Ras Signal Transduction Pathway Research Articles

Hyperinsulinism Gateway Between Diabetes and Cancer: New Roles for Bifunctional Antidiabetic - Anticancer Active Somatostatins, IGF - 1 Inhibitors and Insulin Sensitizers

Hyperinsulinism refers to a condition of abnormally high concentration of circulating insulin in the body commonly seen in obese and type 2 diabetic patients due to insulin resistance. Elevated circulating insulin is the main factor linking obesity, diabetes and cancer and an important mechanism of cancer therapeutic resistance and failure. The insulin receptor subfamily which is made up of the insulin receptor (IR), the type 1 IGF receptor (IGF-1) which binds insulin-like growth factors I and II, and the orphan insulin receptor-related receptor (IRRR) have been identified and linked to the process of carcinogenesis, as cancer cells require insulin for growth. IR receptors are overexpressed in various cancers and insulin activates growth mostly through these receptors and their interplays through mainly the PI3K/AKT/mTOR, MAPK and RAS signal transduction pathways. Estrogens and androgens synergistic effects worsen hyperinsulinism and accelerates its carcinogenesis properties while progesterone and Sex Hormone Binding Globulins exert modulatory effects. This review examined the various factor interplays in hyperinsulinism that links diabetes with cancer and the biomarkers involved. It equally identified the potentials and therapeutic roles for Somatostatins, IGF – 1 inhibitors and Insulin sensitizing antidiabetics in preventing and reversing the cancer linked trends.

97 (PB087) - The ULK1 inhibitor ENV-201 impairs tumor growth in KRAS-driven flank xenografts as a single agent and in combination with the KRAS inhibitor adagrasib

Background: Lung cancer is the leading cancer cause of death in the US, and non-small cell lung cancers (NSCLCs) account for 85% of those cases. Among NSCLCs, mutations resulting in ectopic activation of the RAS signal transduction pathway account for at least a quarter of cases, and the activating KRASG12Cmutation is found in 11% of all NSCLC patient samples. When treated with RAS pathway-inhibiting therapeutics, KRAS-driven tumors have been shown to become dependent on autophagy for continued survival. Simultaneous targeting of both the RAS pathway and autophagy therefore presents an opportunity to block multiple mechanisms of tumor growth and survival. Materials and methods: We have demonstrated the added benefit of KRAS inhibition and autophagy inhibition in an animal model, using the NCI-H2122 cell line (homozygous for the activating KRASG12C mutation) as flank xenografts in NOD/SCID mice. ENV-201 is a selective and potent antagonist of the autophagy-initiating kinase, ULK1, one which also inhibits the related ULK2. Mice were treated via daily oral dosing of (1) vehicle, (2) ENV-201 (20 mg/kg), (3) the KRASG12C-specific inhibitor adagrasib (Mirati Therapeutics, 25 mg/kg), or (4) combination of both ENV-201 and adagrasib at the same doses. Treatment lasted 30 days, body weight was monitored daily, and tumor size was measured three times a week. All animal work was carried out at and met necessary approvals at Crown Bioscience. Results: Both ENV-201 and adagrasib treatments resulted in significant tumor growth inhibition when either drug was used as a single agent (32% or 55% TGI, respectively). Significant additive tumor growth inhibition (78%) was also observed when animals were treated with both drugs in combination. Conclusions: ENV-201-mediated ULK1 inhibition thus shows promise as both a single agent or in combination with a growth factor/RAS/MAPK pathway-targeting cancer treatment. Conflict of interest: Corporate-sponsored Research: All authors are employees of Endeavor BioMedicines.

Abstract 6051: Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance

Abstract Neuroblastoma is a childhood cancer originating from embryonic neuronal progenitor cells and is the most common cancer diagnosed in infants. Although the majority of patients respond to initial chemotherapy, most high-risk patients suffer relapse due to therapy resistance. Here, we generated single-cell transcriptome and bulk whole-genome sequencing data of diagnosis and post-therapy samples from 23 patients diagnosed with high-risk neuroblastoma. We found two distinct adrenergic populations in the patient samples. Most tumor cells showed a mature sympathoblast phenotype whereas a small fraction was in an undifferentiated state with activation of protein translation, unfolded protein, and oxidative phosphorylation pathways. We found that therapy-resistant tumor cells in these two subpopulations had distinct characteristics. The more mature resistant subpopulation upregulated genes associated with epithelial-to-mesenchymal transition, including TWIST1, PLCB1 and CD276. The undifferentiated resistant subpopulation upregulated Ras signal transduction and TP53 pathway genes. Analysis of the immune microenvironment revealed that most tumor-associated macrophages became more immunosuppressive post-therapy via multiple newly gained signaling interactions including the complement signaling pathway. We discovered a limited infiltration of T lymphocytes in the tumor microenvironment, and chemotherapy induced an effector state with upregulated mTOR signaling and metabolism. Overall, our study revealed subpopulations of tumor cells in neuroblastoma that responded differently to induction chemotherapy. Our findings uncovered distinct molecular signatures of the resistant cells and their interactions with the immune microenvironment, paving the way for developing novel therapies for high-risk neuroblastoma. Citation Format: Yasin Uzun, Liron D. Grossmann, Chia-Hui Chen, Anusha Thadi, Chi-Yun Wu, Peng Gao, Dinh Diep, Lea Surrey, Daniel Martinez, Tasleema Patel, Qi Qiu, Sarah Johnson, Wenbao Yu, Shane Drabings, Changya Chen, Yuxuan Hu, Gregory Chen, Derek A. Oldridge, Kun Zhang, Hao Wu, Kathrin Bernt, Nancy Zhang, John M. Maris, Kai Tan. Longitudinal single-cell sequencing of high-risk neuroblastoma tumors reveals intrinsic and extrinsic mechanisms of therapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6051.

Interaction between Ras and Bcl2L12 in B cells suppresses IL-10 expression

The pathogenesis of recurrent tonsillitis is to be further investigated. B cell-derived interleukin (IL)-10 plays a critical role in immune regulation. Ras activation plays an important role in cancer and many immune disorders. This study aims to investigate the role of Ras activation in down regulating IL-10 expression in tonsillar B cells. Surgically removed tonsil tissues were collected from patients with recurrent acute tonsillar inflammation; B cells were isolated from the tonsillar tissues by flow cytometry sorting to be analyzed by the Ras-specific enzyme-linked immunosorbent assay and pertinent immunological approaches. We found that, compared to peripheral B cells (pBC), B cells isolated from the tonsillar tissues with recurrent inflammation (tBC) showed higher Ras activation, lower IL-10 expression and higher Bcl2L12 expression. Bcl2L12 formed a complex with GAP (GTPase activating protein) to prevent Ras from deactivating. The Ras activation triggered the MAPK/Sp1 pathway to promote the Bcl2L12 expression in B cells. Bcl2L12 prevented the IL-10 expression in tBCs, that was counteracted by inhibition of Ras or the Ras signal transduction pathway. In conclusion, Bcl2L12 interacts with Ras activation to compromise immune tolerance in the tonsils by inhibiting the IL-10 expression in tBCs. Inhibition of Bcl2L12 can restore the IL-10 expression in tBCs.

Collagen VI (CVI) antibodies regulate macrophages differentiation through FcgammaRIIb signal transduction pathway

We previously reported that monoclonal antibody against collagen VI (CVI mAb) regressing atherosclerotic plaque through inducing the shift of macrophage differentiation, namely from M1 to M2, and ensuing the macrophages engulf the plaque lipids and transfer the lipids to HDL through restoring lipid transfer protein ABCA1 expression. Here we report the cellular signal transduction on the CVI mAb inhibition of oxLDL‐induced M1 differentiation. Using purified human CD14+ cells, we find that oxLDL induces M1 differentiation is JNK and ERK MAPKs dependent. And CVI mAb inhibits M1, while activates M2 differentiation, though oxLDL also activates M2 differentiation in some way, but the M1/M2 ratio are significantly reversed by CVI mAb. The molecular mechanism behind these is that CVI mAb activates macrophage FcgammaRIIb and its downstream SHIP and Dok phosphatases, and inhibits Syk and Ras signal transduction pathway, which are MAPKs upstream kinases, so that CVI mAb inhibits ERK, JNK, and p38 MAPKs activation, and ensuing M1 differentiation. Thus, we found the signal transduction pathway of CVI mAb inhibition of oxLDL‐induced M1 differentiation: CVI mAb→FcgammaRIIb→SHIP→Dok→Syk and Ras→ERK and JNK MAPKs→M1. These results might give a new clue on control the lipids‐induced inflammatory reaction on the large blood vessel and therapeutic method to prevent atherosclerosis.Support or Funding InformationThis work was supported by the National Natural Science Foundation of China (grants 81772074, 81974045), the Natural Science Foundation of Guangdong, China (grant 2012B050600002), the Guangdong Province Talent Recruitment Foundation and the Guangdong Innovative Research Team Program (No. 201001Y0104675344), Project of Guangzhou city scientific plan (201804020002).

Pediatric Acute Myeloid Leukemia (AML): From Genes to Models Toward Targeted Therapeutic Intervention.

This review aims to provide an overview of the current knowledge of the genetic lesions driving pediatric acute myeloid leukemia (AML), emerging biological concepts, and strategies for therapeutic intervention. Hereby, we focus on lesions that preferentially or exclusively occur in pediatric patients and molecular markers of aggressive disease with often poor outcome including fusion oncogenes that involve epigenetic regulators like KMT2A, NUP98, or CBFA2T3, respectively. Functional studies were able to demonstrate cooperation with signaling mutations leading to constitutive activation of FLT3 or the RAS signal transduction pathways. We discuss the issues faced to faithfully model pediatric acute leukemia in mice. Emerging experimental evidence suggests that the disease phenotype is dependent on the appropriate expression and activity of the driver fusion oncogenes during a particular window of opportunity during fetal development. We also highlight biochemical studies that deciphered some molecular mechanisms of malignant transformation by KMT2A, NUP98, and CBFA2T3 fusions, which, in some instances, allowed the development of small molecules with potent anti-leukemic activities in preclinical models (e.g., inhibitors of the KMT2A–MENIN interaction). Finally, we discuss other potential therapeutic strategies that not only target driver fusion-controlled signals but also interfere with the transformed cell state either by exploiting the primed apoptosis or vulnerable metabolic states or by increasing tumor cell recognition and elimination by the immune system.

JMML genomics and decisions.

Juvenile myelomonocytic leukemia (JMML) is a unique clonal hematopoietic disorder of early childhood characterized by hyperactivation of the RAS signal transduction pathway. Approximately 90% of patients harbor molecular alteration in 1 of 5 genes (PTPN11, NRAS, KRAS, NF1, CBL), which define genetically and clinically distinct JMML subtypes. Three subtypes, PTPN11- , NRAS-, and KRAS-mutated JMML, are characterized by heterozygous somatic gain-of-function mutations in non syndromic children, while two subtypes, JMML in neurofibromatosis type 1 and in JMML in children with CBL syndrome, are characterized by germ line RAS disease and acquired biallelic inactivation of the respective tumor suppressor genes in hematopoietic cells. In addition to the initiating RAS pathway lesion, secondary genetic alterations within and outside of the RAS pathway are detected in about half the patients. Most recently, genome-wide DNA methylation profiles identified distinct methylation signatures correlating with clinical and genetic features and highly predictive of outcome. JMML is a stem cell disorder, and most JMML patients require allogeneic stem cell transplantation for long-term survival. However, spontaneous disease regression is noted in the majority of children with CBL-mutated JMML and in some NRAS-mutated cases. In the absence of 1 of the 5 canonical RAS pathway alteration, rare mutations in other RAS genes and non-JMML myeloproliferative disorders need to be excluded. Understanding the genetic basis of myeloproliferative disorders in early childhood will greatly improve clinical decision making.

Abstract 4540: Use of SIAH E3 ubiquitin ligase as a biomarker in pancreatic adenocarcinoma

Abstract The dismal prognosis of patients diagnosed with pancreatic cancer points to our limited arsenal of effective anticancer therapies. Oncogenic K-RAS pathway activation is commonly detected in pancreatic cancer. These activations confer drug resistance, drive aggressive tumor growth and metastasis, and ultimately contribute to poor clinical outcome. Currently, there is no curative treatments for pancreatic cancers. Thus, early detection, biomarker discovery, and identifying novel means of countervailing hyperactive K-RAS signals are urgently needed. Seven-In-Absentia Homolog (SIAH), E3 ubiquitin ligase, is an essential downstream component of the RAS signal transduction pathway. SIAH is a member of a highly evolutionarily conserved family of RING finger E3 ubiquitin ligases. Our previous immunohistochemistry (IHC) studies have shown SIAH is a tumor-specific biomarker and SIAH expression is progressively increased as pancreatic tumor progresses from pancreatic intraepithelial neoplasia (PanIN) to pancreatic adenocarcinoma (PDCA) at late stages. In this study, we expand our observations by comparing the expression patterns of SIAH and several newly identified SIAH-interacting proteins using Mayo clinic pancreatic cancer SPORE tissue microarrays (TMA) in a hope of identifying new vulnerability and novel prognostic biomarkers in pancreatic cancer. We hypothesized that SIAH and SIAH-interacting proteins may serve as useful biomarkers and will help to assess effectiveness of anticancer treatment, disease progress, predict clinical outcome and patient survival in the clinical settings. Moreover, we are evaluating SIAH's role in the regulation of the SIAH-interacting proteins and validating the potential mechanisms of these dynamic interactions identified in vitro using pancreatic cancer patients TMA. Ultimately, we aim to explore the new cancer vulnerability, develop new therapeutic strategies, and validate novel prognostic biomarkers for pancreatic cancer patients. This study is performed in a double blind control settings and we are currently awaiting the disclosure of the clinical information from the pathologist. Citation Format: Elizaevta Svyatova. Use of SIAH E3 ubiquitin ligase as a biomarker in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4540.

Abstract 3403: SIAH and SIAH-interacting proteins as potential prognostic and predictive biomarkers in pancreatic adenocarcinoma

Abstract Introduction: Seven-In-Absentia Homolog (SIAH), an E3 ubiquitin ligase, is an essential downstream component of the RAS signal transduction pathway. SIAH, is a member of a highly evolutionarily conserved family of RING finger E3 ubiquitin ligases. Our previous published work has shown that SIAH is an essential signaling “gatekeeper” in the oncogenic K-RAS signal transduction cascade and SIAH is a new, effective and logical anti-K-RAS drug target against pancreatic cancer. Our previous immunohistochemistry studies have shown SIAH is a tumor-specific biomarker and SIAH expression progressively increased as pancreatic tumors progress from PanINs to pancreatic adenocarcinoma (PDCA) at late stages. In this study, we expand our observations by comparing the expression patterns of SIAH and several newly identified SIAH-interacting proteins using the Mayo Clinic pancreatic cancer tissue microarrays (TMA) in hope to identify new vulnerability and novel prognostic biomarkers in pancreatic cancer. We hypothesize that SIAH and SIAH-interacting proteins may serve as useful biomarkers helping to assess effectiveness of the anticancer treatment, disease progress and predict clinical outcome and patient survival in the clinical settings. Methods: In this double blind controlled study two hundred pancreatic adenocarcinoma patients at different stages of disease progression who had underwent surgical resection at the Mayo Clinic, Rochester, Minnesota. Tissue microarrays were generated using de-identified patient tumor biospecimens. 12x distinct TMA sets (with ∼ 700 TMA cores) were stained with 13x antibodies corresponding to our newly identified SIAH-interacting proteins and the IHC staining was scored by the Mayo Clinic GI expert and pancreatic cancer pathologist. This resulted in approximately 9000 histological cores and slides that were then analyzed. The correlation with respect to SIAH and SIAH-interacting proteins as a new prognostic and predictive biomarker were scored and analyzed using Excel and standard biostatistical softwares. Results: This study is performed in a double blind control settings and we are currently awaiting the disclosure of the clinical information from the pathologist for the final evaluation of SIAH and SIAH-interacting proteins with respect to disease progression, outcome and survival. Here we report the promising interactions have been observed between SIAH and several putative biomarkers in pancreatic cancer. Conclusion: As we previously reported, SIAH/SIAH-interacting proteins may serve as useful prognostic biomarkers to predict cancer disease progression and patient survival in clinical settings. At the end of this study, we hope to establish and validate the prognostic value of the SIAH-complex as new biomarkers and a reliable readout for oncogenic K-RAS activation, pancreatic cancer cell dissemination and metastasis. Citation Format: Elizaevta Svyatova. SIAH and SIAH-interacting proteins as potential prognostic and predictive biomarkers in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3403. doi:10.1158/1538-7445.AM2015-3403

Evaluation of somatic mutations in tibial pseudarthrosis samples in neurofibromatosis type 1

BackgroundTibial pseudarthrosis is associated with neurofibromatosis type 1 (NF1) and there is wide clinical variability of the tibial dysplasia in NF1, suggesting the possibility of genetic modifiers. Double inactivation of...

Focus formation: a cell-based assay to determine the oncogenic potential of a gene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals(1). In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)(2). When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci(1) that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

SiRNA blocking the RAS signalling pathway and inhibits the growth of oesophageal squamous cell carcinoma in nude mice.

The aim of this study was to study RAS-siRNA blocking RAS pathway and suppressing cell growth in human oesophageal squamous cell carcinoma in nude mice. The methods in this study was to construct RAS-siRNA expression vector, establish 40 oesophageal squamous cell carcinoma xenograft animal models and divided them into five groups: control group, siRNA control group, RAS-siRNA group, paclitaxel group and RAS-siRNA and paclitaxel group. We observed tumour growth in nude mice, studied histology by HE staining, tumour growth inhibition by TUNEL assay and detected the RAS, MAPK and cyclin D1 protein expression by immunohistochemistry and western blot. We have obtained the following results: (i) successfully established animal models; (ii) nude mice in each group after treatment inhibited tumour volume was significantly reduced compared with the control group (p < 0.05); (iii) compared with the control group, the number of apoptotic cells were significantly increased in the siRNA control group and the RAS-siRNA group, and the number of apoptosis cells in the paclitaxel and RAS-siRNA group is significantly most than the paclitaxel group and RAS-siRNA group (p < 0.05); and (iv) after treatment, RAS, MAPK and cyclin D1 expression in five groups was decreasing gradually. After adding paclitaxel, the protein expression in the paclitaxel and RAS-siRNA group was significantly lower than that of paclitaxel group, negative control and paclitaxel group (p < 0.05). We therefore conclude that RAS-siRNA can block the RAS signal transduction pathway, reduce the activity of tumour cells, arrest tumour cell cycle, promote apoptosis, inhibit cell proliferation and increase tumour cell sensitivity to chemotherapeutic drugs.

Ras activation mediates WISP-1-induced increases in cell motility and matrix metalloproteinase expression in human osteosarcoma

WISP-1 is a cysteine-rich protein that belongs to the CCN (Cyr61, CTGF, Nov) family of matrix cellular proteins. Osteosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. However, the effect of WISP-1 on migration activity in human osteosarcoma cells is mostly unknown. In this study, we first found that the expression of WISP-1 in osteosarcoma patients was significantly higher than that in normal bone and corrected with tumor stage. Exogenous treatment of osteosarcoma cells with WISP-1 promoted cell motility and matrix metalloproteinase (MMP)-2 and MMP-9 expression. In addition, the Ras and Raf-1 inhibitor or siRNA abolished WISP-1-induced cell migration and MMP expression. On the other hand, activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after WISP-1 treatment was demonstrated, and WISP-1-induced expression of MMPs and migration activity were inhibited by the specific inhibitor, and mutant of MEK, ERK, and NF-κB cascades. Taken together, our results indicated that WISP-1 enhances the migration of osteosarcoma cells by increasing MMP-2 and MMP-9 expression through the integrin receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.

A Serrate-Notch-Canoe complex mediates glial-neuroepithelial cell interactions essential during Drosophila optic lobe development

It is firmly established that interactions between neurons and glia are fundamental across species for the correct establishment of a functional brain. Here, we found that the glia of the Drosophila larval brain display an essential non-autonomous role during the development of the optic lobe. The optic lobe develops from neuroepithelial cells that proliferate by dividing symmetrically until they switch to asymmetric/differentiative divisions that generate neuroblasts. The proneural gene lethal of scute (l'sc) is transiently activated by the epidermal growth factor receptor (EGFR)-Ras signal transduction pathway at the leading edge of a proneural wave that sweeps from medial to lateral neuroepithelium, promoting this switch. This process is tightly regulated by the tissue-autonomous function within the neuroepithelium of multiple signaling pathways, including EGFR-Ras and Notch. This study shows that the Notch ligand Serrate (Ser) is expressed in the glia and it forms a complex in vivo with Notch and Canoe, which colocalize at the adherens junctions of neuroepithelial cells. This complex is crucial for interactions between glia and neuroepithelial cells during optic lobe development. Ser is tissue-autonomously required in the glia where it activates Notch to regulate its proliferation, and non-autonomously in the neuroepithelium where Ser induces Notch signaling to avoid the premature activation of the EGFR-Ras pathway and hence of L'sc. Interestingly, different Notch activity reporters showed very different expression patterns in the glia and in the neuroepithelium, suggesting the existence of tissue-specific factors that promote the expression of particular Notch target genes or/and a reporter response dependent on different thresholds of Notch signaling.

A Comprehensive Overview of Targeted Therapy in Metastatic Renal Cell Carcinoma

Chemotherapy and immunotherapy failed to deliver decisive results in the systemic treatment of metastatic renal cell carcinoma. Agents representing the current standards operate on members of the RAS signal transduction pathway. Sunitinib (targeting vascular endothelial growth factor), temsirolimus (an inhibitor of the mammalian target of rapamycin - mTOR) and pazopanib (a multi-targeted receptor tyrosine kinase inhibitor) are used in the first line of recurrent disease. A combination of bevacizumab (inhibition of angiogenesis) plus interferon α is also first-line therapy. Second line options include everolimus (another mTOR inhibitor) as well as tyrosine kinase inhibitors for patients who previously received cytokine. We review the results of clinical investigations focusing on survival benefit for these agents. Additionally, trials focusing on new agents, including the kinase inhibitors axitinib, tivozanib, dovitinib and cediranib and monoclonal antibodies including velociximab are also discussed. In addition to published outcomes we also include follow-up and interim results of ongoing clinical trials. In summary, we give a comprehensive overview of current advances in the systemic treatment of metastatic renal cell carcinoma.

Identification of yeast genes that confer resistance to chitosan oligosaccharide (COS) using chemogenomics

BackgroundChitosan oligosaccharide (COS), a deacetylated derivative of chitin, is an abundant, and renewable natural polymer. COS has higher antimicrobial properties than chitosan and is presumed to act by disrupting/permeabilizing the cell membranes of bacteria, yeast and fungi. COS is relatively non-toxic to mammals. By identifying the molecular and genetic targets of COS, we hope to gain a better understanding of the antifungal mode of action of COS.ResultsThree different chemogenomic fitness assays, haploinsufficiency (HIP), homozygous deletion (HOP), and multicopy suppression (MSP) profiling were combined with a transcriptomic analysis to gain insight in to the mode of action and mechanisms of resistance to chitosan oligosaccharides. The fitness assays identified 39 yeast deletion strains sensitive to COS and 21 suppressors of COS sensitivity. The genes identified are involved in processes such as RNA biology (transcription, translation and regulatory mechanisms), membrane functions (e.g. signalling, transport and targeting), membrane structural components, cell division, and proteasome processes. The transcriptomes of control wild type and 5 suppressor strains overexpressing ARL1, BCK2, ERG24, MSG5, or RBA50, were analyzed in the presence and absence of COS. Some of the up-regulated transcripts in the suppressor overexpressing strains exposed to COS included genes involved in transcription, cell cycle, stress response and the Ras signal transduction pathway. Down-regulated transcripts included those encoding protein folding components and respiratory chain proteins. The COS-induced transcriptional response is distinct from previously described environmental stress responses (i.e. thermal, salt, osmotic and oxidative stress) and pre-treatment with these well characterized environmental stressors provided little or any resistance to COS.ConclusionsOverexpression of the ARL1 gene, a member of the Ras superfamily that regulates membrane trafficking, provides protection against COS-induced cell membrane permeability and damage. We found that the ARL1 COS-resistant over-expression strain was as sensitive to Amphotericin B, Fluconazole and Terbinafine as the wild type cells and that when COS and Fluconazole are used in combination they act in a synergistic fashion. The gene targets of COS identified in this study indicate that COS’s mechanism of action is different from other commonly studied fungicides that target membranes, suggesting that COS may be an effective fungicide for drug-resistant fungal pathogens.

Dynamin II Involves in Cell Migration and Actin Formation of NIH3T3 Cells

It was previously reported that in Ras transformed NIH3T3 cells, dynamin II acts as an intermediate messenger in the Ras signal transduction pathway leading to membrane ruffling and cell migration. However, these results do not provide sufficient evidence of a relationship between dynamin II and the Ras signal transduction pathway leading to membrane ruffling and cell migration. The results showed that a dynamin II association with myosin II as a signaling molecule is involved in NIH3T3 cell migration through the Ras/PI3K signaling pathway, and is associated with the p85 subunit of PI3K. Confocal microscopy also revealed co-localization between dynamin II and paxillin after PDGF stimulation. In addition, immunofluorescence results showed that dynamin II was colocalized with the actin filament. After stimulating the NIH3T3 cells with PDGF and treating them with an actin inhibitor, such as Cytochalasin D, it was observed that dynamin II with the myosin II complex inhibited binding to the actin. Therefore, dynamin II is localized in focal adhesion when cell migration is triggered and binds to the actin filament component, suggesting that it is a good candidate nanomolecule to regulate the cell attachment and migration to the materials such as implants etc.

CCL2 increases MMP-9 expression and cell motility in human chondrosarcoma cells via the Ras/Raf/MEK/ERK/NF-κB signaling pathway

Chondrosarcoma is a type of highly malignant tumor with a potent capacity to invade locally and cause distant metastasis. Chondrosarcoma shows a predilection for metastasis to the lungs. Chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1), belongs to the CC chemokine family that is associated with the disease status and outcomes of cancers. However, the effect of CCL2 on migration activity in human chondrosarcoma cells is mostly unknown. Here we found that CCL2 increased the migration and expression of matrix metalloproteinase (MMP)-9 in human chondrosarcoma cells. CCL2-mediated migration and MMP-9 up-regulation were attenuated by CCR2, Ras, Raf-1, and MEK inhibitor. Activation of the Ras, Raf-1, MEK, ERK, and NF-κB signaling pathway after CCL2 treatment was demonstrated, and CCL2-induced expression of MMP-9 and migration activity were inhibited by the specific inhibitor, and mutant of Ras, Raf-1, MEK, ERK, and NF-κB cascades. Taken together, our results indicated that CCL2 enhances the migration of chondrosarcoma cells by increasing MMP-9 expression through the CCR2 receptor, Ras, Raf-1, MEK, ERK, and NF-κB signal transduction pathway.

Abstract 2335: Oncogenic K-RAS activation promotes cell motility, invasion, tumor growth and cancer metastasis via its most downstream signaling module, the SIAH2-dependent proteolytic machinery, in the highly aggressive and metastatic human cancer cells

Abstract Hyperactivation of EGFR/HER2/K-RAS is often associated with reduced cell/focal adhesion, altered cell junctions, increased cell motility, and enhanced invasion/metastasis in a large percentage of human cancers. Finding novel measures that inhibit activated ERBB/K-RAS signals and, thus, reverse tumorigenesis and metastasis remains an important goal in cancer biology. Instead of targeting an upstream component in the EGFR/HER2/K-RAS pathway, we have targeted the most downstream signaling module, called the SIAH-dependent proteolytic machinery. SIAH/SINA are evolutionarily conserved RING E3 ligases that act as an essential “gatekeeper” signaling module in the RAS signal transduction pathway. We have demonstrated that proper SIAH function is required for K-RAS signal transduction. SIAH is a novel and tumor-specific biomarker for proliferating cells in human cancer. Furthermore, we have shown that SIAH2 inhibition can successfully block both tumorigenesis and metastasis in eleven of the most aggressive human cancer cell lines known in athymic nude mice. These observations highlight the importance of SIAH2 in K-RAS-dependent tumorigenesis and metastasis in human cancer. To elucidate the molecular mechanism of SIAH2 action in human cancer cells, a proteomic approach was taken to identify novel SIAH2-interacting proteins in three highly aggressive human cancer cell lines carrying K-RAS mutations (MiaPaCa, A549 and MDA-MB-435). From these studies, three focal adhesion LIM domain proteins (LPXN, TRIP6 and FHL2) were found to interact with SIAH2 endogenously in these human cancer cells. Here, we provide extensive biochemical evidence demonstrating that SIAH2 interacts with, ubiquitinates and degrades TRIP6/FHL2/LPXN in a dose-dependent fashion, both exogenously and endogenously. This study clearly demonstrates that LPXN, TRIP6 and FHL2 are bona fide SIAH2 substrates in vivo. Ectopic expression of LPXN/TRIP6/FHL2 successfully rescued the defects in cell motility and tumor growth observed in these SIAH2-deficient human cancer cells. These focal adhesion LIM domain proteins may, therefore, represent a novel class of SIAH2 substrates (logical RAS targets) that function downstream of SIAH2 and modulate focal adhesion, increased cell motility, invasion, tumorigenesis and metastasis in response to active ERBB/K-RAS signals. The identification of SIAH2 as an important regulator of focal adhesion and cell motility downstream of K-RAS pathway may help to explain the well-documented changes in cell adhesion, aggressive tumor growth, invasion and metastasis in response to K-RAS pathway activation in the most lethal forms of human cancers. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2335. doi:10.1158/1538-7445.AM2011-2335