Reduced Tumor Cell Proliferation Research Articles

Abstract P3-03-09: Dual targeting of BCL2 and MDM2 as a novel therapeutic strategy in ER+ breast cancer model

Abstract Background: Apoptosis is the therapeutic goal following the administration of anti-cancer drugs. BCL2 and p53 are two critical nodes in the apoptosis signaling pathway. BCL2 is overexpressed in approximately 80% of primary ER+ breast cancer (BC) patients (Merino D et al. Oncogene 2016). Venetoclax is a BCL2 selective inhibitor found to be active in clinical trials for solid tumors, including ER+ BC breast cancer (Lok SW et al. Cancer Discovery 2019). MDM2/MDM4 is a proliferating protein involved in tumorigenesis via degradation of p53. In our ER+ BC breast cancer cohort (Avera Cancer Institute, Sioux Falls, South Dakota), we observed TP53 and MDM2/4 alterations in 25% and 14% of patients respectively. Aim: Here, we hypothesize that concomitant p53 activation (by an MDM2 inhibitor, e.g., AMG232) and BCL2 inhibition (by venetoclax) induce maximum apoptosis and markedly reduce tumor cell proliferation in ER+/TP53 WT BC cells. Methods: We analyzed the expression pattern of 129 ER+ BC breast cancer patients whose tumors were subjected to comprehensive genomic profiling [FoundationOne]. We tested the anti-proliferative and apoptotic activity of venetoclax alone, AMG 232 alone, and a combination of venetoclax plus AMG 232 along with fulvestrant in ER+ BC breast cancer cells (MCF7, Zr-75-1, & MDA-MB415). Results: 1) AMG 232 binds the MDM2 protein, blocks MDM2-p53 interaction, and induces p53 expression and activity, 2) p53 effector molecule p21 is robustly induced following the treatment of AMG 232, 3) the anti-proliferative activity of venetoclax or AMG232 was observed by 3D-ON-TOP clonogenic assay and real-time monitoring in an IncuCyte Zoom, 4) ER+ /TP53 wild-type (WT) cell lines exhibited an increase in annexin V positivity (initiation of apoptotic activity) following venetoclax or AMG 232 treatment, 5) importantly, a combination of venetoclax plus AMG232 robustly induced the apoptotic markers e.g. cleaved-CASPASE3/7 and cleaved PARP1 protein expression in all ER+ BC cell lines, 6) high mitochondrial depolarization was also observed following the combination of treatment, and 7) similar to protein expression data, mRNA data also showed that pro-apoptotic/cell cycle inhibitor transcripts such as p21 and PUMA mRNA expression were significantly increased and pro-survival transcripts such as survivin and stathmin mRNA expression were significantly decreased following the combination treatment. Conclusion: Our data reveal the potential of simultaneously targeting these two apoptosis regulators and provides a rational basis for clinical testing of this therapeutic approach for ER+ BC breast cancer patients that retain WT-TP53. Citation Format: Pradip De, Jennifer Carlson Aske, Poulin Pocha, Michael Sulaiman, Ethan Thompson, Nandini Dey. Dual targeting of BCL2 and MDM2 as a novel therapeutic strategy in ER+ breast cancer model [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P3-03-09.

Abstract P5-13-05: Gene expression changes with neoadjuvant hormonal treatment

Abstract Breast cancers (BC) expressing estrogen and/or progesterone receptors, referred to as hormone receptor (HR)-positive represent the largest molecular subset of breast tumors. Fortunately, estrogen receptor expression on the cell surface serves as a drug target for endocrine therapy. In HR+, human epidermal growth factor receptor-2 (HER2) negative BC, neoadjuvant endocrine therapy (NET) is being increasingly used. While NET has been shown to reduce tumor cell proliferation causing apoptosis, up to 20% of tumors do not respond. Currently there are no validated predictive gene signatures distinguishing responders from non-responders to NET. Furthermore, there are limited data on the effects of different NET regimens in vivo and how they vary between responders and non-responders. Methods: We conducted a retrospective translational study in patients who were treated with NET at our institution. This study was approved by our institutional review board. Paired pre-treatment and surgical samples were collected from 24 patients along with clinico-pathological information. Responders were defined as those with a preoperative endocrine prognostic index (PEPI) score of 0. RNA isolation was performed using the Roche HighPure FFPET RNA Isolation spin-column kit. Purified RNA was hybridized with the PanCancer Code Set and finally the cartridge was scanned on the nCounter Digital Analyzer. Raw counts from each gene were imported into the nSolver Analysis. Log2 normalized data obtained from Nanostring nCounter analysis was analyzed for differentially expressed genes between pre- and post-treatment samples. The bioinformatics analysis was carried out using nCounter analysis software. Results: The median age at diagnosis was 64 years and NET used was anastrozole alone or in combination with fulvestrant. At baseline the majority of tumors were invasive ductal carcinomas (n=18), grade II (n=18), with a Ki67% >15% (n=16). Median duration of treatment with NET was 5 months (range 2-10 months). Nine tumors were defined as responders to NET and 15 were non-responders. In all of the 24 paired samples, we found 123 genes with a statistically significant change in expression (defined by greater than two-fold change and a false discovery rate adjusted p-value <0.05) in the surgical specimen compared to pre-treatment biopsy. Genes in the Janus kinase-signal transducers and activators of transcription (Jak-STAT) signaling pathway, mitogen-activated protein kinase (MAPK) signaling pathway, RAS signaling, cytosine-cytosine receptor interaction and the Phosphatidylinositol-3-kinase/Akt/mammalian target of rapamycin (PI3K-Akt) signaling pathway were found to be upregulated whereas genes in cell cycle pathway were downregulated in the surgical specimens after NET. Comparing pre-treatment samples of responders to non-responders 25 genes involving PI3K-Akt, Notch signaling and Fanconi anemia pathway were differentially expressed. Conclusions: In our retrospective translational study we found that NET upregulates genes in the Jak-STAT, MAPK, RAS, cytosine-cytosine receptor interaction and PI3K-Akt signaling pathways and downregulates cell cycle pathway genes independent of type or duration of NET. We also found multiple genes involving the PI3K-Akt and Notch pathway were differentially expressed in pre-treatment specimens among responders compared to non-responders. While the sample size comparing responders versus non-responders was inadequate, our results suggest some early trends which warrant further investigation. Citation Format: Darien E Reed, Ritu Pandey, Jennifer M Segar, Michele Chu, Lauren LeBeau, Pavani Chalasani. Gene expression changes with neoadjuvant hormonal treatment [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P5-13-05.

The role of melatonin on miRNAs modulation in triple-negative breast cancer cells

Melatonin, a hormone secreted by pineal gland, exerts antimetastatic effects by reducing tumor cell proliferation, migration and invasion. MicroRNAs (miRNAs) are small, non-coding RNAs that play a crucial role in regulation of gene expression and biological processes of the cells. Herein, we search for a link between the tumor/metastatic-suppressive actions of melatonin and miRNA expression in triple-negative breast cancer cells. We demonstrated that melatonin exerts its anti-tumor actions by reducing proliferation, migration and c-Myc expression of triple negative breast cancer cells. By using Taqman-based assays, we analyzed the expression levels of a set of miRNAs following melatonin treatment of triple negative breast cancer cells and we identified 17 differentially expressed miRNAs, 6 down-regulated and 11 up-regulated. We focused on the anti-metastatic miR-148b and the oncogenic miR-210 both up-regulated by melatonin treatment and studied the effect of their modulation on melatonin-mediated impairment of tumor progression. Surprisingly, when miR-148b or miR-210 were depleted in triple-negative breast cancer cells, using a specific miR-148b sponge or anti-miR-210, melatonin effects on migration inhibition and c-myc downregulation were still visible suggesting that the increase of miR-148b and miR-210 expression observed following melatonin treatment was not required for the efficacy of melatonin action. Nevertheless, ours results suggest that melatonin exhibit a compound for metastatic trait inhibition, especially in MDA-MB-231 breast cancer cells even if a direct link between modulation of expression of certain proteins or miRNAs and melatonin effects has still to be established.

Nobiletin Attenuates Cell Proliferation by Modulating the Activating Protein-1 Signaling Pathway in 7,12-Dimethylbenz[a]anthracene-Induced Mammary Carcinogenesis.

Breast cancer is a widespread disease that affects women globally. Diagnostic processes and remedial approaches to breast carcinogenesis have improved in recent decades, but continuous survival of patients with breast carcinogenesis is still lacking due to increased cell proliferation. The aim of the present work is to explore the anticell proliferative effects of nobiletin (NOB) against 7,12-dimethylbenz[a]anthracene (DMBA)-treated mammary tumorigenesis in rats. We stimulate mammary carcinogenesis using oral gavage of DMBA (25 mg/kg body weight) mixed with olive oil (1 mL). This results in reduced body weight; increased liver marker enzymes such as alkaline phosphatase, acid phosphatase, aspartate aminotransferase, and alanine aminotransferase; and cell proliferative markers such as c-Jun, proliferating cell nuclear antigen, c-Fos, cyclin D1, and activating protein-1 (AP-1) in the DMBA-treated cancer-bearing animals. NOB administration improved body weight, significantly reduced hepatic marker enzymes, and altered histopathological changes. Furthermore, NOB efficiently reduced tumor cell proliferation markers in DMBA-induced mammary carcinogenesis. Overall, these results suggest that NOB has an anticell proliferative effect on DMBA-induced mammary cancer via modulation of the AP-1 signaling pathway.

Gigantol Targets Cancer Stem Cells and Destabilizes Tumors via the Suppression of the PI3K/AKT and JAK/STAT Pathways in Ectopic Lung Cancer Xenografts.

Lung cancer has long been recognized as an important world heath concern due to its high incidence and death rate. The failure of treatment strategies, as well as the regrowth of the disease driven by cancer stem cells (CSCs) residing in the tumor, lead to the urgent need for a novel CSC-targeting therapy. Here, we utilized proteome alteration analysis and ectopic tumor xenografts to gain insight on how gigantol, a bibenzyl compound from orchid species, could attenuate CSCs and reduce tumor integrity. The proteomics revealed that gigantol affected several functional proteins influencing the properties of CSCs, especially cell proliferation and survival. Importantly, the PI3K/AKT/mTOR and JAK/STAT related pathways were found to be suppressed by gigantol, while the JNK signal was enhanced. The in vivo nude mice model confirmed that pretreatment of the cells with gigantol prior to a tumor becoming established could decrease the cell division and tumor maintenance. The results indicated that gigantol decreased the relative tumor weight with dramatically reduced tumor cell proliferation, as indicated by Ki-67 labeling. Although gigantol only slightly altered the epithelial-to-mesenchymal and angiogenesis statuses, the gigantol-treated group showed a dramatic loss of tumor integrity as compared with the well-grown tumor mass of the untreated control. This study reveals the effects of gigantol on tumor initiation, growth, and maintain in the scope that the cells at the first step of tumor initiation have lesser CSC property than the control untreated cells. This study reveals novel insights into the anti-tumor mechanisms of gigantol focused on CSC targeting and destabilizing tumor integrity via suppression of the PI3K/AKT/mTOR and JAK/STAT pathways. This data supports the potential of gigantol to be further developed as a drug for lung cancer.

Modulation of EPS8 functions by phosphorylation

Objectives Epidermal growth factor receptor pathway substrate 8 (EPS8) is a scaffolding protein involved in regulating cell proliferation, actin dynamics, and receptor trafficking in human cells. EPS8 expression is increased in a range of human cancers, including head and neck squamous cell carcinoma (HNSCC). Previous studies have indicated that overexpression of EPS8 enhances mitogenesis and migration of tumor cells and is sufficient to convert nontumorigenic cells to a tumorigenic phenotype. The nonreceptor tyrosine kinase Src is reported to phosphorylate EPS8 at 4 tyrosine residues, although the impact of this on EPS8 function is unknown. The purpose of this study was to investigate the role of tyrosine phosphorylation of EPS8 at Src target sites in modulating biochemical functions, cell growth, and motility in HNSCC. Study Design Expression plasmids encoding EPS8 with amino acid substitutions to phenylalanine (F) at the 4 putative Src phosphorylation sites (Y485 F, Y525 F, Y602 F, Y774 F), and all 4 combined (4 F), were prepared by site-directed mutagenesis. To evaluate the effect of unphosphorylated EPS8 on downstream signals and biologic behavior, plasmids were transduced into a model cell line expressing a normal endogenous level of EPS8. Additionally, cells were treated with dasatinib, a Src inhibitor, to block phosphorylation of Src substrates. Expression of downstream targets was evaluated by Western blotting. Wound closure and proliferation assays were used to investigate the impact of these mutations on cell motility and growth. Results FOXM1, AURKA, and AURKB levels were decreased in cells expressing the nonphosphorylatable 4 F and Y602 F EPS8 mutants, whereas cells harboring the Y485 F, Y525 F, and Y774 F EPS8 mutants showed no differences in the expression of these proteins compared with controls. Moreover, dasatinib treatment resulted in a significant decrease in the expression of EPS8 downstream targets. In addition, both Y602 F and 4 F mutants elicited a significant reduction in tumor cell proliferation and migration. Conclusions The nonphosphorylatable Y602 F-EPS8 and 4 F-EPS8 mutants decreased the expression of EPS8 downstream targets, as well as reducing tumor cell growth and motility, implying a crucial role for phosphorylation of EPS8, principally at Y602, in mediating protumorigenic signal transduction.

Abstract B43: p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of PTHrP-mediated calcium signaling

Abstract Purpose: An unbiased approach to discover candidate cancer genes in pancreatic ductal adenocarcinoma (PDAC) identified the p120 catenin gene (Ctnnd1) as one of the top 20 driver genes, and further analysis revealed that P120CTN loss correlated with reduced survival. Results presented herein used genetically engineered mouse models (GEMMs) to show that P120CTN is a potent metastasis suppressor in PDAC. Methods: We employed orthotopic injection, tail vein metastasis assays, and mouse hospital preclinical trials to show that deletion or pharmacologic inhibition of Parathyroid Hormone Related Protein (PTHrP/PTHLH), a signaling element downstream of P120CTN, delayed tumor development and metastatic outgrowth. Finally, we generated a novel GEMM of Pthlh deletion to demonstrate in the autochthonous KPC model that loss of PTHrP phenocopies anti-PTHrP therapy by blocking both primary and metastatic tumor growth. Results: We have generated a mouse model to delete the gene Ctnnd1, whose gene product p120 catenin (P120CTN) is necessary for E-cadherin stability, resulting in enhanced epithelial-to-mesenchymal transition (EMT) and metastasis in KPC animals. Specifically, we show that KPC-p120ctncKO mice have a dramatically enhanced metastatic phenotype relative to KPC controls, suggesting that P120CTN is a critical factor in metastatic cell dissemination. An unbiased screen of tumor cells isolated from these mice identified misregulated calcium signaling through the Parathyroid Hormone Related Protein (PTHrP) as a previously unappreciated contributor to EMT and metastasis. Genetic deletion of the gene that codes for PTHrP in orthotopic transplantation experiments showed significantly reduced tumor growth and metastasis, establishing PTHrP as an oncogenic and prometastatic secreted peptide. Furthermore, treatment with anti-PTHrP monoclonal antibodies reduced tumor cell proliferation and migration in vitro, demonstrating that anti-PTHrP therapies may be of clinical benefit. Importantly, we generated KPC-PthlhcKO mice and showed that they have significantly reduced primary and metastatic tumor burden and increased survival relative to KPC controls. In parallel experiments, we treated KPC mice in a preclinical trial with anti-PTHrP neutralizing antibodies, which delayed both primary and metastatic tumor growth. Finally, analysis of human samples demonstrated that increased PTHLH expression is associated with significantly decreased survival, and that a subset of patients have PTHLH genomic amplifications. Conclusions: This novel work has demonstrated the importance of the previously unappreciated role that PTHrP-mediated calcium signaling plays in pancreatic cancer cellular plasticity and metastasis, and future studies will look to determine the efficacy of anti-PTHrP monoclonal antibodies with a view towards translation in human clinical trials. Citation Format: Jason R. Pitarresi, Robert J. Norgard, Ben Z. Stanger, Anil K. Rustgi. p120 catenin loss drives pancreatic cancer EMT and metastasis through activation of PTHrP-mediated calcium signaling [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr B43.

Abstract C036: Discovery of IACS-13909, an allosteric SHP2 inhibitor that overcomes multiple mechanisms underlying osimertinib resistance

Abstract Osimertinib, a third generation EGFR inhibitor, is a front-line therapy for EGFR mutated non-small lung cancer (NSCLC). The long-term effectiveness of osimertinib is limited by acquired resistance. Clinically identified resistance mechanisms include EGFR-dependent mechanisms such as mutations on EGFR that preclude drug binding, and EGFR-independent activation of the MAPK pathway, for instance via activation of alternate RTKs. It has also been noted that frequently a tumor from a single patient harbors more than one resistance mechanism, and the plasticity between the multiple resistance mechanisms will restrict the effectiveness of therapies targeting a single node of the oncogenic signaling network. SHP2 (Src homology 2 domain-containing phosphatase) is a phosphatase that mediates the signaling of multiple RTKs and is required for full activation of the MAPK pathway. Here we report IACS-13909 - a specific and potent allosteric inhibitor of SHP2 - suppresses the signaling of RTK/MAPK pathway. IACS-13909 potently impedes the proliferation of tumors with a broad spectrum of RTKs as the oncogenic driver. Importantly, in NSCLC models with acquired resistance to osimertinib, IACS-13909 administered as a single agent or in combination with osimertinib potently reduces tumor cell proliferation in vitro and in vivo. Together, our findings provide preclinical evidence for using a SHP2 inhibitor as a therapeutic strategy in acquired EGFR inhibitor-resistant NSCLC. Currently, a compound that potently inhibits SHP2 has been selected as the clinical development candidate and is undergoing IND-enabling studies with a projected first-in-human target of early 2020. Citation Format: Yuting Sun, Brooke A Meyers, Sarah B Johnson, Angela L Harris, Barbara Czako, Jason B Cross, Paul G Leonard, Faika Mseeh, Maria E Di Francesco, Connor A Parker, Qi Wu, Christopher A Bristow, Jason P Burke, Caroline C Carrillo, Christopher L Carroll, Qing Chang, Ningping Feng, Sonal Gera, Gao Guang, Justin Kwang-Lay Huang, Yongying Jiang, Zhijun Kang, Jeffrey J Kovacs, Xiaoyan Ma, Pijus K Mandal, Timothy McAfoos, Robert A Mullinax, Michael D Peoples, Vandhana Ramamoorthy, Sahil Seth, Erika Suzuki, Christopher Conrad Williams, Simon S Yu, Andy M Zuniga, Giulio F Draetta, Joseph R Marszalek, Timothy P Heffernan, Nancy E Kohl, Philip Jones. Discovery of IACS-13909, an allosteric SHP2 inhibitor that overcomes multiple mechanisms underlying osimertinib resistance [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C036. doi:10.1158/1535-7163.TARG-19-C036

Thiamine mimetics sulbutiamine and benfotiamine as a nutraceutical approach to anticancer therapy

Malignant cells frequently demonstrate an oncogenic-driven reliance on glycolytic metabolism to support their highly proliferative nature. Overexpression of pyruvate dehydrogenase kinase (PDK) may promote this unique metabolic signature of tumor cells by inhibiting mitochondrial function. PDKs function to phosphorylate and inhibit pyruvate dehydrogenase (PDH) activity. Silencing of PDK expression has previously been shown to restore mitochondrial function and reduce tumor cell proliferation. High dose Vitamin B1, or thiamine, possesses antitumor properties related to its capacity to reduce PDH phosphorylation and promote its enzymatic activity, presumably through PDK inhibition. Though a promising nutraceutical approach for cancer therapy, thiamine’s low bioavailability may limit clinical effectiveness. Here, we have demonstrated exploiting the commercially available lipophilic thiamine analogs sulbutiamine and benfotiamine increases thiamine’s anti-cancer effect in vitro. Determined by crystal violet proliferation assays, both sulbutiamine and benfotiamine reduced thiamine’s millimolar IC50 value to micromolar equivalents. HPLC analysis revealed that sulbutiamine and benfotiamine significantly increased intracellular thiamine and TPP concentrations in vitro, corresponding with reduced levels of PDH phosphorylation. Through an ex vitro kinase screen, thiamine’s activated cofactor form thiamine pyrophosphate (TPP) was found to inhibit the function of multiple PDK isoforms. Attempts to maximize intracellular TPP by exploiting thiamine homeostasis gene expression resulted in enhanced apoptosis in tumor cells. Based on our in vitro evaluations, we conclude that TPP serves as the active species mediating thiamine’s inhibitory effect on tumor cell proliferation. Pharmacologic administration of benfotiamine, but not sulbutiamine, reduced tumor growth in a subcutaneous xenograft mouse model. It remains unclear if benfotiamine’s effects in vivo are associated with PDK inhibition or through an alternative mechanism of action. Future work will aim to define the action of lipophilic thiamine mimetics in vivo in order to translate their clinical usefulness as anticancer strategies.

EXTH-04. BLOCKADE OF NRF2/GLUTATHIONE METABOLISM AS A SYNTHETIC LETHALITY APPROACH FOR IDH1-MUTATED GLIOMA

Abstract BACKGROUND Mutations in isocitrate dehydrogenase (IDH1/2) are frequent genetic abnormalities in human malignancies. IDH1/2-mutated cancers are a recently defined disease entity with distinctive patterns of tumor cell biology, metabolism and resistance to therapy. Molecular targeting approaches against this disease cluster remain limited. METHODS We investigated the redox homeostasis in IDH1 mutant-transduced cells and patient-derived brain tumor initiating cells. The importance of antioxidant genes was confirmed through COX regression analysis on a large cohort of lower grade glioma. We investigated the biologic impact of Nuclear factor erythroid 2-related factor 2 (NRF2) on the glutathione de novo synthesis in IDH1-mutated cells. Finally, we evaluated the value of targeting NRF2/glutathione metabolic pathway as a potential synthetic lethality approach for IDH1-mutated cell in vitro and in vivo. RESULTS We discovered that acquisition of cancer-associated IDH1 mutants results in constitutive activation of NRF2-governed cytoprotective pathways through decoupling of NRF2 from its E3 ligase Kelch-like ECH-associated protein 1. NRF2 mediated the transcriptional activation of GCLC, GCLM and SLC7A11, which strengthens the glutathione de novo synthesis, and relieves the metabolic burden derived from IDH1 mutant neomorphic activity. Blockade of the NRF2/glutathione metabolic pathway synergizes with the elevated intrinsic reactive oxygen species, which results in overwhelming oxidative damage in IDH1-mutated cells, as well as a substantial reduction in tumor cell proliferation and xenograft expansion. CONCLUSION Our findings suggest that blockade of the NRF2/glutathione synthetic pathway is a novel targeting strategy for IDH1-mutated malignancies.

Upregulated expression of eIF3C is associated with malignant behavior in renal cell carcinoma.

Eukaryotic initiation factor 3c (eIF3C) is involved in the initiation of protein translation. Aberrant eIF3C expression has been reported in different types of human cancer. The present study aimed to assess the role of eIF3C in the malignant behavior of renal cell carcinoma invitro and invivo. eIF3C expression was assessed in 16 pairs of renal cell carcinoma (RCC) and matched distant normal tissues, and in RCC cell lines using immunohistochemistry. Subsequently, eIF3C was depleted using lentiviral short hairpin RNA and cell proliferation, cell cycle distribution and apoptosis of these eIF3C‑depleted cells were examined. Additionally, tumor cell xenograft assays in nude mice, Affymetrix microarrays and ingenuity pathway analyses were performed. eIF3C expression was upregulated in RCC tissues and cell lines. Depletion of eIF3C reduced tumor cell proliferation and arrested them at the G1 stage, thus promoting their apoptosis invitro. Depletion of eIF3C also inhibited the formation and growth of tumor cell xenografts in nude mice. In addition, depletion of eIF3C altered the expression levels of 994 differentially expressed genes in RCC cells (516 genes were upregulated and 478genes were downregulated). The expression levels of phosphorylated‑AKT, c‑JUN and NFKB inhibitor α were lower in the shorth hairpin RNA eIF3C‑transfected RCC cells compared with in the control group. In conclusion, the present study demonstrated that upregulated eIF3C expression contributed to the development and progression of RCC. Future studies should further evaluate whether eIF3C could be used as a potential strategy for RCC targeting therapy.

Expression of Tumor-mediated CD137 ligand in human colon cancer indicates dual signaling effects

ABSTRACTCD137-targeting immune therapy, which activates anti-tumor T effector cell responses, seems to be an attractive concept in clinical oncology. Recent evidence has demonstrated that tumor cells besides T cells and antigen-presenting cells are able to express CD137 and CD137L. Here we aimed to identify CD137/CD137L expression in established colon cancer cell lines and primary tumors (UICC stages I-IV) from patients with documented long-term follow-up. CD137/CD137L expression was highly upregulated in early to late-stage tumors while the inverse was observed in patient-derived peripheral blood mononuclear cells. High CD137L expression within primary tumors was mediated by tumor cells and significantly correlated with the occurrence of distant metastases and shortened survival in advanced stages of disease (UICC stage IV). Interestingly, induced tumor cell signaling via CD137L on its surface in vitro resulted in dual effects: (i) reduced tumor cell proliferation suggesting inhibitory signaling in all investigated cancers and (ii) increased epithelial-to-mesenchymal transition signaling events. Taken together CD137/CD137L expression was stage-dependently upregulated with shortened survival in patients with highly CD137L-expressing tumors. Our clinical and experimental data suggest that colon cancer cells predominantly express CD137L and thereby have negative impact on overall survival through a process of reverse signaling. Beside agonistic CD137 antibody therapy to foster T effector cell responses, CD137L-mediated intervention strategies may become instrumental to circumvent relapsed tumor growth through induced epithelial-to-mesenchymal transition and consecutive metastases formation.

Systemic delivery of a Gli inhibitor via polymeric nanocarriers inhibits tumor-induced bone disease

Solid tumors frequently metastasize to bone and induce bone destruction leading to severe pain, fractures, and other skeletal-related events (SREs). Osteoclast inhibitors such as bisphosphonates delay SREs but do not prevent skeletal complications or improve overall survival. Because bisphosphonates can cause adverse side effects and are contraindicated for some patients, we sought an alternative therapy to reduce tumor-associated bone destruction. Our previous studies identified the transcription factor Gli2 as a key regulator of parathyroid hormone-related protein (PTHrP), which is produced by bone metastatic tumor cells to promote osteoclast-mediated bone destruction. In this study, we tested the treatment effect of a Gli antagonist GANT58, which inhibits Gli2 nuclear translocation and PTHrP expression in tumor cells. In initial testing, GANT58 did not have efficacy in vivo due to its low water solubility and poor bioavailability. We therefore developed a micellar nanoparticle (NP) to encapsulate and colloidally stabilize GANT58, providing a fully aqueous, intravenously injectable formulation based on the polymer poly(propylene sulfide)135-b-poly[(oligoethylene glycol)9 methyl ether acrylate]17 (PPS135-b-POEGA17). POEGA forms the hydrophilic NP surface while PPS forms the hydrophobic NP core that sequesters GANT58. In response to reactive oxygen species (ROS), PPS becomes hydrophilic and degrades to enable drug release. In an intratibial model of breast cancer bone metastasis, treatment with GANT58-NPs decreased bone lesion area by 49% (p<.01) and lesion number by 38% (p<.05) and resulted in a 2.5-fold increase in trabecular bone volume (p<.001). Similar results were observed in intracardiac and intratibial models of breast and lung cancer bone metastasis, respectively. Importantly, GANT58-NPs reduced tumor cell proliferation but did not alter mesenchymal stem cell proliferation or osteoblast mineralization in vitro, nor was there evidence of cytotoxicity after repeated in vivo treatment. Thus, inhibition of Gli2 using GANT58-NPs is a potential therapy to reduce bone destruction that should be considered for further testing and development toward clinical translation.

Paracrine Crosstalk between Fibroblasts and ER+ Breast Cancer Cells Creates an IL1β-Enriched Niche that Promotes Tumor Growth.

SummaryBreast cancer-induced activated fibroblasts support tumor progression. However, the role of normal fibroblasts in tumor progression remains controversial. In this study, we used modified patient-derived organoid cultures and demonstrate that constitutively secreted cytokines from normal breast fibroblasts initiate a paracrine signaling mechanism with estrogen receptor-positive (ER+) breast cancer cells, which results in the creation of an interleukin (IL)-1β-enriched microenvironment. We found that this paracrine signaling mechanism is shared between normal and activated fibroblasts. Interestingly, we observed that in reconstructed tumor microenvironment containing autologous ER+ breast cancer cells, activated fibroblasts, and immune cells, tamoxifen is more effective in reducing tumor cell proliferation when this paracrine signaling is blocked. Our findings then suggest that ER+ tumor cells could create a growth-promoting environment without activating stromal fibroblasts and that in breast-conserving surgeries, normal fibroblasts could be a significant modulator of tumor recurrence by enhancing the proliferation of residual breast cancer cells in the tumor-adjacent breast tissue.

Stromal iodothyronine deiodinase 2 (DIO2) promotes the growth of intestinal tumors in ApcΔ716 mutant mice.

Iodothyronine deiodinase 2 (DIO2) converts the prohormone thyroxine (T4) to bioactive T3 in peripheral tissues and thereby regulates local thyroid hormone (TH) levels. Although epidemiologic studies suggest the contribution of TH to the progression of colorectal cancer (CRC), the role of DIO2 in CRC remains elusive. Here we show that Dio2 is highly expressed in intestinal polyps of Apc Δ716 mice, a mouse model of familial adenomatous polyposis and early stage sporadic CRC. Laser capture microdissection and in situ hybridization analysis show almost exclusive expression of Dio2 in the stroma of Apc Δ716 polyps in the proximity of the COX‐2‐positive areas. Treatment with iopanoic acid, a deiodinase inhibitor, or chemical thyroidectomy suppresses tumor formation in Apc Δ716 mice, accompanied by reduced tumor cell proliferation and angiogenesis. Dio2 expression in Apc Δ716 polyps is strongly suppressed by treatment with the COX‐2 inhibitor meloxicam. Analysis of The Cancer Genome Atlas data shows upregulation of DIO2 in CRC clinical samples and a close association of its expression pattern with the stromal component, consistently with almost exclusive expression of DIO2 in the stroma of human CRC as revealed by in situ hybridization. These results indicate essential roles of stromal DIO2 and thyroid hormone signaling in promoting the growth of intestinal tumors.

N-Acetyltransferase 10 Enhances Doxorubicin Resistance in Human Hepatocellular Carcinoma Cell Lines by Promoting the Epithelial-to-Mesenchymal Transition.

Background N-Acetyltransferase 10 (NAT10) has been reported to be expressed at high levels in hepatocellular carcinoma (HCC); however, its role in chemoresistance is unclear. This study is aimed at investigating whether NAT10 regulates the epithelial-mesenchymal transition (EMT) and chemoresistance in HCC. Methods HCC cell lines (Huh-7, Bel-7402, SNU387, and SNU449) were treated with remodelin, an inhibitor of NAT10, or transfected with small inhibitory RNAs (siRNAs) targeting NAT10 or Twist. The EMT was induced by hypoxia. The CCK-8 assay was used to quantify cell viability, the EdU incorporation assay to assess cell proliferation. siRNA knockdown efficiency and epithelial/mesenchymal marker expression were assessed by western blotting. Results Knockdown of NAT10 using siRNA or inhibition of NAT10 using remodelin increased the sensitivity of HCC cell lines to doxorubicin; similar effects were observed in cells transfected with the Twist siRNA. Inhibition of NAT10 using remodelin also reversed the ability of doxorubicin to induce the EMT in HCC cells. Furthermore, inhibiting NAT10 reversed the hypoxia-induced EMT. Finally, we confirmed that combining doxorubicin with remodelin delayed tumor growth and reduced tumor cell proliferation in a mouse xenograft model of HCC. Conclusions NAT10 may contribute to chemoresistance in HCC by regulating the EMT. The mechanism by which NAT10 regulates the EMT and doxorubicin sensitivity in HCC cells merits further investigation.

The FOXM1 Inhibitor RCM-1 Decreases Carcinogenesis and Nuclear β-Catenin.

The oncogenic transcription factor FOXM1 has been previously shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple cancer types. A small-molecule compound, Robert Costa Memorial drug-1 (RCM-1), has been recently identified from high-throughput screen as an inhibitor of FOXM1 in vitro and in mouse model of allergen-mediated lung inflammation. In the present study, we examined antitumor activities of RCM-1 using tumor models. Treatment with RCM-1 inhibited tumor cell proliferation as evidenced by increased cell-cycle duration. Confocal imaging of RCM-1-treated tumor cells indicated that delay in cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM-1 reduced the formation and growth of tumor cell colonies in the colony formation assay. In animal models, RCM-1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76-9, melanoma B16-F10, and human H2122 lung adenocarcinoma. RCM-1 decreased FOXM1 protein in the tumors, reduced tumor cell proliferation, and increased tumor cell apoptosis. RCM-1 decreased protein levels and nuclear localization of β-catenin, and inhibited protein-protein interaction between β-catenin and FOXM1 in cultured tumor cells and in vivo Altogether, our study provides important evidence of antitumor potential of the small-molecule compound RCM-1, suggesting that RCM-1 can be a promising candidate for anticancer therapy.

Abstract 2208: Targeted inhibition of FGF/PDGF/VEGF signaling enhances nanoparticle taxane response in preclinical gastric cancer models

Abstract Background: Gastric adenocarcinoma (GAC) remains the third most common cause of cancer-related deaths worldwide. Most available systemic chemotherapy options for advanced GAC have limited efficacy. Nab-paclitaxel (NPT), a nanoparticle albumin-bound taxane formulation, has shown significant activity in preclinical GAC studies. Overexpression of multiple growth factors and their receptors such as FGF/FGFR, PDGF/PDGFR and VEGF/VEGFR, promote angiogenesis in several cancers including GAC that leads to tumor progression and metastasis. Dovitinib is a small molecule RTK inhibitor that potentially inhibits the activity of FGFR, PDGFR and VEGFR. We evaluated the antitumor efficacy of dovitinib and its ability to enhance nab-paclitaxel response in preclinical GAC models. Methods: In vitro cell proliferation and protein expression were measured by WST-1 assay and Immunoblotting. In vivo tumor growth and animal survival studies were performed in NOD/SCID mice using human GAC MKN-45 cells. Results: Nab-paclitaxel and dovitinib had in vitro growth inhibitory effect on several GAC associated cell lines tested, with additive effects in combination. Immunoblot analysis revealed that dovitinib treatment caused a decrease in the expression of phospho-FGFR, phospho-AKT, phospho-ERK; and an increase in the expression of apoptosis-related proteins cleaved PARP-1 and cleaved caspase-3. Nab-paclitaxel activity correlated with the increased expression of phospho-stathmin. In subcutaneous GAC xenografts, NPT and dovitinib demonstrated inhibition in tumor growth, while NPT+dovitinib had an additive effect. Net tumor growth was 533.7 mm3 in controls, 394.8 mm3 after oxaliplatin, 135.8 mm3 after NPT, 128 mm3 after dovitinib, 98.9 mm3 after oxaliplatin+dovitinib and -41.8 mm3 (tumor regression) after NPT+dovitinib. Tumor tissue analysis revealed that dovitinib reduced tumor vasculature, while nab-paclitaxel reduced tumor cell proliferation. In a peritoneal dissemination model, animal survival compared to controls (23 days) remained unchanged after monotherapy with oxaliplatin (24 days) or dovitinib (25 days) but increased significantly after NPT monotherapy (42 days, a 83% increase). Combination of dovitinib with NPT exhibited a further increase in animal survival (66 days, a 187% increase), while the combination of dovitinib with oxaliplatin had no survival benefit (25 days). Conclusion: These findings demonstrate that the antitumor effect of nab-paclitaxel can be significantly enhanced by the FGFR/PDGFR/VEGFR pathway inhibitor dovitinib in preclinical GAC models. The data support the clinical relevance of this therapeutic combination for advanced GAC patients. Citation Format: Niranjan Awasthi, Kate Crawford, Erin Bontrager, Sazzad Hassan, Urs von Holzen, Margaret A. Schwarz, Roderich E. Schwarz. Targeted inhibition of FGF/PDGF/VEGF signaling enhances nanoparticle taxane response in preclinical gastric cancer models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2208.

Abstract 5075: Naproxen blocks spontaneous lung adenoma progression to adenocarcinoma in Kras-G12V mice

Abstract The aim of the present study was to investigate the effects of a non-steroidal anti-inflammatory drug (NSAID), naproxen, on a spontaneous mouse model of lung adenocarcinoma. Lung cancer is the most frequently diagnosed cancer and the primary cause of cancer-related deaths worldwide. Inflammation plays an important role in the development and progression of lung and several other cancers. The association between NSAID use and lung cancer risk suggests a beneficial effect on patients’ lung cancer progression. Preventing lung cancer can help to significantly reduce cancer-related mortality. Compared with other NSAIDs, naproxen is relatively safer in terms of cardiovascular risk. Eight-week-old transgenic KrasG12V male and female mice (n=20) or littermate wild-type (n=5) mice were fed (AIN76A) diets containing naproxen (0 ppm or 400 ppm) in modified AIN76-A diet for 28 weeks. At 36 weeks of age, mice were euthanized. Lungs were collected and evaluated for lung tumor incidence and multiplicity, and were saved in formalin for histopathological identification of adenoma and adenocarcinoma. By 12 weeks of age, KrasG12V mice developed visible lung tumors that enlarged in size and progressed to adenocarcinoma by 24-36 weeks of age. Lung tumor incidence was observed in 100% of KrasG12V mice. Dietary administration of naproxen did not show any overt-toxicities. No significant changes were observed in body weight gain or any major organ’s (liver, kidney, and pancreas) gross morphology or weight in mice fed with naproxen compared with mice fed control diet. Due to reduced tumor burden, lungs of the naproxen-fed mice weighed less (230.6± 12.2 mg, p=0.019) (Mean±SEM) than those of the control group (384.0±68.1 mg). Importantly, mice fed control diet developed 19.8±0.96 total lung tumors (2.5±0.3 adenoma, 17.3±0.7 adenocarcinoma). The results suggest that naproxen inhibits total lung tumor formation by ~52% (9.4±0.85;p&amp;lt;0001) and adenocarcinoma by ~64% (6.1±0.6; p&amp;lt;0001), compared with control diet. However, we observed no significant difference in the number of adenomas in mice fed with control diet and mice fed naproxen diet. These data suggest that naproxen delays the progression of lung adenoma to adenocarcinoma. Biomarker analysis of lung tumors from mice exposed to naproxen showed significantly reduced tumor cell proliferation (PCNA, Cyclin D1) and increased apoptosis (p21, Caspase-3). These results support a chemopreventive role of naproxen in spontaneous-induced lung adenocarcinoma formation. (Supported by the Kerley-Cade Chair Endowment &amp; partly by the NCI-PREVENT program HHSN261201500038i) Citation Format: Gaurav Kumar, Venkateshwar Madka, Craig Logsdon, Anil Singh, Nicole Stratton, Stanley Lightfoot, Altaf Mohammed, Chinthalapally V. Rao. Naproxen blocks spontaneous lung adenoma progression to adenocarcinoma in Kras-G12V mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5075.

Abstract 1998: Selinexor effectively inhibits tumor growth in a triple negative breast cancer brain metastasis mouse model

Abstract Introduction: Breast cancer brain metastasis (BCBM) is a devastating disease and remains largely incurable. Patients with triple negative breast cancer (TNBC) develop brain metastasis at a significantly higher incidence (46%) than patients with other breast cancer subtypes. Median survival after brain metastases in TNBC can be as short as 5 months. Selinexor is a first-in-class oral SINE (Selective Inhibitor of Nuclear Export) drug that inhibits XPO1 (exportin-1/ CRM1) activity and exhibits remarkable anti-cancer activity in advanced clinical trials. Moreover, selinexor can penetrate the blood-brain barrier and shows promising anti-tumor activity in animal models of glioblastoma and central nervous system lymphoma. Previous studies have shown that most TNBC cell lines tested in vitro and in vivo are sensitive to selinexor-induced growth inhibition and cell death. To explore the possibility of using selinexor as an effective treatment for TNBC brain metastasis, a mouse model was established and tested. Methods: TNBC cell line HCC1806 (ATCC# CRL-2335) was transformed with RediFect Red-Fluc-Puromycin and selected using puromycin to generate HCC1806-Fluc cells that expressed both puromycin resistance gene and a luciferase gene. Twenty-four nu/nu mice were inoculated intracranially with 1 x 104 HCC1806-Fluc cells. Four days post implant, mice were allocated to three groups of eight mice and treated with either vehicle, selinexor 10 mg/kg, or selinexor 15 mg/kg through oral gavage, three times per week (Mon, Wed, Fri). Body weight and condition of animals were recorded daily, and tumors were imaged twice per week (Tue and Thu). Brain tissues were collected at the end of study and analyzed by immunohistochemistry (IHC). Results: HCC1806-Fluc cells injected intracranially successfully implanted in the brains of host mice. All mice survived the duration of the study (day 16 after treatment initiation) and no significant difference in body weight was observed between different treatment groups. Mice treated with selinexor had a significant reduction in tumor area relative to vehicle controls. Tumor growth inhibition (TGI) was 103% for selinexor at 10 mg/kg and 97% for selinexor at 15 mg/kg compared to vehicle controls. IHC showed reduced tumor cell proliferation, decreased expression of Myc and Survivin, and increased nuclear expression of p21 and p27 in tumor samples from the brain of mice treated with selinexor as compared to that of controls. Decreased expression of HDAC1 and HDAC5 was also observed in brain tumor samples from selinexor-treated mice. Conclusions: Oral intake of selinexor effectively inhibited TNBC cell growth in a mouse model of brain metastasis. The anti-cancer effect is likely achieved through multiple signaling pathways, including cell cycle regulation, down-regulation of oncogenes and HDACs. Citation Format: Hua Chang, Marsha Crochiere, Sophie Debler, Trinayan Kashyap, Thaddeus J. Unger, Erkan Baloglu, William Senapedis, Sharon Shacham, Yosef Landesman. Selinexor effectively inhibits tumor growth in a triple negative breast cancer brain metastasis mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1998.