Impaired Tumor Cell Proliferation Research Articles

BCAT2 promotes melanoma progression by activating lipogenesis via the epigenetic regulation of FASN and ACLY expressions.

Melanoma is the most lethal skin cancer originating from the malignant transformation of epidermal melanocyte. The dysregulation of cellular metabolism is a hallmark of cancer, including in melanoma. Aberrant branched-chain amino acids (BCAA) metabolism and related enzymes has been greatly implicated in the progression of multiple types of cancer, whereas remains far from understood in melanoma. Herein, we reported that the critical BCAA metabolism enzyme branched-chain amino acid transaminase 2 (BCAT2) is an oncogenic factor in melanoma by activating lipogenesis via the epigenetic regulation of fatty acid synthase (FASN) and ATP-citrate lyase (ACLY) expressions. Firstly, we found that BCAT2 expression was prominently increased in melanoma, and highly associated with clinical stage. Then, it was proved that the deficiency of BCAT2 led to impaired tumor cell proliferation, invasion and migration in vitro, and tumor growth and metastasis in vivo. Further, RNA sequencing technology and a panel of biochemical assays demonstrated that BCAT2 regulated de novo lipogenesis via the regulation of the expressions of both FASN and ACLY. Mechanistically, the inhibition of BCAT2 suppressed the generation of intracellular acetyl-CoA, mitigating P300-dependent histone acetylation at the promoter of FASN and ACLY, and thereby their transcription. Ultimately, zinc finger E-box binding homeobox 1 (ZEB1) was identified as the upstream transcriptional factor responsible for BCAT2 up-regulation in melanoma. Our results demonstrate that BCAT2 promotes melanoma progression by epigenetically regulating FASN and ACLY expressions via P300-dependent histone acetylation. Targeting BCAT2 could be exploited as a promising strategy to restrain tumor progression in melanoma.

Effective Reprogramming of Patient-Derived M2-Polarized Glioblastoma-Associated Microglia/Macrophages by Treatment with GW2580.

Targeting immunosuppressive and pro-tumorigenic glioblastoma (GBM)-associated macrophages and microglial cells (GAM) has great potential to improve patient outcomes. Colony-stimulating factor-1 receptor (CSF1R) has emerged as a promising target for reprograming anti-inflammatory M2-like GAMs. However, treatment data on patient-derived, tumor-educated GAMs and their influence on the adaptive immunity are lacking. CD11b+-GAMs freshly isolated from patient tumors were treated with CSF1R-targeting drugs PLX3397, BLZ945, and GW2580. Phenotypical changes upon treatment were assessed using RNA sequencing, flow cytometry, and cytokine quantification. Functional analyses included inducible nitric oxide synthase activity, phagocytosis, transmigration, and autologous tumor cell killing assays. Antitumor effects and changes in GAM activation were confirmed in a complex patient-derived 3D tumor organoid model serving as a tumor avatar. The most effective reprogramming of GAMs was observed upon GW2580 treatment, which led to the downregulation of M2-related markers, IL6, IL10, ERK1/2, and MAPK signaling pathways, while M1-like markers, gene set enrichment indicating activated MHC-II presentation, phagocytosis, and T-cell killing were substantially increased. Moreover, treatment of patient-derived GBM organoids with GW2580 confirmed successful reprogramming, resulting in impaired tumor cell proliferation. In line with its failure in clinical trials, PLX3397 was ineffective in our analysis. This comparative analysis of CSF1R-targeting drugs on patient-derived GAMs and human GBM avatars identified GW2580 as the most powerful inhibitor with the ability to polarize immunosuppressive GAMs to a proinflammatory phenotype, supporting antitumor T-cell responses while also exerting a direct antitumor effect. These data indicate that GW2580 could be an important pillar in future therapies for GBM.

Abstract 12: Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression

Abstract KEAP1 negatively regulates the transcription factor NRF2, which regulates the response to oxidative stress. Mutations in the KEAP1-NRF2 pathway occur in up to a third of non-small cell lung cancer (NSCLC) cases, leading to constitutive activation of NRF2 and resistance to therapy in patients. To study the role of NRF2 activation in lung cancer, we developed murine alleles of the KEAP1 and NRF2 mutations found in human NSCLC and performed a thorough interrogation of how these mutations affect tumor initiation and progression. Following targeted activation of these alleles in the lung with adenoviral-Cre, immunohistochemical analysis of Nrf2 and its target Nqo1 revealed that the Keap1R554Q/R554Q mutant was the most activating towards Nrf2, followed by the Nrf2D29H/+ and Keap1R554Q/+ mutants. We found that chronic Nrf2 stabilization by Keap1 or Nrf2 mutation was not sufficient to induce tumorigenesis, even in the absence of tumor suppressors p53 or Lkb1. We also found that constitutive Nrf2 activation promotes lung tumor initiation and early progression of KrasG12D cells to low-grade tumors. To analyze the influence of Keap1/Nrf2 mutation on later progression, we crossed these mutations into the KrasG12D; Trp53fl/fl model of lung adenocarcinoma. Surprisingly, we found that Keap1R554Q/R554Q mutation blocked progression to advanced-grade tumors. We also found that advanced-grade tumors downregulated Nrf2 expression and activity, suggesting that excess Nrf2 activation is detrimental to tumor progression. Next, to test whether the progression block in the Keap1R554Q/R554Q model was dependent on Nrf2, we performed single copy deletion of Nrf2. We found that lowering Nrf2 levels in this model rescued the impairment in tumor progression. Finally, using a NSCLC cell culture system of NRF2 overexpression, we found that high NRF2 levels impaired cell proliferation, viability, and anchorage-independent growth. In our in vitro studies, we observed that NRF2 expression was rapidly downregulated to basal levels to restore proliferation. Our findings indicate that NRF2 promotes lung tumor initiation and early progression but impairs late progression in a dosage-dependent manner. Our preliminary data also suggests that NRF2 hyperactivation is detrimental to tumor cell proliferation. Collectively, our results establish the context-dependence and threshold for NRF2 during the tumorigenic process. Future work will include interrogating the mechanism of NRF2 downregulation in vivo and use of additional cell lines and systems to investigate how NRF2 overexpression can impair tumor cell proliferation. Citation Format: Janine M. DeBlasi, Aimee Falzone, Samantha Caldwell, Yun Pyo Kang, Nicolas Prieto-Farigua, Justin R. Prigge, Edward E. Schmidt, Iok In Christine Chio, Florian A. Karreth, Gina M. DeNicola. Distinct Nrf2 signaling thresholds mediate lung tumor initiation and progression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 12.

TMIC-62. INHIBITION OF TUMOR-ASSOCIATED COL1A1 MATRIX ARRESTS GLIOMA MESENCHYMAL TRANSFORMATION AND REPROGRAMS THE TUMOR MICROENVIRONMENT

Abstract Tumor mesenchymal transformation (MT) is a hallmark of high-grade gliomas. The mesenchymal state is associated with specific changes related to cell adhesion, migration, and the extracellular matrix. Collagen 1a1 (COL1A1) is a main component of the extracellular matrix in gliomas, and its expression correlates inversely with patient survival. However, the cellular and molecular mechanisms of the tumor-associated COL1A1 matrix in gliomas remains elusive. Our study integrates histopathological features, spatially resolved transcriptomics, cellular dynamics and microenvironment alterations associated with MT in high-grade gliomas. Using deep learning analysis of mouse and human glioma histological samples we identified that the density of areas of MT, named oncostreams, correlates with tumor aggressiveness. Spatial transcriptomics analysis, using laser capture microdissection, identified a signature enriched in extracellular matrix related proteins, in which COL1A1 appeared as a key determinant of mesenchymal organization. Correspondingly, human and mouse high-grade gliomas showed prominent alignment of collagen fibers along these mesenchymal fascicles and higher COL1A1 expression compared to low-grade gliomas. Moreover, RNA fluorescent multiplex assays identified at single cell level that different cells within glioma tumors contribute to COL1A1 expression, including neoplastic cells and perivascular non-neoplastic cells such as ACTA2+, CYR61+ and FAP+. Inhibition of COL1A1 using genetically engineered mouse models decreased areas of mesenchymal transformation and increased survival. COL1A1 downregulation impaired tumor cell proliferation and remodeled the tumor microenvironment by reducing CD68+ macrophages/microglia cells, CD31+ endothelial cells, ACTA2+, CYR61+ and FAP+ perivascular cells, and increased GFAP+ astrocytes infiltration withing the tumor mass. Further studies, using ex-vivo glioma explants demonstrated that CO1A1 downregulation decreased collective invasion of the normal brain, supporting its importance in tumor progression. We propose that COL1A1 expression is a valuable marker for diagnosis, and COL1A1 depletion within glioma tumors is a promising direct or complementary therapeutic approach to reprogram mesenchymal transformation, and halt tumor growth.

Terbinafine prevents colorectal cancer growth by inducing dNTP starvation and reducing immune suppression

Existing evidence indicates that gut fungal dysbiosis might play a key role in the pathogenesis of colorectal cancer (CRC). We sought to explore whether reversing the fungal dysbiosis by terbinafine, an approved antifungal drug, might inhibit the development of CRC. A population-based study from Sweden identified a total of 185 patients who received terbinafine after their CRC diagnosis and found that they had a decreased risk of death (hazard ratio= 0.50) and metastasis (hazard ratio= 0.44) compared with patients without terbinafine administration. In multiple mouse models of CRC, administration of terbinafine decreased the fungal load, the fungus-induced myeloid-derived suppressor cell (MDSC) expansion, and the tumor burden. Fecal microbiota transplantation from mice without terbinafine treatment reversed MDSC infiltration and partially restored tumor proliferation. Mechanistically, terbinafine directly impaired tumor cell proliferation by reducing the ratio of nicotinamide adenine dinucleotide phosphate (NADP+) to reduced form of nicotinamide adenine dinucleotide phosphate (NADPH), suppressing the activity of glucose-6-phosphate dehydrogenase (G6PD), resulting in nucleotide synthesis disruption, deoxyribonucleotide (dNTP) starvation, and cell-cycle arrest. Collectively, terbinafine can inhibit CRC by reversing fungal dysbiosis, suppressing tumor cell proliferation, inhibiting fungus-induced MDSC infiltration, and restoring antitumor immune response.

Abstract 2476: Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy

Abstract Glioblastomas multiforme (GBMs) are the most lethal tumors of the brain. Tumoral mesenchymal transformation is a hallmark of GBMs associated with alterations in cellular morphology and dynamic organization. However, little is known about the mechanisms that control this pathological process. Here, we report a comprehensive spatiotemporal study integrating novel intra-tumoral histopathological structures, ‘oncostreams’, with tumor dynamic properties, microenvironment assets and spatial molecular features. Cellular analyses of genetic engineered mouse models of glioma identified that oncostreams are heterogenous structures formed by elongated and aligned neoplastic cells enriched in non-neoplastic cells such as ACTA2+ mesenchymal like cells and CD68+ tumor associated microglia/macrophages (TAM). Deep learning analysis of H&E glioma histological samples from mouse and human gliomas identified that oncostream density correlates with tumor aggressiveness. To determine whether oncostreams fascicles are characterized by a specific gene expression profile, we performed transcriptomic analysis using laser capture microdissection coupled to RNA-sequencing. We found that oncostreams are defined by a transcriptomic signature enriched in mesenchymal genes. Network analyses identified that COL1A1 is a critical gene that regulates oncostream organization and function. Correspondingly, human and mouse high-grade gliomas with high oncostream densities showed prominent alignment of collagen fibers along these fascicles and higher COL1A1 expression compared to low-grade gliomas. To evaluate the functional role of COL1A1 in oncostream formation we generated a COL1A1-deficient GEMM of glioma. We observed that COL1A1 inhibition decreased oncostream formation, impaired tumor cell proliferation and remodeled the tumor microenvironment by diminishing CD68+ TAM cells, CD31+ endothelial vascular proliferation and ACTA2+ perivascular mesenchymal cells, thus increasing animal survival. Further studies, using time lapse confocal imaging in ex vivo glioma explants, and intravital imaging in vivo demonstrated that oncostreams are organized collective dynamic structures present at the tumor core and the invasive tumor border. Oncostreams dynamics increased the intra-tumoral spread of cells within the tumor and foster glioma aggressiveness through collective invasion of the normal brain parenchyma. The analysis of glioma invasion in COL1A1 knockdown tumors exhibited a reduction in collective migration patterns, strongly supporting its importance in tumor progression. We propose that oncostreams represent a novel pathological marker of potential value for diagnosis and COL1A1 depletion within oncostreams is a promising approach and reprogram mesenchymal transformation to reduce the tumor malignancy. Citation Format: Andrea Comba, Syed Faisal, Patrick J. Dunn, Anna E. Argento, Todd C. Hollon, Wajd N. Al-Holou, Maria L. Varela, Daniel B. Zamler, Gunnar L. Quass, Pierre F. Apostolides, Christine E. Brown, Phillip E. E. Kish, Alon Kahana, Celina G. Kleer, Sebastien Motsch, Maria G. Castro, Pedro R. Lowenstein. Spatiotemporal analyses of preclinical glioma models reveal ‘oncostreams’ as dynamic fascicles regulating tumor mesenchymal transformation, invasion, and malignancy [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 2476.

Design, synthesis and antitumor activity study of PARP-1/HDAC dual targeting inhibitors

Both poly(ADP-ribose)polymerase-1 (PARP-1) and histone deacetylase (HDAC) are important antitumor targets and have attracted extensive attention. In this work, a total of fourteen PARP-1/HDAC dual targeting inhibitors were designed and synthesized using either benzopyrazole or benzimidazole as core structures. Two leading compounds 1–8-6 and 1–8-7 were proven to be dual targeting inhibitors of PARP-1 and HDAC6, and showed high antiproliferative activities against six human cancer cell lines with IC50 values in micromole range. Moreover, compounds 1–8-6 and 1–8-7 could impair tumor cell proliferation in 48 h and 72 h with much higher potency than co-treatment of Olaparib and Tubastatin A. 1–8-6 displayed remarkable anti-migration and anti-angiogenesis activities. Meanwhile, western blot experiment result showed that 1–8-6 was able to heighten expression level of acetylated α-tubulin with marginal effects to acetylated histones H3 and H4. Finally, docking simulation work showed that 1–8-6 could fit into the active sites of PARP-1 and HDAC6. All results indicated that 1–8-6 is a promising candidate for further preclinical studies.

Abstract OT2-11-07: Solti-1905. Elacestrant in preoperative setting, a window of opportunity study (ELIPSE trial)

Abstract Background.Patients with hormone receptor-positive/HER2-negative (HR+/HER2-) breast cancer (BC) are mainly treated with therapies that target estrogen receptor (ER) signaling to impair tumor cell proliferation. Several drugs targeting the ER pathway are currently used in the clinical practice including aromatase inhibitors (AI) that limit the production of estradiol, selective ER modulators (SERMs) that compete with estradiol for binding ER, and selective ER degraders (SERDs) that induce ER degradation. Despite the availability of these drugs, finding new ET remains a clinical unmet need since some patients do not initially respond to these treatments or become resistant to available endocrine agents. Elacestrant is an orally bioavailable drug that acts as SERD in breast tissue promoting degradation of ER and inhibiting breast cancer cell proliferation. Several clinical studies have confirmed the tolerable safety profile of elacestrant in BC (Bardia A et al. ASCO, 2021) and a phase III trial investigating elacestrant versus standard ET in metastatic BC patients is ongoing for patients treated with at least 1 prior ET and a CDK4/6 inhibitor for advanced BC (EMERALD; NCT03778931). In this study, we aim to investigate the biological effect of elacestrant on cell proliferation in patients with ER+/HER2- resectable BC. Study design. ELIPSE is prospective, multicenter, single-arm, window-of-opportunity study designed to evaluate the biological effect of elacestrant in treatment naïve patients with ER+/HER2- resectable BC. The study population consists of postmenopausal women with clinically negative axillary lymph node (cN0) and whose primary tumors are≥ 1.5 cm by ultrasound with Ki67 ≥10% locally assessed. Patients will receive 400 mg of elacestrant in monotherapy orally, once a day, continuously. After 4 weeks of treatment, surgery will be performed in accordance with local practice. Two biopsies of the same lesion will be obtained as mandatory: a baseline sample and a surgical sample (tumor core-biopsy is permitted if surgery is not performed after 4 weeks of treatment). Plasma samples for biomarkers will be collected at baseline, surgery and end of study visit. The primary objective is to evaluate the Complete Cell Cycle Arrest rate (defined as Ki67 ≤ 2.7%) by central assessment after 4 weeks of elacestrant therapy. Secondary endpoints include:. 1) safety,. 2) correlation of biological activity determined by changes in: Ki67 as a continuous variable, intrinsic PAM50 subtypes, proliferative signatures, tumor cellularity and tumor-infiltrating lymphocytes (CelTIL, Nuciforo P et al., Ann Oncol, 2018) in tumor samples taken before and at the end of treatment, and. 3) analysis of ctDNA dynamics after treatment. As of July 2021, 7 patients have been enrolled in 4 sites in Spain. NCT04797728. Acknowledgement. This study is financially supported by Radius Pharmaceuticals, Inc. Citation Format: María Vidal, Montserrat Muñoz, Mireia Margeli, Xavier González, Kepa Amillano, Rodrigo Sánchez-Bayona, Fernando Salvador, Tomás Pascual, Aleix Prat, Meritxell Bellet. Solti-1905. Elacestrant in preoperative setting, a window of opportunity study (ELIPSE trial) [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr OT2-11-07.

New inhibitors of cathepsin V impair tumor cell proliferation and elastin degradation and increase immune cell cytotoxicity

Cathepsin V is a human lysosomal cysteine peptidase with specific functions during pathological processes and is as such a promising therapeutic target. Peptidase inhibitors represent powerful pharmacological tools for regulating excessive proteolytic activity in various diseases. Cathepsin V is highly related to cathepsin L but differs in tissue distribution, binding site morphology, substrate specificity, and function. To validate its therapeutic potential and extend the number of potent and selective cathepsin V inhibitors, we used virtual high-throughput screening of commercially available compound libraries followed by an evaluation of kinetic properties to identify novel potent and selective cathepsin V inhibitors. We identified the ureido methylpiperidine carboxylate derivative, compound 7, as a reversible, selective, and potent inhibitor of cathepsin V. It also exhibited the most preferable characteristics for further evaluation with in vitro functional assays that simulate the processes in which cathepsin V is known to play an important role. Compound 7 exerted significant effects on cell proliferation, elastin degradation, and immune cell cytotoxicity. The latter was increased because compound 7 impaired conversion of immunosuppressive factor cystatin F to its active monomeric form. Taken together, our results present novel potent inhibitors of cathepsin V and provide new hit compounds for detailed development and optimization. Further, we demonstrate that cathepsin V is a potential target for new approaches to cancer therapy.

Combination of tucatinib and neural stem cells secreting anti-HER2 antibody prolongs survival of mice with metastatic brain cancer

Brain metastases are a leading cause of death in patients with breast cancer. The lack of clinical trials and the presence of the blood-brain barrier limit therapeutic options. Furthermore, overexpression of the human epidermal growth factor receptor 2 (HER2) increases the incidence of breast cancer brain metastases (BCBM). HER2-targeting agents, such as the monoclonal antibodies trastuzumab and pertuzumab, improved outcomes in patients with breast cancer and extracranial metastases. However, continued BCBM progression in breast cancer patients highlighted the need for novel and effective targeted therapies against intracranial metastases. In this study, we engineered the highly migratory and brain tumor tropic human neural stem cells (NSCs) LM008 to continuously secrete high amounts of functional, stable, full-length antibodies against HER2 (anti-HER2Ab) without compromising the stemness of LM008 cells. The secreted anti-HER2Ab impaired tumor cell proliferation invitro in HER2+ BCBM cells by inhibiting the PI3K-Akt signaling pathway and resulted in a significant benefit when injected in intracranial xenograft models. In addition, dual HER2 blockade using anti-HER2Ab LM008 NSCs and the tyrosine kinase inhibitor tucatinib significantly improved the survival of mice in a clinically relevant model of multiple HER2+ BCBM. These findings provide compelling evidence for the use of HER2Ab-secreting LM008 NSCs in combination with tucatinib as a promising therapeutic regimen for patients with HER2+ BCBM.

Pharmacological Inhibition of CA-IX Impairs Tumor Cell Proliferation, Migration and Invasiveness.

Carbonic anhydrase IX (CA-IX) plays a pivotal role in regulation of pH in tumor milieu catalyzing carbonic acid formation by hydrating CO2. An acidification of tumor microenvironment contributes to tumor progression via multiple processes, including reduced cell-cell adhesion, increased migration and matrix invasion. We aimed to assess whether the pharmacological inhibition of CA-IX could impair tumor cell proliferation and invasion. Tumor epithelial cells from breast (MDA-MB-231) and lung (A549) cancer were used to evaluate the cytotoxic effect of sulfonamide CA-IX inhibitors. Two CA-IX enzyme blockers were tested, SLC-0111 (at present in phase Ib clinical trial) and AA-06-05. In these cells, the drugs inhibited cell proliferation, migration and invasion through shifting of the mesenchymal phenotype toward an epithelial one and by impairing matrix metalloprotease-2 (MMP-2) activity. The antitumor activity was elicited via apoptosis pathway activation. An upregulation of p53 was observed, which in turn regulated the activation of caspase-3. Inhibition of proteolytic activity was accompanied by upregulation of the endogenous tissue inhibitor TIMP-2. Collectively, these data confirm the potential use of CA-IX inhibitors, and in particular SLC-0111 and AA-06-05, as agents to be further developed, alone or in combination with other conventional anticancer drugs.

Histone deacetylase inhibition promotes intratumoral CD8+ T-cell responses, sensitizing murine breast tumors to anti-PD1

Histone deacetylase (HDAC) inhibitors impair tumor cell proliferation and alter gene expression. However, the impact of these changes on anti-tumor immunity is poorly understood. Here, we showed that the class I HDAC inhibitor, entinostat (ENT), promoted the expression of immune-modulatory molecules, including MHCII, costimulatory ligands, and chemokines on murine breast tumor cells in vitro and in vivo. ENT also impaired tumor growth in vivo-an effect that was dependent on both CD8+ T cells and IFNγ. Moreover, ENT promoted intratumoral T-cell clonal expansion and enhanced their functional activity. Importantly, ENT sensitized normally unresponsive tumors to the effects of PD1 blockade, predominantly through increases in T-cell proliferation. Our findings suggest that class I HDAC inhibitors impair tumor growth by enhancing the proliferative and functional capacity of CD8+ T cells and by sensitizing tumor cells to T-cell recognition.

MicroRNA-708 activation by glucocorticoid receptor agonists regulate breast cancer tumorigenesis and metastasis via downregulation of NF-κB signaling.

Therapeutic administration of glucocorticoids (GCs) is frequently used as add-on chemotherapy for palliative purposes during breast cancer treatment. Recent studies have shown that GC treatment induces microRNA-708 in ovarian cancer cells, resulting in impaired tumor cell proliferation and metastasis. However, the regulatory functions of GCs on miR-708 and its downstream target genes in human breast cancer cells (BCCs) are poorly understood. In this study, we found that treatment with either the synthetic GC dexamethasone (DEX) or the natural GC mimic, antcin A (ATA) significantly increased miR-708 expression by transactivation of glucocorticoid receptor alpha (GRα) in MCF-7 and MDA-MB-231 human BCCs. Induction of miR-708 by GR agonists resulted in inhibition of cell proliferation, cell-cycle progression, cancer stem cell (CSC)-like phenotype and metastasis of BCCs. In addition, GR agonist treatment or miR-708 mimic transfection remarkably inhibited IKKβ expression and suppressed nuclear factor-kappaB (NF-κB) activity and its downstream target genes, including COX-2, cMYC, cyclin D1, Matrix metalloproteinase (MMP)-2, MMP-9, CD24, CD44 and increased p21CIP1 and p27KIP1 that are known to be involved in proliferation, cell-cycle progression, metastasis and CSC marker protein. BCCs xenograft models indicate that treatment with GR agonists significantly reduced tumor growth, weight and volume. Overall, our data strongly suggest that GR agonists induced miR-708 and downstream suppression of NF-κB signaling, which may be applicable as a novel therapeutic intervention in breast cancer treatment.

The JmjN domain as a dimerization interface and a targeted inhibitor of KDM4 demethylase activity.

Histone methylation is regulated to shape the epigenome by modulating DNA compaction, thus playing central roles in fundamental chromatin-based processes including transcriptional regulation, DNA repair and cell proliferation. Histone methylation is erased by demethylases including the well-established KDM4 subfamily members, however, little is known about their dimerization capacity and its impact on their demethylase activity. Using the powerful bimolecular fluorescence complementation technique, we herein show the in situ formation of human KDM4A and KDM4C homodimers and heterodimers in nuclei of live transfectant cells and evaluate their H3K9me3 demethylation activity. Using size exclusion HPLC as well as Western blot analysis, we show that endogenous KDM4C undergoes dimerization under physiological conditions. Importantly, we identify the JmjN domain as the KDM4C dimerization interface and pin-point specific charged residues therein to be essential for this dimerization. We further demonstrate that KDM4A/C dimerization is absolutely required for their demethylase activity which was abolished by the expression of free JmjN peptides. In contrast, KDM4B does not dimerize and functions as a monomer, and hence was not affected by free JmjN expression. KDM4 proteins are overexpressed in numerous malignancies and their pharmacological inhibition or depletion in cancer cells was shown to impair tumor cell proliferation, invasion and metastasis. Thus, the KDM4 dimer-interactome emerging from the present study bears potential implications for cancer therapeutics via selective inhibition of KDM4A/C demethylase activity using JmjN-based peptidomimetics.

Salinomycin inhibits metastatic colorectal cancer growth and interferes with Wnt/β-catenin signaling in CD133+ human colorectal cancer cells.

BackgroundThe polyether antibiotic Salinomycin (Sal) is regarded as an inhibitor of cancer stem cells. Its effectiveness on human colorectal cancer (CRC) cells in vitro has been demonstrated before. The aim of this study was to establish a murine model to investigate the effectiveness of Sal in vivo. Furthermore, we investigated the impact of Sal on Wnt/β-catenin signaling in human CD133+ CRC cells.MethodsThe two murine CRC cell lines MC38 and CT26 were used to analyze the impact of Sal on tumor cell proliferation, viability, migration, cell cycle progression and cell death in vitro. For in vivo studies, CT26 cells were injected into syngeneic BALB/c mice to initiate (i) subcutaneous, (ii) orthotopic, or (iii) metastatic CRC growth. Sal was administered daily, 5-Fluoruracil served as a control. For mechanistic studies, the CD133+and CD133- subpopulations of human CRC cells were separated by flow cytometry and separately exposed to increasing concentrations of Sal. The impact on Wnt/β-catenin signaling was determined by Western blotting and quantitative PCR.ResultsSal markedly impaired tumor cell viability, proliferation and migration, and induced necrotic cell death in vitro. CRC growth in vivo was likewise inhibited upon Sal treatment. Interference with Wnt signaling and reduced expression of the Wnt target genes Fibronectin and Lgr5 indicates a novel molecular mechanism, mediating anti-tumoral effects of Sal in CRC.ConclusionSal effectively impairs CRC growth in vivo. Furthermore, Sal acts as an inhibitor of Wnt/β-catenin signaling. Thus, Salinomycin represents a promising candidate for clinical CRC treatment.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-016-2879-8) contains supplementary material, which is available to authorized users.

LIM kinase inhibitors disrupt mitotic microtubule organization and impair tumor cell proliferation.

The actin and microtubule cytoskeletons are critically important for cancer cell proliferation, and drugs that target microtubules are widely-used cancer therapies. However, their utility is compromised by toxicities due to dose and exposure. To overcome these issues, we characterized how inhibition of the actin and microtubule cytoskeleton regulatory LIM kinases could be used in drug combinations to increase efficacy. A previously-described LIMK inhibitor (LIMKi) induced dose-dependent microtubule alterations that resulted in significant mitotic defects, and increased the cytotoxic potency of microtubule polymerization inhibitors. By combining LIMKi with 366 compounds from the GSK Published Kinase Inhibitor Set, effective combinations were identified with kinase inhibitors including EGFR, p38 and Raf. These findings encouraged a drug discovery effort that led to development of CRT0105446 and CRT0105950, which potently block LIMK1 and LIMK2 activity in vitro, and inhibit cofilin phosphorylation and increase αTubulin acetylation in cells. CRT0105446 and CRT0105950 were screened against 656 cancer cell lines, and rhabdomyosarcoma, neuroblastoma and kidney cancer cells were identified as significantly sensitive to both LIMK inhibitors. These large-scale screens have identified effective LIMK inhibitor drug combinations and sensitive cancer types. In addition, the LIMK inhibitory compounds CRT0105446 and CRT0105950 will enable further development of LIMK-targeted cancer therapy.

Phosphorylation of AKT(Ser473) serves as an independent prognostic marker for radiosensitivity in advanced head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is frequently characterized by high resistance to radiotherapy, which critically depends on both altered signaling pathways within tumor cells and their dynamic interaction with the tumor microenvironment. This study evaluated the prognostic value of the phosphorylation status of AKT on Ser473 and Thr308 for the clinical outcome of patients with advanced HNSCC on radiotherapy. Furthermore, we investigated the impact of AKT(Ser473) phosphorylation [p-AKT(Ser473)] in the context of radioresistance using ex vivo tissue cultures that resemble the complex tissue architecture and paracrine interaction with the tumor microenvironment. In a cohort of 120 patients with advanced HNSCC, who were treated with primary or adjuvant radiotherapy, a significant association was found between relative p-AKT(Ser473) levels and overall survival (p = 0.006) as well as progression-free survival (p = 0.021), while no significant correlation was revealed for relative p-AKT(Thr308) levels. In ex vivo tissue cultures p-AKT(Ser473) levels were increased upon irradiation and treatment with the PI3K inhibitor LY294002 inhibited both basal and irradiation induced AKT(Ser473) phosphorylation. Strikingly, pretreatment with LY294002 sensitized tissue cultures derived from primary and recurrent tumors to radiotherapy as determined by impaired tumor cell proliferation and enhanced DNA damage. In conclusion, phosphorylation status of AKT(Ser473) in tumor specimens serves as a novel biomarker to identify patients with advanced HNSCC at high risk for treatment failure following radiotherapy, and our data from ex vivo tissue cultures support the assumption that pharmacological inhibition of AKT(Ser473) phosphorylation might circumvent radioresistance to improve efficiency and reduce toxicity of current treatment modalities.

Discoidin domain receptor 1 contributes to tumorigenesis through modulation of TGFBI expression.

Discoidin domain receptor 1 (DDR1) is a member of the receptor tyrosine kinase family. The receptor is activated upon binding to its ligand, collagen, and plays a crucial role in many fundamental processes such as cell differentiation, adhesion, migration and invasion. Although DDR1 is expressed in many normal tissues, upregulated expression of DDR1 in a variety of human cancers such as lung, colon and brain cancers is known to be associated with poor prognosis. Using shRNA silencing, we assessed the oncogenic potential of DDR1. DDR1 knockdown impaired tumor cell proliferation and migration in vitro and tumor growth in vivo. Microarray analysis of tumor cells demonstrated upregulation of TGFBI expression upon DDR1 knockdown, which was subsequently confirmed at the protein level. TGFBI is a TGFβ-induced extracellular matrix protein secreted by the tumor cells and is known to act either as a tumor promoter or tumor suppressor, depending on the tumor environment. Here, we show that exogenous addition of recombinant TGFBI to BXPC3 tumor cells inhibited clonogenic growth and migration, thus recapitulating the phenotypic effect observed from DDR1 silencing. BXPC3 tumor xenografts demonstrated reduced growth with DDR1 knockdown, and the same xenograft tumors exhibited an increase in TGFBI expression level. Together, these data suggest that DDR1 expression level influences tumor growth in part via modulation of TGFBI expression. The reciprocal expression of DDR1 and TGFBI may help to elucidate the contribution of DDR1 in tumorigenesis and TGFBI may also be used as a biomarker for the therapeutic development of DDR1 specific inhibitors.

Abstract 5089: Combined treatment of trebananib (AMG 386) with panitumumab in preclinical tumor models.

Abstract Introduction: Cancer therapies that combine agents directly targeting tumor cells with those impacting the tumor stroma hold the promise of improving patient outcomes. In this preclinical study, we examined the effects of inhibiting both EGF signaling in human tumor cells and angiopoietin signaling in the murine stroma. Using tumor xenograft models, we tested a treatment combination of panitumumab, a fully human monoclonal antibody against human EGF receptor, and trebananib, an investigational peptide-Fc fusion protein that prevents binding of angiopoietins 1 and 2 to their receptor, Tie2. We assessed the effect of this combination on tumor growth and explored mechanisms of enhanced efficacy. Methods: Athymic nude mice were injected SC in the right flank with either 1x107 A431 (human epidermoid carcinoma) or 5x106 DLD-1 (colon adenocarcinoma) cells. When tumors were ∼ 200 mm3, 20 μg panitumumab IP and/or 70 μg trebananib SC were given twice weekly. Controls were isotype antibody or human Fc. In mechanism of action studies, DLD-1 tumors (∼ 300-400 mm3) received 2 treatments over 5 days before harvest and processing for paraffin sectioning. To examine endothelial cell proliferation, treated tumors were enzyme-digested and stained with anti-CD31, anti-CD45, and anti-BrdU antibodies. The percentage of BrdU-positive tumor-associated endothelial cells (CD31high/CD45neg) was determined by flow cytometry. Results: In both tumor models, combined treatment with panitumumab and trebananib resulted in significantly greater tumor growth inhibition (p≤0.014) than treatment with either single agent alone. All treatments were well tolerated; no significant weight loss was observed. Mechanistic studies revealed that combination treatment did not alter the single-agent activity of either treatment: histological analyses showed a significant reduction in estimated blood vessel area within viable tumor after treatment with either trebananib (p=0.0006) or panitumumab (p=0.012) alone; this reduction was not affected by combination treatment. Similarly, BrdU incorporation assays demonstrated that single-agent panitumumab significantly impaired tumor cell proliferation (p=0.0003), whereas single-agent trebananib significantly reduced endothelial cell proliferation (p<0.0001). Combined treatment did not change the effects on cell proliferation seen with either single agent alone. Conclusions: This study demonstrates that combined treatment with agents targeting EGF signaling in the tumor and angiopoietin signaling in the stroma results in significantly enhanced antitumor activity. Neither agent appears to impact the biochemical activities of the other, but instead gives rise to an orthogonal enhancement of antitumor activities. These findings support further clinical testing of agents that target the tumor stroma in combination with direct tumor-targeting therapies. Citation Format: James V. Bready, Paula Kaplan-Lefko, Jodi Moriguchi, Marc Payton, Stephen Kaufman, Jonathan Oliner, Robert Radinsky, Angela Coxon. Combined treatment of trebananib (AMG 386) with panitumumab in preclinical tumor models. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5089. doi:10.1158/1538-7445.AM2013-5089

Abstract A65: ShcA is required for the TGFβ-induced repression of Chordin-like-1 in ErbB2-expressing breast cancer cells

Abstract Clinical data and transgenic mouse models have provided clear evidence of a critical role for the adaptor protein ShcA in mammary tumor formation and metastasis downstream of several oncogenes. Indeed, we have demonstrated that stable silencing of ShcA expression in ErbB2-expressing breast cancer cells impairs tumor outgrowth and metastasis in vivo. In addition, we have identified a requirement for an intact PTB domain and the three tyrosine residues in ShcA for mediating TGFβ-induced migration and invasion of ErbB2-expressing breast cancer cells. Together, these observations position the ShcA adaptor as an important signaling node that facilitates cooperation between the ErbB2 and TGFβ signaling pathways in breast cancer progression. To understand how ShcA signaling contributes to TGFβ-induced gene expression changes, we generated a doxycycline-inducible system to deplete ShcA levels from ErbB2-expressing breast cancer cells. The inducible silencing of ShcA in pre-established ErbB2-positive tumors resulted in impaired tumor cell proliferation and survival in vivo. Gene expressing profiling was then performed on cells with an inducible loss of ShcA expression to identify ShcA-dependent transcriptional responses downstream of active TGFβ and ErbB2 signaling in breast cancer cells. ShcA was found to mediate the suppression of Chordin-like 1, a BMP antagonist. Conditioned media taken from TGFβ-stimulated ErbB2-expressing cells treated with doxycycline to achieve reduced ShcA levels was confirmed to contain increased levels of Chordin-like-1 compared with unstimulated controls. This same conditioned media was efficient at inhibiting the BMP-stimulated phosphorylation of Smad proteins 1/5/8 in ErbB2-expressing cells. Furthermore, this TGFβ upregulation of Chordin-like 1 production resulting from ShcA loss correlated with decreased activation of Smad1/5/8 in ShcA-deficient tumor cells in vivo, and may represent a transcriptional mechanism through which ShcA coordinates ErbB2 and TGFβ initiated tumor growth and invasion. Citation Format: Jason J. Northey, Zhifeng Dong, Chanele Cyr-Depauw, Sean Cory, Peter M. Siegel. ShcA is required for the TGFβ-induced repression of Chordin-like-1 in ErbB2-expressing breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr A65.