Regulation of multidrug resistance in cancer cells by hyaluronan

Multidrug resistance in cancer cells is often due to ATP-dependent efflux pumps, but is also linked to alterations in cell survival and apoptotic signaling pathways. We have found previously that perturbation of hyaluronan-tumor cell interaction by treatment with hyaluronan oligosaccharides suppresses the phosphoinositide 3-kinase/Akt cell survival signaling pathway in cancer cells and reduces tumor growth in vivo. Here we find that these oligomers suppress both the MAP kinase and phosphoinositide 3-kinase pathways in multidrug resistant tumor cells and sensitize these cells to a variety of chemotherapeutic drugs. On the other hand, increased hyaluronan production induces resistance in drug-sensitive tumor cells. Likewise, increased expression of emmprin, which is a glycoprotein that is present on the surface of most malignant cancer cells and that stimulates hyaluronan production, also induces increased resistance. Thus, perturbation of hyaluronan signaling may provide a dual therapeutic role, since it has intrinsic suppressive effects on tumor growth as well as sensitizing cancer cells to chemotherapeutic agents.     

EGFR and c-Met Cross Talk in Glioblastoma and Its Regulation by Human Cord Blood Stem Cells

Receptor tyrosine kinases (RTK) and their ligands control critical biologic processes, such as cell proliferation, migration, and differentiation. Aberrant expression of these receptor kinases in tumor cells alters multiple downstream signaling cascades that ultimately drive the malignant phenotype by enhancing tumor cell proliferation, invasion, metastasis, and angiogenesis. As observed in human glioblastoma (hGBM) and other cancers, this dysregulation of RTK networks correlates with poor patient survival. Epidermal growth factor receptor (EGFR) and c-Met, two well-known receptor kinases, are coexpressed in multiple cancers including hGBM, corroborating that their downstream signaling pathways enhance a malignant phenotype. The integration of c-Met and EGFR signaling in cancer cells indicates that treatment regimens designed to target both receptor pathways simultaneously could prove effective, though resistance to tyrosine kinase inhibitors continues to be a substantial obstacle. In the present study, we analyzed the antitumor efficacy of EGFR inhibitors erlotinib and gefitinib and c-Met inhibitor PHA-665752, along with their respective small hairpin RNAs (shRNAs) alone or in combination with human umbilical cord blood stem cells (hUCBSCs), in glioma cell lines and in animal xenograft models. We also measured the effect of dual inhibition of EGFR/c-Met pathways on invasion and wound healing. Combination treatments of hUCBSC with tyrosine kinase inhibitors significantly inhibited invasion and wound healing in U251 and 5310 cell lines, thereby indicating the role of hUCBSC in inhibition of RTK-driven cell behavior. Further, the EGFR and c-Met localization in glioma cells and hGBM clinical specimens indicated that a possible cross talk exists between EGFR and c-Met signaling pathway.     

Hepatocyte growth factor installs a survival platform for colorectal cancer cell invasive growth and overcomes p38 MAPK-mediated apoptosis

Hepatocyte growth factor (HGF) induces invasive growth, a biological program that confers tumor cells the capability to invade and metastasize by integrating cell proliferation, motility, morphogenesis, and survival. We here demonstrate that HGFR activation promotes survival of colorectal carcinoma (CRC) cells exposed to conditions that mimic those met during tumor progression, i.e. nutrient deprivation or substrate detachment, and following chemotherapeutic treatment. In all these conditions, a sustained activation of p38 MAPK delivers a main death signal that is overcome by cell treatment with HGF. HGF-driven survival requires the engagement of the PI3K/Akt/mTOR/p70S6K and ERK MAPK transduction pathways. Abrogation of p38 MAPK activity prevents CRC cell apoptosis also when these transduction pathways are inhibited, and treatment with HGF further increases survival. Engagement of these signaling cascades is also needed for HGF to induce CRC cell scattering, morphogenesis, motility and invasion. Activation of p38 MAPK signaling is therefore a main apoptotic switch for CRC cells in the stressful conditions encountered during tumor progression. Conversely, HGF orchestrates several biochemical pathways, which allow cell survival in these same conditions and promote the biological responses required for tumor invasive growth. Both p38 MAPK and HGF/HGFR signaling constitute potential molecular targets for inhibiting colorectal carcinogenesis.     

Low Dose Geldanamycin Inhibits Hepatocyte Growth Factor- and Hypoxia-Stimulated Invasion of Cancer Cells

Hepatocyte growth factor (HGF) receptor Met and hypoxia-inducible factor-1 (HIF-1) signaling pathways are commonly activated in aggressive tumors and promote progression. Since both Met and HIF-1α proteins are heat shock protein (Hsp) 90 clients, Hsp90 inhibitors might be expected to positively impact tumor progression. Here, we systematically evaluated the inhibitory effects of the prototypical Hsp90 inhibitor geldanamycin (GA) on cellular processes involved in invasion and angiogenesis in T24 bladder cancer cells stimulated with HGF and chemical hypoxia. First, we demonstrated the positive feedback loop between Met and HIF-1 pathways, which serves to sustain and amplifies their signaling in T24 cells. GA down-regulated Met by inhibiting new protein maturation, thereby dampening HGF signaling. HGF and chemical hypoxia with CoCl2 cooperatively promoted in vitro invasion and vascular endothelial growth factor (VEGF) secretion, while CoCl2 but not HGF activated urokinase-type plasminogen activator and matrix metalloproteinase 2, both of which promote invasion and angiogenesis. Low dose GA (100 nmol/L) inhibited these processes by suppressing both HGF and HIF-1 pathways. Notably, brief GA pretreatment inhibited in vitro invasion and VEGF secretion induced by HGF as effectively as did continuous treatment. Moreover, we found that GA inhibited activation of focal adhesion kinase, focal adhesion assembly, and actin reorganization induced by HGF and integrin engagement by extracellular matrix. Thus, GA widely suppresses extrinsic stimuli-induced signaling that contribute to tumor invasion and angiogenesis in this bladder carcinoma model, suggesting the utility of Hsp90 inhibitors in preventing tumor progression and metastasis.     

Melanoma chondroitin sulfate proteoglycan enhances FAK and ERK activation by distinct mechanisms

Melanoma chondroitin sulfate proteoglycan (MCSP) is an early cell surface melanoma progression marker implicated in stimulating tumor cell proliferation, migration, and invasion. Focal adhesion kinase (FAK) plays a pivotal role in integrating growth factor and adhesion-related signaling pathways, facilitating cell spreading and migration. Extracellular signal-regulated kinase (ERK) 1 and 2, implicated in tumor growth and survival, has also been linked to clinical melanoma progression. We have cloned the MCSP core protein and expressed it in the MCSP-negative melanoma cell line WM1552C. Expression of MCSP enhances integrin-mediated cell spreading, FAK phosphorylation, and activation of ERK1/2. MCSP transfectants exhibit extensive MCSP-rich microspikes on adherent cells, where it also colocalizes with alpha4 integrin. Enhanced activation of FAK and ERK1/2 by MCSP appears to involve independent mechanisms because inhibition of FAK activation had no effect on ERK1/2 phosphorylation. These results indicate that MCSP may facilitate primary melanoma progression by enhancing the activation of key signaling pathways important for tumor invasion and growth.     

Multi-scale agent-based brain cancer modeling and prediction of TKI treatment response: Incorporating EGFR signaling pathway and angiogenesis

ABSTRACT: BACKGROUND: The epidermal growth factor receptor (EGFR) signaling pathway and angiogenesis in brain cancer act as an engine for tumor initiation, expansion and response to therapy. Since the existing literature does not have any models that investigate the impact of both angiogenesis and molecular signaling pathways on treatment, we propose a novel multi-scale, agent-based computational model that includes both angiogenesis and EGFR modules to study the response of brain cancer under tyrosine kinase inhibitors (TKIs) treatment. RESULTS: The novel angiogenesis module integrated into the agent-based tumor model is based on a set of reaction--diffusion equations that describe the spatio-temporal evolution of the distributions of micro-environmental factors such as glucose, oxygen, TGFalpha, VEGF and fibronectin. These molecular species regulate tumor growth during angiogenesis. Each tumor cell is equipped with an EGFR signaling pathway linked to a cell-cycle pathway to determine its phenotype. EGFR TKIs are delivered through the blood vessels of tumor microvasculature and the response to treatment is studied. CONCLUSIONS: Our simulations demonstrated that entire tumor growth profile is a collective behaviour of cells regulated by the EGFR signaling pathway and the cell cycle. We also found that angiogenesis has a dual effect under TKI treatment: on one hand, through neo-vasculature TKIs are delivered to decrease tumor invasion; on the other hand, the neo-vasculature can transport glucose and oxygen to tumor cells to maintain their metabolism, which results in an increase of cell survival rate in the late simulation stages.     

INHBA gene silencing inhibits gastric cancer cell migration and invasion by impeding activation of the TGF-β signaling pathway

Gastric cancer (GC) is the fourth largest cancer in the world, with a 5-year survival rate of <30%. Thus, this study intends to investigate the effects of inhibin βA (INHBA) gene silencing on the migration and invasion of GC cells via the transforming growth factor-β (TGF-β) signaling pathway. Initially, this study determined the expression of INHBA and the TGF-β signaling pathway-related genes in GC tissues. After that, to assess the effect of INHBA silencing on GC progression, GC cells were transfected with short hairpin RNAs that targeted INHBA in order to detect the expression of INHBA and the TGF-β signaling pathway-related genes, as well as cell migration, invasion, and proliferation abilities. Finally, a tumor xenograft model in nude mice was constructed to verify the effect that the silencing of INHBA had on tumor growth. Highly expressed INHBA and activated TGF-β signaling pathways were observed in GC tissues. In response to shINHBA-1 and shINHBA-2, the TGF-β signaling pathway was inhibited in GC cells, whereas the GC cell migration, invasion, proliferation, and tumor growth were significantly dampened. On the basis of the observations and findings of this study, INHBA gene silencing inhibited the progression of GC by inactivating the TGF-β signaling pathway, which provides a potential target in the treatment of GC.     

Molecular targets and signaling pathways regulated by interleukin (IL)-24 in mediating its antitumor activities

Cancer remains a major health issue in the world and the effectiveness of current therapies is limited resulting in disease recurrence and resistance to therapy. Therefore to overcome disease recurrence and have improved treatment efficacy there is a continued effort to develop and test new anticancer drugs that are natural or synthetic - (conventional chemotherapeutics, small molecule inhibitors) and biologic (antibody, tumor suppressor genes, oligonucleotide) product. In parallel, efforts for identifying molecular targets and signaling pathways to which cancer cells are “addicted” are underway. By inhibiting critical signaling pathways that is crucial for cancer cell survival, it is expected that the cancer cells will undergo a withdrawal symptom akin to “de-addiction” resulting in cell death. Thus, the key for having an improved and greater control on tumor growth and metastasis is to develop a therapeutic that is able to kill tumor cells efficiently by modulating critical signaling pathways on which cancer cells rely for their survival.Currently several small molecule inhibitors targeted towards unique molecular signaling pathways have been developed and tested in the clinic. Few of these inhibitors have shown efficacy while others have failed. Thus, targeting a single molecule or pathway may be insufficient to completely block cancer cell proliferation and survival. It is therefore important to identify and test an anticancer drug that can inhibit multiple signaling pathways in a cancer cell, control growth of both primary and metastatic tumors and is safe.One biologic agent that has the characteristics of serving as a potent anticancer drug is interleukin (IL)-24. IL-24 suppresses multiple signaling pathways in a broad-spectrum of human cancer cells leading to tumor cell death, inhibition of tumor angiogenesis and metastasis. Additionally, combining IL-24 with other therapies demonstrated additive to synergistic antitumor activity. Clinical testing of IL-24 as a gene-based therapeutic for the treatment of solid tumors demonstrated that IL-24 is efficacious and is safe. The unique features of IL-24 support its further development as an anticancer drug for cancer treatment.In this review we summarize the current understanding on the molecular targets and signaling pathways regulated by IL-24 in mediating its anticancer activity.     

Tumor mechanics and metabolic dysfunction

Desmosplasia is a characteristic of most solid tumors and leads to fibrosis through abnormal extracellular matrix (ECM) deposition, remodeling, and posttranslational modifications. The resulting stiff tumor stroma not only compromises vascular integrity to induce hypoxia and impede drug delivery, but also promotes aggressiveness by potentiating the activity of key growth, invasion, and survival pathways. Intriguingly, many of the protumorigenic signaling pathways that are mechanically activated by ECM stiffness also promote glucose uptake and aerobic glycolysis, and an altered metabolism is a recognized hallmark of cancer. Indeed, emerging evidence suggests that metabolic alterations and an abnormal ECM may cooperatively drive cancer cell aggression and treatment resistance. Accordingly, improved methods to monitor tissue mechanics and metabolism promise to improve diagnostics and treatments to ameliorate ECM stiffening and elevated mechanosignaling may improve patient outcome. Here we discuss the interplay between ECM mechanics and metabolism in tumor biology and suggest that monitoring these processes and targeting their regulatory pathways may improve diagnostics, therapy, and the prevention of malignant transformation.     

The TLR3, PI3K,survin, FasL,and Fas genes as major risk factors of occurrence and development of cervical cancer disease

To investigate the role of TLR3/PI3K signals in the occurrence and development of cervical cancer disease, TLR3–siRNA was used to block key signaling pathways involved in cervical cancer metastasis that are pivotal to metastatic tumor cells but not to normal cells under ordinary physiologic conditions. Results show that tumor U14 cell growth, migration and invasion in TLR3–siRNA treatment group were significantly decreased. Through LY294002 suppressing targeted gene, the LY294002 treatment specifically and significantly knocked down the expressions of tumor TLR3 and PI3K proteins in cervical cancer mice. Furthermore, expressions of tumor Survivin and FasL proteins were markedly suppressed, whereas expressions of Fas protein were upregulated in LY294002 treatment group mice. LY294002 treatment suppressed tumor growth and increased the thymus and spleen indeces and survival days of cervical cancer mice. This study demonstrates that TLR3–siRNA and LY294002 treatments can markedly suppress cervical cancer cell invasion and tumor growth and increase survival life by silencing targeted genes.     

Direct inhibition of myosin II effectively blocks glioma invasion in the presence of multiple motogens

Anaplastic gliomas, the most common and malignant of primary brain tumors, frequently contain activating mutations and amplifications in promigratory signal transduction pathways. However, targeting these pathways with individual signal transduction inhibitors does not appreciably reduce tumor invasion, because these pathways are redundant; blockade of any one pathway can be overcome by stimulation of another. This implies that a more effective approach would be to target a component at which these pathways converge. In this study, we have investigated whether the molecular motor myosin II represents such a target by examining glioma invasion in a series of increasingly complex models that are sensitive to platelet-derived growth factor, epidermal growth factor, or both. Our results lead to two conclusions. First, malignant glioma cells are stimulated to invade brain through the activation of multiple signaling cascades not accounted for in simple in vitro assays. Second, even though there is a high degree of redundancy in promigratory signaling cascades in gliomas, blocking tumor invasion by directly targeting myosin II remains effective. Our results thus support our hypothesis that myosin II represents a point of convergence for signal transduction pathways that drive glioma invasion and that its inhibition cannot be overcome by other motility mechanisms.     

Targeting Notch signaling pathway to overcome drug resistance for cancer therapy

Chemotherapy is an important therapeutic strategy for cancer treatment and remains the mainstay for the management of human malignancies; however, chemotherapy fails to eliminate all tumor cells because of intrinsic or acquired drug resistance, which is the most common cause of tumor recurrence. Recently, emerging evidences suggest that Notch signaling pathway is one of the most important signaling pathways in drug-resistant tumor cells. Moreover, down-regulation of Notch pathway could induce drug sensitivity, leading to increased inhibition of cancer cell growth, invasion, and metastasis. This article will provide a brief overview of the published evidences in support of the roles of Notch in drug resistance and will further summarize how targeting Notch by “natural agents” could become a novel and safer approach for the improvement of tumor treatment by overcoming drug resistance.     

Autocrine functions of VEGF in breast tumor cells: Adhesion,survival, migration and invasion

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

The role of VEGF and EGFR inhibition: implications for combining anti-VEGF and anti-EGFR agents

Multiple cellular pathways influence the growth and metastatic potential of tumors. This creates heterogeneity, redundancy, and the potential for tumors to bypass signaling pathway blockade, resulting in primary or acquired resistance. Combining therapies that inhibit different signaling pathways has the potential to be more effective than inhibition of a single pathway and to overcome tumor resistance. Vascular endothelial growth factor (VEGF) and epidermal growth factor receptor (EGFR) inhibitors have become key therapies in several tumor types. Close relationships between these factors exist: VEGF signaling is up-regulated by EGFR expression and, conversely, VEGF up-regulation independent of EGFR signaling seems to contribute to resistance to EGFR inhibition. Therefore, inhibition of both pathways could improve antitumor efficacy and overcome resistance to EGFR inhibition. Preclinical studies have shown that VEGF and EGFR inhibitors can have additive effects and that combined inhibition is effective in EGFR inhibitor-resistant cell lines. Clinical trials have also produced promising data: combining the anti-VEGF monoclonal antibody bevacizumab with the anti-EGFR antibody cetuximab or the EGFR tyrosine kinase inhibitor erlotinib increases benefit compared with either of these anti-EGFR agents alone or combined with chemotherapy. The potential of this novel approach to anticancer therapy will be elucidated by large, ongoing clinical trials.     

Widespread deregulation of phosphorylation-based signaling pathways in multiple myeloma cells: opportunities for therapeutic intervention

Multiple myeloma (MM) is a neoplasm of plasma cell origin that is largely confined to the bone marrow (BM). Chromosomal translocations and other genetic events are known to contribute to deregulation of signaling pathways that lead to transformation of plasma cells and progression to malignancy. However, the tumor stroma may also provide trophic support and enhance resistance to therapy. Phosphorylation of proteins on tyrosine, serine and threonine residues plays a pivotal role in cell growth and survival. Therefore, knowing the status of phosphorylation-based signaling pathways in cells may provide key insights into how cell growth and survival is promoted in tumor cells. To provide a more comprehensive molecular analysis of signaling disruptions in MM, we conducted a kinome profile comparison of normal plasma cells and MM plasma cells as well as their surrounding cells from normal BM and diseased BM. Integrated pathway analysis of the profiles obtained reveals deregulation of multiple signaling pathways in MM cells but also in surrounding bone marrow blood cells compared to their normal counterparts. The deregulated kinase activities identified herein, which include the mTOR (mammalian target of rapamycin)/p70S6K and ERK1/2 (extracellular signal-regulated kinases 1 and 2) pathways, are potential novel molecular targets in this lethal disease.     

Autocrine functions of VEGF in breast tumor cells

Vascular endothelial growth factor A (VEGF-A) is well known for its key roles in blood vessel growth. Although most studies on VEGF and VEGF receptors have been focused on their functions in angiogenesis and in endothelial cells, the role of VEGF in cancer biology appears as an emerging area of importance. In this context, the presence of VEGF receptors in tumor cells strongly suggests that VEGF-A also promotes a wide range of functions, both in vitro and in vivo, all autocrine functions on tumor cells, including adhesion, survival, migration and invasion. Ultimately, refining our knowledge of VEGF signaling pathways in tumor cells should help us to understand why the current used treatments targeting the VEGF pathway in cancer are not universally effective in inhibiting metastasis tumors, and it should also provide new avenues for future therapies.     

RNA interference-directed knockdown of urokinase plasminogen activator and urokinase plasminogen activator receptor inhibits prostate cancer cell invasion, survival, and tumorigenicity in vivo

The invasive ability of tumor cells plays a key role in prostate cancer metastasis and is a major cause of treatment failure. Urokinase plasminogen activator-(uPA) and its receptor (uPAR)-mediated signaling have been implicated in tumor cell invasion, survival, and metastasis in a variety of cancers. This study was undertaken to investigate the biological roles of uPA and uPAR in prostate cancer cell invasion and survival, and the potential of uPA and uPAR as targets for prostate cancer therapy. uPA and uPAR expression correlates with the metastatic potential of prostate cancer cells. Thus, therapies designed to inhibit uPA and uPAR expression would be beneficial. LNCaP, DU145, and PC3 are prostate cancer cell lines with low, moderate, and high metastatic potential, respectively, as demonstrated by their capacity to invade the extracellular matrix. In this study we utilized small hairpin RNAs (shRNAs), also referred to as small interfering RNAs, to target human uPA and uPAR. These small interfering RNA constructs significantly inhibited uPA and uPAR expression at both the mRNA and protein levels in the highly metastatic prostate cancer cell line PC3. Our data demonstrated that uPA-uPAR knockdown in PC3 cells resulted in a dramatic reduction of tumor cell invasion as indicated by a Matrigel invasion assay. Furthermore, simultaneous silencing of the genes for uPA and uPAR using a single plasmid construct expressing shRNAs for both uPA and uPAR significantly reduced cell viability and ultimately resulted in the induction of apoptotic cell death. RNA interference for uPA and uPAR also abrogated uPA-uPAR signaling to downstream target molecules such as ERK1/2 and Stat 3. In addition, our results demonstrated that intratumoral injection with the plasmid construct expressing shRNAs for uPA and uPAR almost completely inhibited established tumor growth and survival in an orthotopic mouse prostate cancer model. These findings uncovered evidence of a complex signaling network operating downstream of uPA-uPAR that actively advances tumor cell invasion, proliferation, and survival of prostate cancer cells. Thus, RNA interference-directed targeting of uPA and uPAR is a convenient and novel tool for studying the biological role of the uPA-uPAR system and raises the potential of its application for prostate cancer therapy.