Key Signaling Pathways In Cancer Research Articles

Abstract 5962: Novel xCT-targeted biologics significantly suppress metastasis, improve survival, and sensitize primary tumors to chemotherapy in TNBC xenograft models

Abstract Background: Triple negative breast cancer (TNBC) is a highly heterogeneous subset of breast cancer, which has the worst prognosis and lacks specifically targeted therapeutic options. The TNBC subtype termed M/MSL is enriched with cells of high mesenchymal state and increased stemness, and is particularly resistant to current therapies1,2. But this cancer cell phenotype is susceptible to ferroptosis, hence targeting these aggressive tumors with ferroptosis inducers such as xCT inhibitors (xCTi) is an attractive alternative. The cystine/glutamate exchanger xCT/SLC7A11 plays a key role in maintaining redox homeostasis in cancer cells. xCT is expressed on subsets of most solid tumors, including stem/progenitor and metastatic tumor cells. xCT inhibition yields a variety of anti-proliferative phenotypes including death by ferroptosis3. xCT knockout mice exhibit normal development and lifespan. Altogether, targeting xCT outside of CNS may potentially have widespread clinical utility in cancer treatment. Results: We present preclinical data demonstrating development of novel, first-in-class xCT-targeted biotherapeutics (AX68c, AX76c) using studies in TNBC cell lines in vitro and in xenograft. We find that these agents have highly favorable pharmacokinetics, with FcRn-dependent serum half-lives greatly exceeding those of known small molecule xCTi. In vitro, AX68c or AX76c reduces glutathione levels in TNBC cells in accord with inhibition of xCT function. Analysis of anti-proliferative effects in a panel of TNBC cell lines revealed particular potency against those of M/MSL phenotype (IC50s 0.3-1uM). We further tested the susceptibility of the mesenchymal phenotype to AX68c and found that EMT induction in epithelial cancer cells significantly increased sensitivity in a ferroptosis-dependent manner. In wildtype and immunodeficient mice, AX68c is well tolerated at steady-state serum concentrations that are 15-300 fold of its in vitro IC50s in tested TNBC cells. In orthotopic MDA-MB-231 xenografts, AX68c treatment significantly reduced tumor metastasis and sensitized primary tumors to cisplatin and paclitaxel. Bulk RNAseq and GSEA analysis on primary tumors revealed treatment associated alterations in key cancer signaling pathways such as TGFβ, INFγ and TNFα/NFκB. In MDA-MB-436 orthotopic xenografts, AX68c alone led to significantly improved survival primarily through abrogating metastasis. Combining AX68c and cisplatin further prolonged survival through concurrent suppression of metastasis and primary tumor growth. Conclusions: We conclude that our anti-xCT biologics may provide novel targeted therapies for M/MSL-subtype TNBC and metastatic tumors with a large mesenchymal component. 1. Viswanathan, et al., Nature, 2017; 2. Wang, et al., npj Precision Oncology, 2021; 3. Koppula, et al., Protein and Cell, 2021 (review). Citation Format: He Gong, Jun Li, Cyrus Becker, Luika Timmerman, Shaheen Sikandar, Yong Huang. Novel xCT-targeted biologics significantly suppress metastasis, improve survival, and sensitize primary tumors to chemotherapy in TNBC xenograft models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5962.

Molecular Pathways of Rosmarinic Acid Anticancer Activity in Triple-Negative Breast Cancer Cells: A Literature Review.

Breast cancer is the most frequent type of cancer in women. Oncogenic transcription factors promote the overproduction of cellular adhesion molecules and inflammatory cytokines during cancer development. Cancer cells exhibit significant upregulation of antiapoptotic proteins, resulting in increased cell survival, tumor growth, and metastasis. Research on the cell cycle-mediated apoptosis pathway for drug discovery and therapy has shown promising results. In fact, dietary phytoconstituents have been extensively researched for anticancer activity, providing indirect protection by activating endogenous defense systems. The role of polyphenols in key cancer signaling pathways could shed light on the underlying mechanisms of action. For instance, Rosmarinic Acid, a polyphenol constituent of many culinary herbs, has shown potent chemoprotective properties. In this review, we present recent progress in the investigation of natural products as potent anticancer agents, with a focus on the effect of Rosmarinic Acid on triple-negative BC cell lines resistant to hormone therapy. We highlight a variety of integrated chemical biology approaches aimed at utilizing relevant mechanisms of action that could lead to significant clinical advances in BC treatment.

M2 macrophage inhibits the antitumor effects of Lenvatinib on intrahepatic cholangiocarcinoma.

The relationship between the tumor microenvironment and the network of key signaling pathways in cancer plays a key role in the occurrence and development of tumors. Tumor-associated macrophages (TAMs) are important inflammatory cells in the tumor microenvironment and play an important role in tumorigenesis and progression. Macrophages in malignant tumors, mainly the M2 subtype, promote tumor progression by producing cytokines and down-regulating anti-inflammatory immune responses. Several articles have investigated the effect of macrophages on the sensitivity of cancer chemotherapeutic agents, but few such articles have been reported in cholangiocarcinoma, so we investigated the effect of M2 macrophage on the sensitivity of cholangiocarcinoma cells to Lenvatinib compared to M1. THP-1 monocytes were polarized to M0 macrophage by phorbol 12-myristate 13-acetate (PMA) and then induced to differentiate into M1 and M2 macrophages by LPS, IFN-γ and IL-4 and IL-13, respectively. Macrophages and cholangiocarcinoma cells were co-cultured prior to 24 hours of Lenvatinib administration, cancer cell apoptosis was detected by western-blot, FACS analysis of Annexin V and PI staining. Furthermore, we use xCELLigence RTCA SP Instrument (ACEA Bio-sciences) to monitor cell viability of Lenvatinib administration in co-culture of cholangiocarcinoma cells and macrophages. After tumorigenesis in immunodeficient mice, Lenvatinib was administered, and the effects of M2 on biological characteristics of cholangiocarcinoma cells were investigated by immuno-histochemistry. mRNA and protein expression of M1 and M2 markers confirmed the polarization of THP-1 derived macrophages, which provided a successful and efficient model of monocyte polarization to TAMs. Lenvatinib-induced apoptosis of cholangiocarcinoma cells was significantly reduced when co-cultured with M2 macrophage, whereas apoptosis of cholangiocarcinoma cells co-cultured with M1 macrophage was increased. In the CDX model, Lenvatinib-induced cancer cell apoptosis was markedly reduced, and proliferative cells increased in the presence of M2 macrophages. Angiogenesis related factors was significantly increased in cholangiocarcinoma cells co-cultured with M2. Compared with M1, M2 macrophages can inhibit the anti-tumor effect of Lenvatinib on cholangiocarcinoma through immune regulation, which may be related to the tumor angiogenesis factor effect of M2 macrophage.

Colon Adenocarcinoma Quantitative Proteomics Reveals Dysregulation in Key Cancer Signaling Pathways and a Candidate Protein Marker Panel

Colorectal cancer (CRC) is reportedly the second leading cause of cancer death worldwide. By the end of the decade, there will likely be more than one million fatalities worldwide from this cancer, with an estimated 2.2 million additional cases. We need new ways of thinking about cancer research. One approach is to deploy systems science using quantitative proteomics to obtain postgenomic and functional insights into cancer. The present study compares the tissue proteome of CRC (n = 10) with the matched peritumoral controls (n = 10) in samples obtained from the Indian subcontinent. When compared with the controls, a list of 22 substantially altered protein candidates was identified, which were associated with the growth, survival, and metastasis of the tumor. A list of the unique peptides from top significant proteins, including olfactomedin-4, alanyl aminopeptidase, and grancalcin was further validated using a parallel reaction monitoring-based targeted proteomics approach. In addition, biological pathway analysis showed perturbation in key biological processes, including dysregulation in purine metabolism, MYC targets in cancer, DNA repair, and replication, and leukocyte transendothelial migration, among others. The protein panel reported herein is also shown to be dysregulated in CRC and warrants further research toward understanding pathobiology, diagnostics, and therapeutics development in CRC.

Epigenetic effects of high-fat diet on intestinal tumorigenesis in C57BL/6J-ApcMin/+ mice

Obesity and obesogenic diets might partly accelerate cancer development through epigenetic mechanisms. To determine these early effects, we investigated the impact of three days of a high-fat diet on epigenomic and transcriptomic changes in Apc Min/+ murine intestinal epithelia. ChIP-Seq and RNA-Seq were performed on small intestinal epithelia of WT and Apc Min/+ male mice fed high-fat diet (HFD) or low-fat diet (LFD) for three days to identify genomic regions associated with differential H3K27ac levels as a marker of variant enhancer loci (VELs) as well as differentially expressed genes (DEGs). Regarding epigenetic and transcriptomic changes, diet type (LFD vs. HFD) showed a significant impact, and genotype (WT vs.Apc Min/+) showed a small impact. Compared to LFD, HFD resulted in 1306 gained VELs, 230 lost VELs, 133 upregulated genes, and 127 downregulated genes in WT mice, with 1056 gained VELs, 371 lost VELs, 222 upregulated genes, and 182 downregulated genes in Apc Min/+ mice. Compared to the WT genotype, the Apc Min/+ genotype resulted in zero changed VELs for either diet type group, 21 DEGs for LFD, and 48 DEGs for HFD. Most gained VELs, and upregulated genes were associated with lipid metabolic processes. Gained VELs were also associated with Wnt signaling. Downregulated genes were associated with antigen presentation and processing. Three days of HFD-induced epigenomic and transcriptomic changes involving metabolic and immunologic pathways that may promote tumor growth in the genetically predisposed murine intestine without affecting key cancer signaling pathways.

Cx43 and Associated Cell Signaling Pathways Regulate Tunneling Nanotubes in Breast Cancer Cells.

Simple SummaryThe gap junction protein connexin 43 (Cx43) facilitates direct intercellular communication and displays a complex dichotomous role in cancer. Here, we used breast cancer cell lines and CRISPR/Cas9 technology to dissect some of the poorly understood multimodal functions of Cx43 in this setting. Our data show that Cx43 directly modulates the formation of pro-tumorigenic tunneling nanotubes (TNTs), which are cellular protrusions that connect non-adjacent cells to facilitate cellular communication, including the direct cell–cell transfer of organelles. We identify several important cancer signaling pathways that affect TNT formation and show that this effect is strongly influenced by the presence or absence of Cx43. Finally, we demonstrate that Cx43 can affect TNT formation by modulating the cellular secretome. This work provides important insight into the pro-tumorigenic role of Cx43 and its interconnections with TNTs.Connexin 43 (Cx43) forms gap junctions that mediate the direct intercellular diffusion of ions and small molecules between adjacent cells. Cx43 displays both pro- and anti-tumorigenic properties, but the mechanisms underlying these characteristics are not fully understood. Tunneling nanotubes (TNTs) are long and thin membrane projections that connect cells, facilitating the exchange of not only small molecules, but also larger proteins, organelles, bacteria, and viruses. Typically, TNTs exhibit increased formation under conditions of cellular stress and are more prominent in cancer cells, where they are generally thought to be pro-metastatic and to provide growth and survival advantages. Cx43 has been described in TNTs, where it is thought to regulate small molecule diffusion through gap junctions. Here, we developed a high-fidelity CRISPR/Cas9 system to knockout (KO) Cx43. We found that the loss of Cx43 expression was associated with significantly reduced TNT length and number in breast cancer cell lines. Notably, secreted factors present in conditioned medium stimulated TNTs more potently when derived from Cx43-expressing cells than from KO cells. Moreover, TNT formation was significantly induced by the inhibition of several key cancer signaling pathways that both regulate Cx43 and are regulated by Cx43, including RhoA kinase (ROCK), protein kinase A (PKA), focal adhesion kinase (FAK), and p38. Intriguingly, the drug-induced stimulation of TNTs was more potent in Cx43 KO cells than in wild-type (WT) cells. In conclusion, this work describes a novel non-canonical role for Cx43 in regulating TNTs, identifies key cancer signaling pathways that regulate TNTs in this setting, and provides mechanistic insight into a pro-tumorigenic role of Cx43 in cancer.

Abstract 1513: Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry

Abstract Tumors are increasingly appreciated as complex ecosystems, wherein functional interactions between tumor subclones, as well as components of the microenvironment, contribute to progression and drug resistance. While some studies have focused on soluble factors that mediate these interactions, little is known about the communication between physically neighboring tumor subclones (and their microenvironment). To investigate the nature and impact of such localized interactions, we are assessing the activation status of key cancer signaling pathways in neighboring tumor cells; importantly, we are characterizing these features at the single cell level within physically intact tumors. Triple negative breast cancer (TNBC) exhibits a high degree of intratumor heterogeneity, which has contributed to a lack of effective targeted therapy options. Chemotherapy remains the standard of care; however approximately half of patients have substantial residual disease following chemotherapy, which is associated with a high risk of recurrence. Our objective is to spatially define signaling heterogeneity using patient-derived xenograft (PDX) models before and after chemotherapy treatment, to determine if spatially-defined signaling niches drive chemoresistance in TNBC. We hypothesize that neighborhoods of cells possess specialized phenotypes that mediate chemoresistance, and when disrupted, will inhibit the growth of chemoresistant tumors. We are employing imaging mass cytometry (IMC),a next-generation immunostaining approach that allows for simultaneous measurement of 30-40 biomarkers while retaining the spatial organization of the sample.Wehave constructed an IMC panel of antibodies that combines markers for tissue architecture, tumor and immune cell phenotyping, and signaling pathway activation. Our PDX collection features sequential pairs derived from biopsies taken before and after chemotherapy treatment. IMC of the pre-/post-chemotherapy pairs revealed spatial patterns of pathway activation that emerged following treatment, including increases in PI3K/mTOR and localized MAPK signaling. To complement these studies, we treated PDX models with chemotherapy and analyzed tumors via IMC throughout the course of treatment. Again, we observed the emergence of increased MAPK signaling, localized to discrete neighborhoods within the tumor. Taken together, our findings suggest that unique signaling niches arise following treatment. Our goal is to now determine whether these signaling niches functionally contribute to chemoresistance and if disruption of these niches can inhibit the growth of chemoresistant disease. Citation Format: Amanda L. Rinkenbaugh, Vidya C. Sinha, Gloria V. Echeverria, Xiaomei Zhang, Jiansu Shao, W. Fraser Symmans, Stacy L. Moulder, Helen Piwnica-Worms. Analysis of spatiotemporal phenotypic heterogeneity in chemoresistant triple negative breast cancer using imaging mass cytometry [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1513.

The Microarchitecture of Pancreatic Cancer as Measured by Diffusion-Weighted Magnetic Resonance Imaging Is Altered by T Cells with a Tumor Promoting Th17 Phenotype.

Diffusion-weighted magnetic resonance imaging (DW-MRI) is a diagnostic tool that is increasingly used for the detection and characterization of focal masses in the abdomen, among these, pancreatic ductal adenocarcinoma (PDAC). DW-MRI reflects the microarchitecture of the tissue, and changes in diffusion, which are reflected by changes in the apparent diffusion coefficient (ADC), are mainly attributed to variations in cellular density, glandular formation, and fibrosis. When analyzing the T cell infiltrates, we found an association of a tumor-promoting subpopulation, characterized by the expression of interleukin (IL) 21 and IL26, with high ADC values. Moreover, the presence of IL21+ and IL26+ positive T cells was associated with poor prognosis. Pancreatic cancers—but not healthy pancreatic tissue—expressed receptors for IL21 and IL26, a finding that could be confirmed in pancreatic cell lines. The functionality of these receptors was demonstrated in pancreatic tumor cell lines, which showed phosphorylation of ERK1/2 and STAT3 pathways in response to the respective recombinant interleukins. Moreover, in vitro data showed an increased colony formation of tumor cells. In summary, our data showed an association of IL21+ and IL26+ immune cell infiltration, increased ADC, and aggressive tumor disease, most likely due to the activation of the key cancer signaling pathways ERK1/2 and STAT3 and formation of tumor colonies.

P53-Suppressed Oncogene TET1 Prevents Cellular Aging in Lung Cancer.

The role of transcriptional regulator ten-eleven translocation methylcytosine dioxygenease 1 (TET1) has not been well characterized in lung cancer. Here we show that TET1 is overexpressed in adenocarcinoma and squamous cell carcinomas. TET1 knockdown reduced cell growth in vitro and in vivo and induced transcriptome reprogramming independent of its demethylating activity to affect key cancer signaling pathways. Wild-type p53 bound the TET1 promoter to suppress transcription, while p53 transversion mutations were most strongly associated with high TET1 expression. Knockdown of TET1 in p53-mutant cell lines induced senescence through a program involving generalized genomic instability manifested by DNA single- and double-strand breaks and induction of p21 that was synergistic with cisplatin and doxorubicin. These data identify TET1 as an oncogene in lung cancer whose gain of function via loss of p53 may be exploited through targeted therapy-induced senescence. SIGNIFICANCE: These studies identify TET1 as an oncogene in lung cancer whose gain of function following loss of p53 may be exploited by targeted therapy-induced senescence.See related commentary by Kondo, p. 1751.

Identifying Crosstalk between Raf Kinase Inhibitor Protein and Systemic Lupus Erythematosus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which hyperactive autoantibodies attack and damage healthy tissues and organs. SLE can affect multiple organs, such as the skin, kidneys, joints, and brain. The pathogenesis of SLE is multifaceted and complex, making it difficult to develop targeted therapies to ameliorate symptoms and the onset of disease. A number of signaling pathways have been investigated and shown to be implicated in SLE; however, information regarding the specific pathways involved, at least in part, in the pathogenesis of SLE remains scarce. The role of Raf kinase inhibitor protein (RKIP) in key signaling pathways in cancer is well-studied. However, studies highlighting the role of RKIP in autoimmune diseases and the inflammatory response are emerging. Whereas the induction of RKIP in cancer is associated with improved responses and reduced resistance, the overexpression of RKIP in normal tissues inhibits inflammatory cytokines and chemokines and may also inhibit autoimmunity. In this review, we have analyzed the potential crosstalk between the signaling pathways that regulate SLE and RKIP and whether targeting the pathways that activate the expression of RKIP may prove to be a novel therapeutic tool against the pathogenesis of SLE.

Comprehensive Molecular Characterization of the Hippo Signaling Pathway in Cancer

SUMMARYHippo signaling has been recognized as a key tumor suppressor pathway. Here, we perform a comprehensive molecular characterization of 19 Hippo core genes in 9,125 tumor samples across 33 cancer types using multidimensional “omic” data from The Cancer Genome Atlas. We identify somatic drivers among Hippo genes and the related microRNA (miRNA) regulators, and using functional genomic approaches, we experimentally characterize YAP and TAZ mutation effects and miR-590 and miR-200a regulation for TAZ. Hippo pathway activity is best characterized by a YAP/TAZ transcriptional target signature of 22 genes, which shows robust prognostic power across cancer types. Our elastic-net integrated modeling further reveals cancer-type-specific pathway regulators and associated cancer drivers. Our results highlight the importance of Hippo signaling in squamous cell cancers, characterized by frequent amplification of YAP/TAZ, high expression heterogeneity, and significant prognostic patterns. This study represents a systems-biology approach to characterizing key cancer signaling pathways in the post-genomic era.

The Glucose-Regulated MiR-483-3p Influences Key Signaling Pathways in Cancer.

The hsa-mir-483 gene, located within the IGF2 locus, transcribes for two mature microRNAs, miR-483-5p and miR-483-3p. This gene, whose regulation is mediated by the the CTNNB1/USF1 complex, shows an independent expression from its host gene IGF2. The miR-483-3p affects the Wnt/β-catenin, the TGF-β, and the TP53 signaling pathways by targeting several genes as CTNNB1, SMAD4, IGF1, and BBC3. Accordingly, miR-483-3p is associated with various tissues specific physiological properties as insulin and melanin production, as well as with cellular physiological functions such as wounding, differentiation, proliferation, and survival. Deregulation of miR-483-3p is observed in different types of cancer, and its overexpression can inhibit the pro-apoptotic pathway induced by the TP53 target effectors. As a result, the oncogenic characteristics of miR-483-3p are linked to the effect of some of the most relevant cancer-related genes, TP53 and CTNNB1, as well as to one of the most important cancer hallmark: the aberrant glucose metabolism of tumor cells. In this review, we summarize the recent findings regarding the miR-483-3p, to elucidate its functional role in physiological and pathological contexts, focusing overall on its involvement in cancer and in the TP53 pathway.

Identification of Deregulated Signaling Pathways in Jurkat Cells in Response to a Novel Acylspermidine Analogue-N4-Erucoyl Spermidine.

Natural polyamines such as putrescine, spermidine, and spermine are crucial in the cell proliferation and maintenance in all the eukaryotes. However, the requirement of polyamines in tumor cells is stepped up to maintain tumorigenicity. Many synthetic polyamine analogues have been designed recently to target the polyamine metabolism in tumors to induce apoptosis. N4-Erucoyl spermidine (designed as N4-Eru), a novel acylspermidine derivative, has been shown to exert selective inhibitory effects on both hematological and solid tumors, but its mechanisms of action are unknown. In this study, RNA sequencing was performed to investigate the anticancer mechanisms of N4-Eru-treated T-cell acute lymphoblastic leukemia (ALL) cell line (Jurkat cells), and gene expression was examined through different tools. We could show that many key oncogenes including NDRG1, CACNA1G, TGFBR2, NOTCH1,2,3, UHRF1, DNMT1,3, HDAC1,3, KDM3A, KDM4B, KDM4C, FOS, and SATB1 were downregulated, whereas several tumor suppressor genes such as CDKN2AIPNL, KISS1, DDIT3, TP53I13, PPARG, FOXP1 were upregulated. Data obtained through RNA-Seq further showed that N4-Eru inhibited the NOTCH/Wnt/JAK-STAT axis. This study also indicated that N4-Eru-induced apoptosis could involve several key signaling pathways in cancer. Altogether, our results suggest that N4-Eru is a promising drug to treat ALL.

Regulation of Aquaporin Functional Properties Mediated by the Antioxidant Effects of Natural Compounds.

Some aquaporins (AQPs) have been recently demonstrated to facilitate the diffusion of hydrogen peroxide (H2O2) from the producing cells to the extracellular fluid, and their reactive oxygen species scavenging properties have been defined. Nevertheless, the identification of different AQPs acting as peroxiporins, their functional role in eustress and distress, and the identification of antioxidant compounds able to regulate AQP gating, remain unsolved. This study aims to investigate, in HeLa cells: (1) the expression of different AQPs; (2) the evaluation of naringenin, quercetin, (R)-aloesaponol III 8-methyl ether, marrubiin, and curcumin antioxidant profiles, via α,α-diphenyl-β-picrylhydrazyl assay; (3) the effect of the compounds on the water permeability in the presence and in the absence of oxidative stress; and (4) the effect of pre- and post-treatment with the compounds on the H2O2 content in heat-stressed cells. Results showed that HeLa cells expressed AQP1, 3, 8, and 11 proteins. The oxidative stress reduced the water transport, and both pre- and post-treatment with the natural compounds recovering the water permeability, with the exception of curcumin. Moreover, the pre- and post-treatment with all the compounds reduced the H2O2 content of heat-stressed cells. This study confirms that oxidative stress reduced water AQP-mediated permeability, reversed by some chemical antioxidant compounds. Moreover, curcumin was shown to regulate AQP gating. This suggests a novel mechanism to regulate cell signaling and survival during stress, and to manipulate key signaling pathways in cancer and degenerative diseases.

Webinar | Key signaling pathways in cancer: Emerging trends in immunometabolism

Immune cells divide rapidly when mounting an immune response against a pathogen, for example, or when initiating a wound-healing response. To accommodate the increased energy requirements to mount the immune response, these cells may employ metabolic pathways similar to those engaged by cancer cells—pathways such as aerobic glycolysis. This correlation presents the intriguing possibility that understanding immune cell metabolism will provide new and actionable insights into the behavior of tumor cells. In this webinar, our expert speakers will explore how this phenomenon is being studied and describe how it could enable the development of new strategies in the fight against cancer. View the Webinar

Webinar | Key signaling pathways in cancer: Links to developmental biology

It is notable that the same pathways governing the cell growth, death, and differentiation decisions made during embryonic development are also common drivers of adult malignancy. In this webinar, we will explore the idea that a better understanding of developmental biology signaling pathways will advance our understanding of adult tumors and cancer stem cells as well as boost our ability to create effective therapeutics to fight a broad array of cancers.

Abstract C177: TAS-117, a highly selective non-ATP competitive inhibitor of AKT demonstrated antitumor activity in combination with chemotherapeutic agents and molecular targeted drugs.

Abstract Background: TAS-117 is a potent and highly selective non-ATP competitive small molecule inhibitor of AKT. AKT controls cellular proliferation and resistance to apoptosis, and is known to be activated in patients with relapsed or refractory tumors. Thus the combination of TAS-117 and cytotoxic drugs could be a promising approach to treat cancers. In addition, recent preclinical and clinical studies suggest that the vertical inhibition of key cancer signaling pathways may be an effective way to minimize crosstalk signaling and compensatory feedback activation. Therefore, we evaluated these combination approaches using TAS-117. Materials and Methods: A PTEN negative human ovarian cancer cell line A2780 and HER2 overexpressing human gastric cancer cell lines NCI-N87 and 4-1ST were used. In vitro combination studies were conducted by using CellTiter Glo™ assays. For in vivo studies, cells were subcutaneously transplanted into the side flank of nude mice. TAS-117, S-1, lapatinib, and everolimus were given by daily oral dosing. Carboplatin and irinotecan were given by weekly i.v. bolus injection. Trastuzumab was given by weekly i.p. bolus injection. Tumor volumes and body weights were measured twice a week. Apoptosis induction and pathway inhibition were determined by immunoblotting. Results: The in vitro combination studies revealed that TAS-117 enhanced the cytotoxicity of cisplatin, SN38, and fluorouracil in A2780 cells. In vivo, TAS-117 significantly improved the antitumor effect of carboplatin or irinotecan in an A2780 xenograft model. TAS-117 also enhanced the antitumor effect of S-1 in a 4-1ST xenograft model. With downstream, mTOR inhibitor everolimus, TAS-117 exhibited synergistic growth inhibition in NCI-N87 cells. Interestingly, cleavage of PARP was induced by the combination of TAS-117 and everolimus but not by either single agent alone. Enhanced antitumor activity in combination with TAS-117 and everolimus was confirmed in the NCI-N87 xenograft model. With an upstream, HER2 inhibitor lapatinib, TAS-117 also exhibited synergistic growth inhibition in vitro and enhanced antitumor activity in vivo in the NCI-N87 model. The combinatorial effect of TAS-117 and HER2 blockade was further evaluated using trastuzumab. TAS-117 strongly enhanced the antitumor effect of trastuzumab in a 4-1ST xenograft model. Conclusion: TAS-117 was effective in combination with chemotherapeutic agents including paclitaxel, carboplatin, irinotecan and S-1. Vertical pathway inhibition by TAS-117 with trastuzumab, lapatinib and everolimus were also effective. Our studies provide a strong rationale for clinical evaluation of our highly selective AKT inhibitor TAS-117 in combination with chemotherapeutic agents and molecular targeted drugs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C177. Citation Format: Koji Ichikawa, Tetsuya Abe, Hideki Nagase, Hitoshi Saito, Ryouto Fujita, Megumu Okada, Kazuhiko Yonekura, Toshiyasu Shimomura, Teruhiro Utsugi. TAS-117, a highly selective non-ATP competitive inhibitor of AKT demonstrated antitumor activity in combination with chemotherapeutic agents and molecular targeted drugs. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C177.

Tyrosine Kinase Signal Modulation: A Matter of H2O2 Membrane Permeability?

Abstract H2O2 produced by extracellular NADPH oxidases regulates tyrosine kinase signaling inhibiting phosphatases. How does it cross the membrane to reach its cytosolic targets? Silencing aquaporin-8 (AQP8), but not AQP3 or AQP4, inhibited H2O2 entry into HeLa cells. Re-expression of AQP8 with silencing-resistant vectors rescued H2O2 transport, whereas a C173A-AQP8 mutant failed to do so. Lowering AQP8 levels affected H2O2 entry into the endoplasmic reticulum, but not into mitochondria. AQP8 silencing also inhibited the H2O2 spikes and phosphorylation of downstream proteins induced by epidermal growth factor. These observations lead to the hypothesis that H2O2 does not freely diffuse across the plasma membrane and AQP8 and other H2O2 transporters are potential targets for manipulating key signaling pathways in cancer and degenerative diseases.

Systems Analysis of the NCI-60 Cancer Cell Lines by Alignment of Protein Pathway Activation Modules with “-OMIC” Data Fields and Therapeutic Response Signatures

The NCI-60 cell line set is likely the most molecularly profiled set of human tumor cell lines in the world. However, a critical missing component of previous analyses has been the inability to place the massive amounts of "-omic" data in the context of functional protein signaling networks, which often contain many of the drug targets for new targeted therapeutics. We used reverse-phase protein array (RPPA) analysis to measure the activation/phosphorylation state of 135 proteins, with a total analysis of nearly 200 key protein isoforms involved in cell proliferation, survival, migration, adhesion, etc., in all 60 cell lines. We aggregated the signaling data into biochemical modules of interconnected kinase substrates for 6 key cancer signaling pathways: AKT, mTOR, EGF receptor (EGFR), insulin-like growth factor-1 receptor (IGF-1R), integrin, and apoptosis signaling. The net activation state of these protein network modules was correlated to available individual protein, phosphoprotein, mutational, metabolomic, miRNA, transcriptional, and drug sensitivity data. Pathway activation mapping identified reproducible and distinct signaling cohorts that transcended organ-type distinctions. Direct correlations with the protein network modules involved largely protein phosphorylation data but we also identified direct correlations of signaling networks with metabolites, miRNA, and DNA data. The integration of protein activation measurements into biochemically interconnected modules provided a novel means to align the functional protein architecture with multiple "-omic" data sets and therapeutic response correlations. This approach may provide a deeper understanding of how cellular biochemistry defines therapeutic response. Such "-omic" portraits could inform rational anticancer agent screenings and drive personalized therapeutic approaches.

Genome-wide comparison of the transcriptomes of highly enriched normal and chronic myeloid leukemia stem and progenitor cell populations

The persistence leukemia stem cells (LSCs) in chronic myeloid leukemia (CML) despite tyrosine kinase inhibition (TKI) may explain relapse after TKI withdrawal. Here we performed genome-wide transcriptome analysis of highly refined CML and normal stem and progenitor cell populations to identify novel targets for the eradication of CML LSCs using exon microarrays. We identified 97 genes that were differentially expressed in CML versus normal stem and progenitor cells. These included cell surface genes significantly upregulated in CML LSCs: DPP4 (CD26), IL2RA (CD25), PTPRD, CACNA1D, IL1RAP, SLC4A4, and KCNK5. Further analyses of the LSCs revealed dysregulation of normal cellular processes, evidenced by alternative splicing of genes in key cancer signaling pathways such as p53 signaling (e.g. PERP, CDKN1A), kinase binding (e.g. DUSP12, MARCKS), and cell proliferation (MYCN, TIMELESS); downregulation of pro-differentiation and TGF-β/BMP signaling pathways; upregulation of oxidative metabolism and DNA repair pathways; and activation of inflammatory cytokines, including CCL2, and multiple oncogenes (e.g., CCND1). These data represent an important resource for understanding the molecular changes in CML LSCs, which may be exploited to develop novel therapies for eradication these cells and achieve cure.