Role of hypoxia and glycolysis in the development of multi-drug resistance in human tumor cells and the establishment of an orthotopic multi-drug resistant tumor model in nude mice using hypoxic pre-conditioning

<p>Abstract</p> <p>Background</p> <p>The development of multi-drug resistant (MDR) cancer is a significant challenge in the clinical treatment of recurrent disease. Hypoxia is an environmental selection pressure that contributes to the development of MDR. Many cancer cells, including MDR cells, resort to glycolysis for energy acquisition. This study aimed to explore the relationship between hypoxia, glycolysis, and MDR in a panel of human breast and ovarian cancer cells. A second aim of this study was to develop an orthotopic animal model of MDR breast cancer.</p> <p>Methods</p> <p>Nucleic and basal protein was extracted from a panel of human breast and ovarian cancer cells; MDR cells and cells pre-exposed to either normoxic or hypoxic conditions. Western blotting was used to assess the expression of MDR markers, hypoxia inducible factors, and glycolytic proteins. Tumor xenografts were established in the mammary fat pad of <it>nu/nu </it>mice using human breast cancer cells that were pre-exposed to either hypoxic or normoxic conditions. Immunohistochemistry was used to assess the MDR character of excised tumors.</p> <p>Results</p> <p>Hypoxia induces MDR and glycolysis <it>in vitro</it>, but the cellular response is cell-line specific and duration dependent. Using hypoxic, triple-negative breast cancer cells to establish 100 mm³tumor xenografts in nude mice is a relevant model for MDR breast cancer.</p> <p>Conclusion</p> <p>Hypoxic pre-conditiong and xenografting may be used to develop a multitude of orthotopic models for MDR cancer aiding in the study and treatment of the disease.</p

The emerging roles and therapeutic potential of cyclin-dependent kinase 11 (CDK11) in human cancer.

Overexpression and/or hyperactivation of cyclin-dependent kinases (CDKs) are common features of most cancer types. CDKs have been shown to play important roles in tumor cell proliferation and growth by controlling cell cycle, transcription, and RNA splicing. CDK4/6 inhibitor palbociclib has been recently approved by the FDA for the treatment of breast cancer. CDK11 is a serine/threonine protein kinase in the CDK family and recent studies have shown that CDK11 also plays critical roles in cancer cell growth and proliferation. A variety of genetic and epigenetic events may cause universal overexpression of CDK11 in human cancers. Inhibition of CDK11 has been shown to lead to cancer cell death and apoptosis. Significant evidence has suggested that CDK11 may be a novel and promising therapeutic target for the treatment of cancers. This review will focus on the emerging roles of CDK11 in human cancers, and provide a proof-of-principle for continued efforts toward targeting CDK11 for effective cancer treatment

Therapeutic Efficacy and Safety of Paclitaxel/Lonidamine Loaded EGFR-Targeted Nanoparticles for the Treatment of Multi-Drug Resistant Cancer

The treatment of multi-drug resistant (MDR) cancer is a clinical challenge. Many MDR cells over-express epidermal growth factor receptor (EGFR). We exploit this expression through the development of EGFR-targeted, polymer blend nanocarriers for the treatment of MDR cancer using paclitaxel (a common chemotherapeutic agent) and lonidamine (an experimental drug; mitochondrial hexokinase 2 inhibitor). An orthotopic model of MDR human breast cancer was developed in nude mice and used to evaluate the safety and efficacy of nanoparticle treatment. The efficacy parameters included tumor volume measurements from day 0 through 28 days post-treatment, terminal tumor weight measurements, tumor density and morphology assessment through hematoxylin and eosin staining of excised tumors, and immunohistochemistry of tumor sections for MDR protein markers (P-glycoprotein, Hypoxia Inducible Factor, EGFR, Hexokinase 2, and Stem Cell Factor). Toxicity was assessed by tracking changes in animal body weight from day 0 through 28 days post-treatment, by measuring plasma levels of the liver enzymes ALT (Alanine Aminotransferase) and LDH (lactate dehydrogenase), and by white blood cell and platelet counts. In these studies, this nanocarrier system demonstrated superior efficacy relative to combination (paclitaxel/lonidamine) drug solution and single agent treatments in nanoparticle and solution form. The combination nanoparticles were the only treatment group that decreased tumor volume, sustaining this decrease until the 28 day time point. In addition, treatment with the EGFR-targeted lonidamine/paclitaxel nanoparticles decreased tumor density and altered the MDR phenotype of the tumor xenografts. These EGFR-targeted combination nanoparticles were considerably less toxic than solution treatments. Due to the flexible design and simple conjugation chemistry, this nanocarrier system could be used as a platform for the development of other MDR cancer therapies; the use of this system for EGFR-targeted, combination paclitaxel/lonidamine therapy is an advance in personalized medicine

Exosomes promote pre-metastatic niche formation in ovarian cancer.

Ovarian cancer is one of the most common gynecological malignancies. Upon initial diagnosis, the majority of patients present with widespread metastatic growth within the peritoneal cavity. This metastatic growth occurs in stages, with the formation of a pre-metastatic niche occurring prior to macroscopic tumor cell invasion. Exosomes released by the primary ovarian tumor are small extracellular vesicles which prepare the distant tumor microenvironment for accelerated metastatic invasion. They regulate intercellular communication between tumor cells and normal stroma, cancer-associated fibroblasts, and local immune cells within the tumor microenvironment. In this review, we highlight the emerging roles of ovarian cancer exosomes as coordinators of pre-metastatic niche formation, biomarkers amenable to liquid biopsy, and targets of chemotherapy

MicroRNA Involvement in Osteosarcoma

Osteosarcoma (OS) is the most common primary malignant bone tumor, usually arising in the long bones of adolescents and young adults. While our knowledge of the molecular pathogenesis of OS has increased in recent years, we are still far from a comprehensive understanding of the molecular mechanisms of the disease, such as its tumorigenesis, specific mediators of disease progression, occurrence of chemoresistance, and development of metastasis. After the recent discovery of microRNAs (miRNAs), their critical roles in molecular biological processes have been of great interest in the cancer research field, including research on sarcomas. MiRNAs are highly conserved noncoding RNAs which play important roles as oncogenic or suppressive genes to simultaneously regulate multiple targets. Recent genome-wide screening using miRNA expression profiles has identified specific miRNA expression patterns that are associated with the biological and clinical properties of cancers. Additionally, miRNAs and their target genes or proteins can be potential novel biomarkers or therapeutic targets for cancer. However, there are several challenges that must be addressed in order to translate miRNA-based therapeutics to the clinical setting. In this review, we summarize the current understanding of the roles that miRNAs play in OS, and highlight their potential as biomarkers or therapeutic targets

Transcriptional activation of CBFβ by CDK11p110 is necessary to promote osteosarcoma cell proliferation.

BACKGROUND:Aberrant expression of cyclin-dependent protein kinases (CDK) is a hallmark of cancer. CDK11 plays a crucial role in cancer cell growth and proliferation. However, the molecular mechanisms of CDK11 and CDK11 transcriptionally regulated genes are largely unknown. METHODS:In this study, we performed a global transcriptional analysis using gene array technology to investigate the transcriptional role of CDK11 in osteosarcoma. The promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay were used to identify direct transcriptional targets of CDK11. Clinical relevance and function of core-binding factor subunit beta (CBFβ) were further accessed in osteosarcoma. RESULTS:We identified a transcriptional role of protein-DNA interaction for CDK11p110, but not CDK11p58, in the regulation of CBFβ expression in osteosarcoma cells. The CBFβ promoter luciferase assay, chromatin immunoprecipitation assay, and Gel Shift assay confirmed that CBFβ is a direct transcriptional target of CDK11. High expression of CBFβ is associated with poor outcome in osteosarcoma patients. Expression of CBFβ contributes to the proliferation and metastatic behavior of osteosarcoma cells. CONCLUSIONS:These data establish CBFβ as a mediator of CDK11p110 dependent oncogenesis and suggest that targeting the CDK11- CBFβ pathway may be a promising therapeutic strategy for osteosarcoma treatment

Rhabdomyosarcoma: Advances in Molecular and Cellular Biology.

Rhabdomyosarcoma (RMS) is the most common soft tissue malignancy in childhood and adolescence. The two major histological subtypes of RMS are alveolar RMS, driven by the fusion protein PAX3-FKHR or PAX7-FKHR, and embryonic RMS, which is usually genetically heterogeneous. The prognosis of RMS has improved in the past several decades due to multidisciplinary care. However, in recent years, the treatment of patients with metastatic or refractory RMS has reached a plateau. Thus, to improve the survival rate of RMS patients and their overall well-being, further understanding of the molecular and cellular biology of RMS and identification of novel therapeutic targets are imperative. In this review, we describe the most recent discoveries in the molecular and cellular biology of RMS, including alterations in oncogenic pathways, miRNA (miR), in vivo models, stem cells, and important signal transduction cascades implicated in the development and progression of RMS. Furthermore, we discuss novel potential targeted therapies that may improve the current treatment of RMS

Oleanane triterpenoid CDDO-Me induces apoptosis in multidrug resistant osteosarcoma cells through inhibition of Stat3 pathway.

BackgroundThe activation of signal transducer and activator of transcription 3 (Stat3) pathway correlates with tumor growth, survival, drug resistance and poor prognosis in osteosarcoma. To explore the potential therapeutic values of this pathway, we assessed both the expression and the activation of Stat3 pathway in several pairs of multidrug resistant (MDR) osteosarcoma cell lines, and tissues. To explore the potential therapeutic values of this pathway, we analyzed the ability of the synthetic oleanane triterpenoid, C-28 methyl ester of 2-cyano-3,12-dioxoolen-1,9-dien-28-oic acid (CDDO-Me), to inhibit Stat3 expression and activation as well as its effects on doxorubicin sensitivity in osteosarcoma cells.MethodsExpression of Stat3, phosphorylated Stat3 (pStat3) and Stat3 targeted proteins, including Bcl-XL, Survivin and MCL-1 were determined in drug sensitive and MDR osteosarcoma cell lines and tissues by Western blot analysis. The effect of CDDO-Me on osteosarcoma cell growth was evaluated by MTT and apoptosis by PARP cleavage assay and caspase-3/7 activity.ResultsStat3 pathway was activated in osteosarcoma tissues and in MDR cell lines. CDDO-Me inhibited growth and induced apoptosis in osteosarcoma cell lines. Treatment with CDDO-Me significantly decreased the level of nuclear translocation and phosphorylation of Stat3. The inhibition of Stat3 pathway correlated with the suppression of the anti-apoptotic Stat3 targeted genes Bcl-XL, survivin, and MCL-1. Furthermore, CDDO-Me increased the cytotoxic effects of doxorubicin in the MDR osteosarcoma cell lines.ConclusionsStat3 pathway is overexpressed in MDR osteosarcoma cells. CDDO-Me significantly inhibited Stat3 phosphorylation, Stat3 nuclear translocation and induced apoptosis in osteosarcoma. This study provides the framework for the clinical evaluation of CDDO-Me, either as monotherapy or perhaps even more effectively in combination with doxorubicin to treat osteosarcoma and overcome drug resistance

Targeting EZH2-mediated methylation of H3K27 inhibits proliferation and migration of Synovial Sarcoma in vitro.

Synovial sarcoma is an aggressive soft tissue sarcoma genetically defined by the fusion oncogene SS18-SSX. It is hypothesized that either SS18-SSX disrupts SWI/SNF complex inhibition of the polycomb complex 2 (PRC2) methyltransferase Enhancer of Zeste Homologue 2 (EZH2), or that SS18-SSX is able to directly recruit PRC2 to aberrantly silence target genes. This is of potential therapeutic value as several EZH2 small molecule inhibitors are entering early phase clinical trials. In this study, we first confirmed EZH2 expression in the 76% of human synovial sarcoma samples. We subsequently investigated EZH2 as a therapeutic target in synovial sarcoma in vitro. Knockdown of EZH2 by shRNA or siRNA resulted in inhibition of cell growth and migration across a series of synovial sarcoma cell lines. The EZH2 selective small-molecule inhibitor EPZ005687 similarly suppressed cell proliferation and migration. These data support the hypothesis that targeting EZH2 may be a promising therapeutic strategy in the treatment of synovial sarcoma; clinical trials are initiating enrollment currently