Myriad Of Cancers Research Articles

Abstract 5599: Role of HOX gene expression in differentiation of colon cancer stem cells

Abstract HOX genes encode a highly conserved set of transcription factors critical for stem cell (SC) function and embryonic development. Several HOX genes have also been implicated in a myriad of cancers, including colorectal cancer (CRC). However, how HOX genes play a role in SC regulation and contributes to cancer development is unclear. HOX expression is regulated by WNT, FGF, and retinoic acid (RA) signaling - pathways which also become dysregulated in cancer. Goal: To determine how regulation of HOX genes specifies differentiation of colonic SCs into specialized cell lineages and how dysregulation of HOX genes contributes to CRC development. Hypotheses: i) Each of the different cell types within human colonic epithelium has a distinct HOX gene expression signature; ii) All-trans retinoic acid (ATRA) induced differentiation of CRC cells changes HOX expression that correlates with specific HOX expression signatures in normal colon and CRC cells. Results: Immunostaining shows that specific HOX proteins (e.g., HOXA9 & HOXC9) are selectively expressed in ALDH-positive SCs and expression of these HOX genes is increased in CRC tissues compared to normal epithelium. Bioinformatics analysis predicted that these HOX genes (plus HOXA4, HOXA10, HOXC8) are regulated by RA signaling. Accordingly, we studied how ATRA influences HOX expression in CRC cells (HT29 & SW480). We found ATRA treatment: 1) Decreases proliferation and increases neuroendocrine cell (NEC) differentiation; and 2). Leads to specific changes in HOX gene expression patterns. Using NanoString profiling, we also discovered that LGR5+ SCs, ALDH+ SCs and GLP2R+ NECs have unique HOX signatures. By defining how these HOX gene expression changes in response to ATRA treatment in comparison to the HOX gene signatures of different colonic cell lineages will help us understand the role that HOX genes play in SC differentiation and how dysregulation of HOX gene expression contributes to CRC development. Citation Format: Brian T. Osmond, Caroline O. Facey, Lynn O. Opdenaker, Chi Zhang, Bruce M. Boman. Role of HOX gene expression in differentiation of colon cancer stem cells [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 5599.

Role of Syndecans in Ovarian Cancer: New Diagnostic and Prognostic Biomarkers and Potential Therapeutic Targets

Ovarian cancer (OC) is the eighth cancer both in prevalence and mortality in women and represents the deadliest female reproductive cancer. Due to generally vague symptoms, OC is frequently diagnosed only at a late and advanced stage, resulting in high mortality. The tumor extracellular matrix and cellular matrix receptors play a key role in the pathogenesis of tumor progression. Syndecans are a family of four transmembrane heparan sulfate proteoglycans (PG), including syndecan-1, -2, -3, and -4, which are dysregulated in a myriad of cancers, including OC. Many clinicopathological studies suggest that these proteins are promising diagnostic and prognostic biomarkers for OC. Furthermore, functions of the syndecan family in the regulation of cellular processes make it an interesting pharmacological target for anticancer therapies.

A narrative review of ERBB2 in non-small cell lung carcinoma

The plethora of biomarkers and the availability of targeted treatment have revolutionized the therapeutic profile of non-small cell lung cancer (NSCLC). Erythroblastic oncogene B (ERBB2) has been reported in 1%–4% cases of lung adenocarcinoma and recognized as a prognostic marker in a myriad of cancers like pancreatic, gastric, and breast carcinomas. It is more commonly recognized as human epidermal growth factor receptor 2 (HER2). Molecular characterization of NSCLC based on the mechanism of HER2 activation, including mutation, amplification, and overexpression, has recently been widely adopted owing to the differences in prognosis and predictive outcomes. This narrative review of ERBB2 is intended to describe the molecular biology, historical perspective, clinical profile, and therapeutic options for HER2-activated NSCLC. For the purpose of this review, we performed a comprehensive and detailed search in PubMed, Scopus, and My Cancer Genome databases using the keywords “HER2/neu,” “HER2,” “NSCLC,” “pertuzumab,” “trastuzumab,” and “T-DM1.” A total of 59 articles were included in the review.

TGF-β/activin signaling promotes CDK7 inhibitor resistance in triple-negative breast cancer cells through upregulation of multidrug transporters

Cyclin-dependent kinase 7 (CDK7) is a master regulatory kinase that drives cell cycle progression and stimulates expression of oncogenes in a myriad of cancers. Inhibitors of CDK7 (CDK7i) are currently in clinical trials; however, as with many cancer therapies, patients will most likely experience recurrent disease due to acquired resistance. Identifying targets underlying CDK7i resistance will facilitate prospective development of new therapies that can circumvent such resistance. Here we utilized triple-negative breast cancer as a model to discern mechanisms of resistance as it has been previously shown to be highly responsive to CDK7 inhibitors. After generating cell lines with acquired resistance, high-throughput RNA sequencing revealed significant upregulation of genes associated with efflux pumps and transforming growth factor-beta (TGF-β) signaling pathways. Genetic silencing or pharmacological inhibition of ABCG2, an efflux pump associated with multidrug resistance, resensitized resistant cells to CDK7i, indicating a reliance on these transporters. Expression of activin A (INHBA), a member of the TGF-β family of ligands, was also induced, whereas its intrinsic inhibitor, follistatin (FST), was repressed. In resistant cells, increased phosphorylation of SMAD3, a downstream mediator, confirmed an increase in activin signaling, and phosphorylated SMAD3 directly bound the ABCG2 promoter regulatory region. Finally, pharmacological inhibition of TGF-β/activin receptors or genetic silencing of SMAD4, a transcriptional partner of SMAD3, reversed the upregulation of ABCG2 in resistant cells and phenocopied ABCG2 inhibition. This study reveals that inhibiting the TGF-β/Activin-ABCG2 pathway is a potential avenue for preventing or overcoming resistance to CDK7 inhibitors.

Lipocalin-2 Inhibits Osteosarcoma Cell Metastasis by Suppressing MET Expression via the MEK-ERK Pathway.

Simple SummaryHigher neutrophil-derived cytokine lipocalin-2 (LCN2) expression possesses a versatile role in a myriad of cancers, but little is known about the role of LCN2 on osteosarcoma metastasis. In this study, we demonstrated that higher LCN2 inhibited cellular motility, migration, and invasion of osteosarcoma cells. Moreover, the phosphorylation of extracellular signal-regulated kinase (ERK) 1/2 was decreased by LCN2 knockdown. Conclusively, LCN2 inhibits osteosarcoma cell metastasis by suppressing MET via the mitogen-activated protein kinases/ERK kinase (MEK)–ERK pathway.Higher neutrophil-derived cytokine lipocalin-2 (LCN2) expression possesses a versatile role in a myriad of cancers, but little is known about the role of LCN2 on osteosarcoma metastasis. In this study, we demonstrated that higher LCN2 inhibited cellular motility, migration, and invasion of osteosarcoma cells. Moreover, using RNA sequencing technology, we found that LCN2 repressed MET gene expression in U2OS cells. Manipulation of LCN2 levels influenced the migratory potential of osteosarcoma cells as cellular migration was enhanced by transfecting with vectors containing a constitutively active LCN2 cDNA and recombinant human LCN2. Moreover, the phosphorylation of mitogen-activated protein kinases/extracellular signal-regulated kinase (ERK) kinase (MEK) 1/2 and ERK 1/2 was decreased by LCN2 knockdown. Furthermore, the use of ERK inhibitor (U0126) and activator (tBHQ) confirmed that the pharmaceutic inhibition of MEK–ERK augmented the LCN2-mediated MET suppression and migration of U2OS and HOS cells. Conclusively, LCN2 inhibits osteosarcoma cell metastasis by suppressing MET via the MEK–ERK pathway.

Abstract PO-102: A novel disseminated tumor cell identified in myriad cancer harbors tumor initiating properties

Abstract The identification of a disseminated tumor cell generated by heterotypic cell fusion, a circulating hybrid cell (CHCs) is a biologically relevant analyte of disease across myriad of cancers. Detected at elevated levels in peripheral blood, this novel population can be leveraged to understand mechanisms driving metastatic progression and predict metastatic spread. Our long term goal is to leverage evolving knowledge of mechanisms of metastatic progression to identify biologically relevant tumor markers for clinical disease assessment strategies. We therefore detected and delineated the role of CHCs as a measure of disease across a multitude of solid tumors. Human blood specimens were collected and analyzed with IRB approved protocols. Peripheral blood was obtained from patients with cancer in 14 different organ sites: ampullary adenocarcinoma, breast adenocarcinoma, cholangiocarcinoma, colon adenocarcinoma, esophageal cancer, high grade glioma (pediatric & adult), head and neck squamous cell carcinoma, pancreatic ductal adenocarcinoma, pancreatic neuroendocrine tumor, prostate adenocarcinoma, rectal adenocarcinoma and uveal melanoma and healthy subjects. Peripheral blood mononuclear cells were isolated and prepared for flow cytometry or immunofluorescence microscopy analyses with antibodies to CD45 and panCytokeratin, EpCAM, NKI Beteb, chromogranin A (CHGA)/synaptophysin (SYP) or glial fibrillary acid protein (GFAP). Discrete cellular populations including CHCs and circulating tumor cells (CTCs) were identified by protein expression. Cyclic immunofluorescence (cycIF) techniques were performed on a subset of specimens, for indepth interrogation of phenotypic heterogeneity in both the tumor and among CHCs. CHCs, defined by co-expression of CD45+ and a tumor associated protein were detected in all subjects across all disease sites at statistically significantly higher numbers than in controls samples using both detection paradigms. Furthermore, higher levels of CHCs compared with CTCs (that were CD45-) across disease sites. CycIF analysis of tumor tissue and dissemnated tumor cells from a triple negative breast cancer subject revealed a heterogeneous population of CHCs as well as complex tumor ecosystem. The phenotypic diversity of CHCs included CD44 and androgen receptor (AR) expression, which aligned with those detected in the proliferative tumor compartment. AR expression in triple negative breast cancer is known to correlate with treatment resistance and indeed, our subject rapidly succumb to disease. Taken together, these data suggest that tumors disseminate a heterogeneous population of CHCs that reflect the overall phenotype of viable tumor, supporting the value of phenotypically profiling CHCs for liquid biopsy to anticipate disease evolution and treatment resistance. This study established an isolation and detection platform for measuring CHCs in myriad of cancers and methods of phenotypic profiling CHC heterogeneity to identify discrete subpopulations. Citation Format: Matthew S. Dietz, Thomas Sutton, Brett Walker, Young Hwan Chang, Koei Chin, Melissa H. Wong. A novel disseminated tumor cell identified in myriad cancer harbors tumor initiating properties [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-102.

Abstract 6130: Zebrafish: A prominent tool for cancer drug screening and discovery

Abstract Zebrafish xenotransplants have been exploited to study a myriad of cancers. Due to high genetic and physiological similarities with humans, zebrafish represents a powerful and cost-effective animal model. The main advantages of using this model are transparency, easy manipulation, larger sample size, and short testing period. We have utilized this model system to study lung cancer by performing high throughput drug screening, genetic knockout, xenotransplant of human cancer cells, and circulating tumor cells (CTCs). Among the various applications mentioned, we discuss here,1) Cisplatin resistance in NSCLC (Non-small cell lung cancer). 2) CTC growth with single cell sequencing to identify gene signature of CTCs. 1) NSCLC is the most commonly diagnosed form of lung cancer (>85% of cases). Cisplatin resistance remains a major hurdle in treating advanced NSCLC. To understand the mechanistic basis of how drug resistance evolves we studied cancer progression and group behavior of cisplatin sensitive vs cisplatin resistant cancer cells in zebrafish. Cells were stained with DiI Red (H23-sensitive) and DiI Green dyes (H2009-resistant) respectively. Single culture and co-culture microinjections were done with 50-100 cells and the growth of tumor cells was monitored using live imaging for 72 hours at 30°C. In single culture injections we identified that H2009 cells tend to grow in larger groups where 90% of the cells grow in perivitelline space whereas H23 grow as single cells and are able travel farther in the animal faster. For H23 cells some grow in punctate groups in perivitelline space and about 30% of the cells travel to tail region. Next steps are to establish co-culture phenotypes by injecting different ratios (1:1; 1:2; 1:4; 1:8) of two cell types and assay drugs. 2) CTCs are major players of metastasis which leads to progression of cancer at secondary sites. With disease progression and treatment, it is known that the CTC repertoire changes with enrichment of certain populations of CTCs termed sub-clonal evolution. Therefore, monitoring CTC populations to study changes in gene expression profiles, especially in mutation status -before and after treatment, is a great tool to study development of resistance mechanisms. However, isolating a pure CTC population is not trivial due to their extreme rarity in peripheral blood. We utilize the ANGEL Parsotix technology which works on the differences in physical properties of CTCs and blood cells, like size and deformability. But still we retrieve merely 5-10 CTCs from 8ml of blood. Therefore, we allow these CTCs to grow by xenotransplanting them in zebrafish to get enough population of cells for single cell sequencing. Based on this information we plan to study CTC population genotype evolution -before and after treatment and comparison of gene expression profiles/mutation status of CTCs in primary tumor and metastatic lesions. Citation Format: Saumya Srivastava, Linlin Guo, Atish Mohanty, Michael Nelson, Brian Armstrong, Prakash Kulkarni, Ravi Salgia. Zebrafish: A prominent tool for cancer drug screening and discovery [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 6130.

Abstract 3850: FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function

Abstract Regulatory T cells (Tregs) are critical for the maintenance of immune tolerance and control of immune responses to foreign agents. Tregs depend upon the transcriptional protein FOXP3 for their ontogeny and genetic orchestration of their suppressive mechanisms. While T cells have the ability to target tumor-associated antigens from many cancer subtypes, these T cells are often suppressed in patients with immunologically “silenced” tumors. The tolerizing function of Tregs can limit effective anti-tumor T cell-mediated killing, even in the presence of immunotherapies, including checkpoint inhibition, and their presence predicts reduced survival in patients with a myriad of cancers. Currently, there exists no means of depleting Tregs in patients without also targeting activated effector T cells. In an effort to overcome this, we have specifically inhibited Tregs by targeting FOXP3 through molecular mimicry using peptides designed to inhibit FOXP3 homodimerization. Homodimerization of FOXP3 is mediated by an antiparallel coiled-coil interaction between the leucine-zipper domains of two FOXP3 proteins. A series of single and double hydrocarbon-stapled alpha-helical (SAH) peptides were synthesized using the leucine zipper dimerization domain (DD) interface of FOXP3 (SAH-FOXP3DDs) as a molecular template. SAH-FOXP3DDs, but not native peptides, specifically bind FOXP3's leucine zipper domain with nanomolar affinity. Unlike stapled peptide point mutant controls, lead SAH-FOXP3DDs also showed dose-dependent inhibition of FOXP3 binding to cognate DNA. Lead SAH-FOXP3DDs are cell permeable and nontoxic to T cells as measured by absence of non-specific LDH-release and apoptosis induction following treatment. Flow cytometric analysis of treated Tregs measured changes in expression of a number of Treg/T cell - associated proteins including increases in FOXP3 and CD127 and decreases in CD25, while no changes were observed in treated conventional CD4+ T cells (Tcons) or with cells treated with point-mutant control peptides. In addition, treated Tregs demonstrated mRNA expression changes of FOXP3-regulated genes including Foxp3, CD127, IL2 and Ctla. Treatment of freshly isolated and in vitro expanded Tregs with lead SAH-FOXP3DD compounds inhibited Treg-mediated suppression of Tcon proliferation in response to CD3/CD28 activation, while no effects were measured with a point-mutant control. We believe that SAH-FOXP3DDs will be powerful molecular probes to further our understanding of FOXP3 transcriptional control. Additionally, they may be a foundation for prototype therapeutics to target Tregs in cancer patients in an effort to amplify the endogenous anti-tumor immune response alone or in combination with other cancer immunotherapies. Citation Format: Rachel Eclov, Marie Fefferman, Ravand Samaeekia, James L. LaBelle. FOXP3 dimerization by stapled alpha-helical peptides alters regulatory T cell function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3850.

Sharpening the Edge for Precision Cancer Immunotherapy: Targeting Tumor Antigens through Oncolytic Vaccines.

Cancer immunotherapy represents a promising, modern-age option for treatment of cancers. Among the many immunotherapies being developed, oncolytic viruses (OVs) are slowly moving to the forefront of potential clinical therapeutic agents, especially considering the fact that the first oncolytic virus was recently approved by the Food and Drug Administration for the treatment of melanoma. OVs were originally discovered for their ability to kill cancer cells, but they have emerged as unconventional cancer immunotherapeutics due to their ability to activate a long-term antitumor immune response. This immune response not only eliminates cancer cells but also offers potential for preventing cancer recurrence. A fundamental requirement for the generation of such a strong antitumor T cell response is the recognition of an immunogenic tumor antigen by the antitumor T cell. Several tumor antigens capable of activating these antitumor T cells have been identified and are now being expressed through genetically engineered OVs to potentiate antitumor immunity. With the emergence of novel technologies for identifying tumor antigens and immunogenic epitopes in a myriad of cancers, design of “oncolytic vaccines” expressing highly specific tumor antigens provides a great strategy for targeting tumors. Here, we highlight the various OVs engineered to target tumor antigens and discuss multiple studies and strategies used to develop oncolytic vaccine regimens. We also contend how, going forward, a combination of technologies for identifying novel immunogenic tumor antigens and rational design of oncolytic vaccines will pave the way for the next generation of clinically efficacious cancer immunotherapies.

Antagonizing canonical Wnt signaling pathway by recombinant human sFRP4 purified from E. coli and its implications in cancer therapy

The Wnt signaling pathway plays a predominant role in aberrant proliferation in myriad of cancers. In non-cancerous cells, Wnts are blocked by the secreted frizzled-related proteins (sFRPs) that are generally downregulated in cancer cells. We have purified and characterized bacterially expressed glutathione S-transferase-tagged SFRP4 from a novel clone generated from human cell origin. Cervical cancer (HeLa) and lung cancer (A549) cells, in which Wnt and associated genes were found to be expressed, were treated with the purified recombinant sFRP4, which revealed a significant dose-dependent cell growth inhibition up to 40%. The current investigation on functionality of this bacterially produced recombinant sFRP4 in arresting cancer cell proliferation is the first of its kind, where G2/M phase arrest and early apoptosis were evident. Increase in phosphorylated β-catenin in sFRP4 treatment indicated inhibition of Wnt pathway, which was further confirmed by downregulation of pro-proliferative genes, namely cyclin D1, c-myc, and survivin. Functional activity of recombinant sFRP4 was further exploited in co-therapy module with chemotherapeutic drugs to decipher molecular events. Collectively, our study on purified recombinant sFRP4 from bacterial host holds great promise in targeting Wnt signaling for exploring new strategies to combat cancer.

Oncolytic Viral Therapy Using Reovirus.

Current mainstays in cancer treatment such as chemotherapy, radiation therapy, hormonal manipulation, and even targeted therapies such as Trastuzumab (herceptin) for breast cancer or Iressa (gefitinib) for non-small cell lung cancer among others are limited by lack of efficacy, cellular resistance, and toxicity. Dose escalation and combination therapies designed to overcome resistance and increase efficacy are limited by a narrow therapeutic index. Oncolytic viruses are one such group of new biological therapeutics that appears to have a wide spectrum of anticancer activity with minimal human toxicity. Since the malignant phenotype of tumors is the culmination of multiple mutations that occur in genes eventually leading to aberrant signaling pathways, oncolytic viruses either natural or engineered specifically target tumor cells taking advantage of this abnormal cellular signaling for their replication. Reovirus is one such naturally occurring double-stranded RNA virus that exploits altered signaling pathways (including Ras) in a myriad of cancers. The ability of reovirus to infect and lyse tumors under in vitro, in vivo, and ex vivo conditions has been well documented previously by us and others. The major mechanism of reovirus oncolysis of cancer cells has been shown to occur through apoptosis with autophagy taking place during this process in certain cancers. In addition, the synergistic antitumor effects of reovirus in combination with radiation or chemotherapy have also been demonstrated for reovirus resistant and moderately sensitive tumors. Recent progress in our understanding of viral immunology in the tumor microenvironment has diverted interest in exploring immunologic mechanisms to overcome resistance exhibited by chemotherapeutic drugs in cancer. Thus, currently several investigations are focusing on immune potentiating of reovirus for maximal tumor targeting. This chapter therefore has concentrated on immunologic cell death induction with reovirus as a novel approach to cancer therapy used under in vitro and in vivo conditions, as well as in a clinical setting. Reovirus phase I clinical trials have shown indications of efficacy, and several phase II/III trials are ongoing at present. Reovirus's extensive preclinical efficacy, replication competency, and low toxicity profile in humans have placed it as an attractive anticancer therapeutic for ongoing clinical testing that are highlighted in this chapter.

Abstract 4336: Oncolytic reovirus as a novel therapy for neuroblastoma

Abstract Background: Neuroblastoma (NB), a tumour of neurocrest progenitor cells of the sympathetic nervous system, is the most common extracranial pediatric tumour and accounts for approximately 15 percent of childhood cancer mortality. Even with surgery, radiation therapy, aggressive cytotoxic chemotherapy including autologous stem cell transplantation and more recently immunotherapy, high risk neuroblastoma has only a 30 percent predicted survival rate. Reovirus, a double-stranded RNA virus, has been shown to be effective against a myriad of cancers through its ability to preferentially lyse cancer cells with aberrant Ras pathway signaling. In this study, we investigate the potential of reovirus (serotype 3, strain Dearing) as a novel treatment for neuroblastoma and neuroblastoma tumour initiating cells (nbTIC). Experimental Design/Results: Reovirus induces dramatic cytotoxic effects at a multiplicity of infection (MOI) of 40 within 48h as assessed by WST-1 viability assays for the IMR-32, IMR-5, SK-N-AS, SK-N-SH, LAN-1, LAN-5, and SHEP human neuroblastoma cell lines in vitro. Interestingly the human neuroblastoma tumor-initiating cell lines, NB-12, NB88 and NB122 (kind gift from Dr David Kaplan) were also found to be very sensitive to reovirus-induced cytotoxicity, suggesting a role for reovirus treatment of refractory neuroblastoma. In order to further study reovirus, immunotherapy and neuroblastoma, a syngeneic, immunocompetent murine model (Neuro2a, A/J neuroblastoma model) was utilized to test reovirus-induced cytotoxicity and RV-directed immunotherapy of Neuro2A in vitro and in vivo. Preliminary data suggest that the Neuro2a cells are exquisitely sensitive to reovirus in vitro. Updated results will be presented. Conclusion: These preclinical results suggest that reovirus holds promise as a novel therapeutic for neuroblastoma and that it warrants further investigation in early phase clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4336. doi:10.1158/1538-7445.AM2011-4336