of mRNA for GHRH and GHRH receptor induced by EGF. The inhibition of tumor growth, in vivo, was associated with a substantial reduction in the expression of mRNA and protein levels of EGFR and HER2 in the tumors. GHRH antagonist JV-1-38, significantly decreased the phosphorylated Src levels. The cross-talk between HER and GHRH-R may be impeded by combining drugs acting upon GHRH receptors and HER family members in human advanced prostate cancer.

FGFR3 translocations in bladder cancer: differential sensitivity to HSP90 inhibition based on drug metabolism.

Activating mutations and/or overexpression of FGFR3 are common in bladder cancer, making FGFR3 an attractive therapeutic target in this disease. In addition, FGFR3 gene rearrangements have recently been described that define a unique subset of bladder tumors. Here, a selective HSP90 inhibitor, ganetespib, induced loss of FGFR3-TACC3 fusion protein expression and depletion of multiple oncogenic signaling proteins in RT112 bladder cells, resulting in potent cytotoxicity comparable with the pan-FGFR tyrosine kinase inhibitor BGJ398. However, in contrast to BGJ398, ganetespib exerted pleiotropic effects on additional mitogenic and survival pathways and could overcome the FGFR inhibitor-resistant phenotype of FGFR3 mutant-expressing 97-7 and MHG-U3 cells. Combinatorial benefit was observed when ganetespib was used with BGJ398 both in vitro and in vivo. Interestingly, two additional FGFR3 fusion-positive lines (RT4 and SW480) retained sensitivity to HSP90 inhibitor treatment by the ansamycins 17-AAG and 17-DMAG yet displayed intrinsic resistance to ganetespib or AUY922, both second-generation resorcinol-based compounds. Both cell lines, compared with RT112, expressed considerably higher levels of endogenous UGT1A enzyme; this phenotype resulted in a rapid glucuronidation-dependent metabolism and subsequent efflux of ganetespib from SW780 cells, thus providing a mechanism to account for the lack of bioactivity. Pharmacologic blockade of the molecular chaperone HSP90 represents a promising approach for treating bladder tumors driven by oncogenic gene rearrangements of FGFR3. Furthermore, UDP-glucuronosyltransferase enzyme expression may serve as a predictive factor for clinical response to resorcinol-based HSP90 inhibitors.

Roles of oxidative stress in the development and progression of breast cancer.

Oxidative stress is caused by an imbalance in the redox status of the body. In such a state, increase of free radicals in the body can lead to tissue damage. One of the most important species of free radicals is reactive oxygen species (ROS) produced by various metabolic pathways, including aerobic metabolism in the mitochondrial respiratory chain. It plays a critical role in the initiation and progression of various types of cancers. ROS affects different signaling pathways, including growth factors and mitogenic pathways, and controls many cellular processes, including cell proliferation, and thus stimulates the uncontrolled growth of cells which encourages the development of tumors and begins the process of carcinogenesis. Increased oxidative stress caused by reactive species can reduce the body's antioxidant defense against angiogenesis and metastasis in cancer cells. These processes are main factors in the development of cancer. Bimolecular reactions cause free radicals in which create such compounds as malondialdehyde (MDA) and hydroxyguanosine. These substances can be used as indicators of cancer. In this review, free radicals as oxidizing agents, antioxidants as the immune system, and the role of oxidative stress in cancer, particularly breast cancer, have been investigated in the hope that better identification of the factors involved in the occurrence and spread of cancer will improve the identification of treatment goals.

A mechanistic study on the anti-cancer activity of ethyl caffeate in human ovarian cancer SKOV-3 cells

In the present study, we investigated the effect and molecular mechanism of ethyl caffeate (EC), a natural phenolic compound isolated from Ligularia fischeri, on human ovarian cancer cell proliferation and progression. EC-mediated inhibition of cell proliferation in SKOV-3 cells was accompanied by reduced expression of cell cycle-related proteins such as cyclin-dependent kinases and cyclins, resulting in pRb hypophosphorylation and G1 phase cell cycle arrest. Moreover, EC treatment markedly inhibited cell migration and invasion. These regulatory effects of EC on ovarian cancer cell proliferation, migration and invasion were associated with inactivation of mitogenic signaling pathways such as Akt, ERK and p38MAPK, and down-regulation of cell surface signaling molecules including receptor tyrosine kinases, integrin α3β1 and N-cadherin. Taken together, these findings suggest further evaluation and development of EC for the treatment and prevention of ovarian cancer.

Insulin analogs and cancer: a note of caution.

In view of the lifelong exposure and large patient populations involved, insulin analogs with an increased mitogenic effect in comparison to human insulin may potentially constitute a major health problem, since these analogs may possibly induce the growth of pre-existing neoplasms. At present, the available data suggest that insulin analogs are safe. In line with these findings, we observed that serum of diabetic patients treated with insulin analogs, compared to that of diabetic patients treated with human insulin, did not induce an increased phosphorylation of tyrosine residues of the insulin-like growth factor-I receptor (IGF-IR). However, the classical model of the IGF-IR signaling may be insufficient to explain (all) mitogenic effects of insulin analogs since also non-canonical signaling pathways of the IGF-IR may play a major role in this respect. Although phosphorylation of tyrosine residues of the IGF-IR is generally considered to be the initial activation step within the intracellular IGF-IR signaling pathway, it has been found that cells undergo a signaling switch under hyperglycemic conditions. After this switch, a completely different mechanism is utilized to activate the mitogenic (mitogen-activated protein kinase) pathways of the IGF-IR that is independent from tyrosine phosphorylation of the IGF-IR. At present it is unknown whether activation of this alternative intracellular pathway of the IGF-IR occurs during hyperglycemia in vivo and whether it is stronger in patients treated with (some) insulin analogs than in patients treated with human insulin. In addition, it is unknown whether the insulin receptors (IRs) also undergo a signaling switch during hyperglycemia. This should be investigated in future studies. Finally, relative overexpression of IR isoform A (IR-A) in (pre) cancer tissues may play a key role in the development and progression of human cancers during treatment with insulin (analogs). Further studies are required to unravel whether the IR-A is involved in the development of cancers and whether, in this respect (some) insulin analogs differ from human insulin.

Olive phenolics as c-Met inhibitors: (-)-Oleocanthal attenuates cell proliferation, invasiveness, and tumor growth in breast cancer models.

Dysregulation of the Hepatocyte growth factor (HGF)/c-Met signaling axis upregulates diverse tumor cell functions, including cell proliferation, survival, scattering and motility, epithelial-to-mesenchymal transition (EMT), angiogenesis, invasion, and metastasis. (-)-Oleocanthal is a naturally occurring secoiridoid from extra-virgin olive oil, which showed antiproliferative and antimigratory activity against different cancer cell lines. The aim of this study was to characterize the intracellular mechanisms involved in mediating the anticancer effects of (-)-oleocanthal treatment and the potential involvement of c-Met receptor signaling components in breast cancer. Results showed that (-)-oleocanthal inhibits the growth of human breast cancer cell lines MDA-MB-231, MCF-7 and BT-474 while similar treatment doses were found to have no effect on normal human MCF10A cell growth. In addition, (-)-oleocanthal treatment caused a dose-dependent inhibition of HGF-induced cell migration, invasion and G1/S cell cycle progression in breast cancer cell lines. Moreover, (-)-oleocanthal treatment effects were found to be mediated via inhibition of HGF-induced c-Met activation and its downstream mitogenic signaling pathways. This growth inhibitory effect is associated with blockade of EMT and reduction in cellular motility. Further results from in vivo studies showed that (-)-oleocanthal treatment suppressed tumor cell growth in an orthotopic model of breast cancer in athymic nude mice. Collectively, the findings of this study suggest that (-)-oleocanthal is a promising dietary supplement lead with potential for therapeutic use to control malignancies with aberrant c-Met activity.

Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells

Breast cancer is the leading cause of cancer-related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER-α and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER-α and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen-dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy.

Nicotine increases the resistance of lung cancer cells to cisplatin through enhancing Bcl-2 stability

Background:Nicotine is able to activate mitogenic signalling pathways, which promote cell growth or survival as well as increase chemoresistance of cancer cells. However, the underlying mechanisms are not fully understood.Methods:In this study, we used immunoblotting and immunoprecipitation methods to test the ubiquitination and degradation of Bcl-2 affected by nicotine in lung cancer cells. Apoptotic assay was also used to measure the antagonising effect of nicotine on cisplatin-mediated cytotoxicity.Results:We demonstrated that the addition of nicotine greatly attenuated Bcl-2 ubiquitination and degradation, which further desensitised lung cancer cells to cisplatin-induced cytotoxicity. In this process, Bcl-2 was persistently phosphorylated in the cells cotreated with nicotine and cisplatin. Furthermore, Akt was proven to be responsible for sustained activation of Bcl-2 by nicotine, which further antagonised cisplatin-mediated apoptotic signalling.Conclusions:Our study suggested that nicotine activates its downstream signalling to interfere with the ubiquitination process and prevent Bcl-2 from being degraded in lung cancer cells, resulting in the increase of chemoresistance.

Human β-Cell Proliferation and Intracellular Signaling Part 2: Still Driving in the Dark Without a Road Map

Enhancing β-cell proliferation is a major goal for type 1 and type 2 diabetes research. Unraveling the network of β-cell intracellular signaling pathways that promote β-cell replication can provide the tools to address this important task. In a previous Perspectives in Diabetes article, we discussed what was known regarding several important intracellular signaling pathways in rodent β-cells, including the insulin receptor substrate/phosphatidylinositol-3 kinase/Akt (IRS-PI3K-Akt) pathways, glycogen synthase kinase-3 (GSK3) and mammalian target of rapamycin (mTOR) S6 kinase pathways, protein kinase Cζ (PKCζ) pathways, and their downstream cell-cycle molecular targets, and contrasted that ample knowledge to the small amount of complementary data on human β-cell intracellular signaling pathways. In this Perspectives, we summarize additional important information on signaling pathways activated by nutrients, such as glucose; growth factors, such as epidermal growth factor, platelet-derived growth factor, and Wnt; and hormones, such as leptin, estrogen, and progesterone, that are linked to rodent and human β-cell proliferation. With these two Perspectives, we attempt to construct a brief summary of knowledge for β-cell researchers on mitogenic signaling pathways and to emphasize how little is known regarding intracellular events linked to human β-cell replication. This is a critical aspect in the long-term goal of expanding human β-cells for the prevention and/or cure of type 1 and type 2 diabetes.

Platelet-derived Growth Factor-C (PDGF-C) Induces Anti-apoptotic Effects on Macrophages through Akt and Bad Phosphorylation

PDGF-C, which is abundant in the malignant breast tumor microenvironment, plays an important role in cell growth and survival. Because tumor-associated macrophages (TAMs) contribute to cancer malignancy, macrophage survival mechanisms are an attractive area of research into controlling tumor progression. In this study, we investigated PDGF-C-mediated signaling pathways involved in anti-apoptotic effects in macrophages. We found that the human malignant breast cancer cell line MDA-MB-231 produced high quantities of PDGF-C, whereas benign MCF-7 cells did not. Recombinant PDGF-C induced PDGF receptor α chain phosphorylation, followed by Akt and Bad phosphorylation in THP-1-derived macrophages. MDA-MB-231 culture supernatants also activated macrophage PDGF-Rα. PDGF-C prevented staurosporine-induced macrophage apoptosis by inhibiting the activation of caspase-3, -7, -8, and -9 and cleavage of poly(ADP-ribose) polymerase. Finally, TAMs isolated from the PDGF-C knockdown murine breast cancer cell line 4T1 and PDGF-C knockdown MDA-MB-231-derived tumor mass showed higher rates of apoptosis than the respective WT controls. Collectively, our results suggest that tumor cell-derived PDGF-C enhances TAM survival, promoting tumor malignancy.

The Cyp2c44 Epoxygenase Regulates Epithelial Sodium Channel Activity and the Blood Pressure Responses to Increased Dietary Salt

Hypertension is a major risk factor for cerebral, cardiovascular, and renal disease, and its prevalence and devastating consequences raises a need for new strategies for its early diagnosis and treatment. We show here that lack of a Cyp2c44 epoxygenase causes dietary salt-sensitive hypertension, a common form of the human disease. Cyp2c44(-/-) mice on normal salt diets are normotensive but become hypertensive when fed high salt. Hypertensive Cyp2c44(-/-) mice show a hyperactive kidney epithelial sodium channel (ENaC) and reductions in ERK1/2 and ENaC subunit phosphorylation. The demonstration that amiloride, an ENaC inhibitor, lowers the blood pressure of hypertensive Cyp2c44(-/-) mice identifies a role for the channel in the hypertensive phenotype of the animals. These studies: (a) identify an antihypertensive role for the kidney Cyp2c44 epoxygenase and for its epoxyeicosatrienoic acid (EET) metabolites in the in vivo control of ENaC activity and the activation of mitogenic kinase pathways; (b) provide evidence for a Cyp2c44 epoxygenase, EET-mediated mechanism of ENaC regulation involving an ERK1/2-catalyzed threonine phosphorylation of the channel γ subunit: and (c) characterize a common scientific platform that could explain the seemingly unrelated biological activities attributed to the epoxygenase metabolites in cell proliferation, angiogenesis, channel activity, and blood pressure control. It is expected that these results will serve as a basis for the development of novel strategies for the early diagnosis and treatment of hypertension and of pathophysiologies associated with dysfunctional mitogenic signaling.

From Scourge to Cure: Tumour-Selective Viral Pathogenesis as a New Strategy against Cancer

Tumour mutations corrupt cellular pathways, and accumulate to disrupt, dysregulate, and ultimately avoid mechanisms of cellular control. Yet the very changes that tumour cells undergo to secure their own growth success also render them susceptible to viral infection. Enhanced availability of surface receptors, disruption of antiviral sensing, elevated metabolic activity, disengagement of cell cycle controls, hyperactivation of mitogenic pathways, and apoptotic avoidance all render the malignant cell environment highly supportive to viral replication. The therapeutic use of oncolytic viruses (OVs) with a natural tropism for infecting and subsequently lysing tumour cells is a rapidly progressing area of cancer research. While many OVs exhibit an inherent degree of tropism for transformed cells, this can be further promoted through pharmacological interventions and/or the introduction of viral mutations that generate recombinant oncolytic viruses adapted to successfully replicate only in a malignant cellular environment. Such adaptations that augment OV tumour selectivity are already improving the therapeutic outlook for cancer, and there remains tremendous untapped potential for further innovation.

DUSP1 Is a Novel Target for Enhancing Pancreatic Cancer Cell Sensitivity to Gemcitabine

Pancreatic ductal adenocarcinoma (PDAC) is a deadly cancer with a poor prognosis that is characterized by excessive mitogenic pathway activation and marked chemoresistance to a broad spectrum of chemotherapeutic drugs. Dual specificity protein phosphatase 1 (DUSP1) is a key negative regulator of mitogen activated protein kinases (MAPKs). Yet, DUSP1 is overexpressed in pancreatic cancer cells (PCCs) in PDAC where it paradoxically enhances colony formation in soft agar and promotes in vivo tumorigenicity. However, it is not known whether DUSP1 overexpression contributes to PDAC chemoresistance. Using BxPC3 and COLO-357 human PCCs, we show that gemcitabine activates c-JUN N-terminal kinase (JNK) and p38 mitogen activated protein kinase (p38 MAPK), key kinases in two major stress-activated signaling pathways. Gemcitabine-induced JNK and p38 MAPK activation mediates increased apoptosis, but also transcriptionally upregulates DUSP1, as evidenced by increased DUSP1 mRNA levels and RNA polymerase II loading at DUSP1 gene body. Conversely, shRNA-mediated inhibition of DUSP1 enhances JNK and p38 MAPK activation and gemcitabine chemosensitivity. Using doxycycline-inducible knockdown of DUSP1 in established orthotopic pancreatic tumors, we found that combining gemcitabine with DUSP1 inhibition improves animal survival, attenuates angiogenesis, and enhances apoptotic cell death, as compared with gemcitabine alone. Taken together, these results suggest that gemcitabine-mediated upregulation of DUSP1 contributes to a negative feedback loop that attenuates its beneficial actions on stress pathways and apoptosis, raising the possibility that targeting DUSP1 in PDAC may have the advantage of enhancing gemcitabine chemosensitivity while suppressing angiogenesis.