Lay Summary

Pediatric brain tumors have limited responses to conventional therapeutic option and high mortality rates. Novel approaches to treat these and other forms of cancer involve targeting metabolic mechanisms of cancer cells from which they derive energy. These include aerobic glycolysis or the Warburg effect; named after Otto Warburg who first described it in 1926. Unlike normal cells that are flexible in energy production, depending on availability of oxygen, but typically use a far more efficient process called oxidative phosphorylation, cancer cells mainly produce energy via glycolysis in the cytosol, even under aerobic conditions (availability of sufficient oxygen). Cancer cell’s relative inflexibility in energy production represents a kind of Achilles’ heel and a potential target for therapeutic intervention.  Many studies have shown that the inhibition of glycolysis results in reduced cell proliferation and tumor growth; hence, evidence that the inhibition of biosynthetic pathways associated with glycolysis represents important opportunities for cancer therapeutics while sparing normal cells with alternative mechanisms for energy production. Another important element of this novel strategy is the targeting of the metabolism of non-cancerous cells in the microenvironment of cancer. These cells are induced by changes in the microenvironment to produce fuels used by cancer cells. By also addressing this source of energy for cancer cells, anticancer therapeutics may become more effective. In the proposed research we will evaluate the potential for combination therapies specifically directed at the energy metabolism of cancer cells and cells in the microenvironment that include compounds previously developed and studied in our laboratory. Developing a strategy for the targeting of the energy source for cancer cells may represent a novel therapeutic approach while complement conventional therapies in a synergistic manner.

Executive Summary

Over the past 20 years our laboratory has introduced and applied the principles of a novel paradigm of medicinal discovery based on principles of ethnobotany, evochemistry, and evidence-based methodologies. This paradigm is contrasted with principles and methods of conventional drug discovery. Recent innovations include the development of a supercritical CO₂ extract from Curcuma amada (“CA”) rhizomes that has demonstrated direct and adjuvant anticancer properties in different cancer models and GU-MCT810, a supercritical-ethanoic extract of guggul (Commiphora mukul) gum resin that has been shown to activate AMPK, suppress phosphorylated form of mTOR and modulate energy metabolism in normal and cancer cell line models.

Mango ginger (Curcuma amada Roxb.) is among the less-investigated species of Curcuma for anticancer properties. With CA we have investigated the in vitro and in vivo anticancer mechanisms of human glioblastoma (“U-87MG”) and human alveolar (“SJRH30”) and embryonal (“RD”) rhabdomyosarcoma cell lines. The labdane diterpene, (E)-Labda-8(17),12-diene-15,16 dial (“LDD”) is most abundant compound followed by β-myrcene and other terpenoids. In vitro cytotoxicity with CA was shown to be greater than temozolomide, etoposide, irinotecan and curcumin in the U-87MG cells and greater than vinblastine, cyclophosphamide and curcumin in SJRH30 and RD cells. Synergy analysis with CompuSyn software reveals synergism between CA the chemotherapeutic drugs used in each cell line. In vivo studies in U-87MG and SJRH30 xenograft mice demonstrated comparable or greater efficacy in reduction of tumor growth and increased survival compared to the chemotherapeutic drugs (irinotecan and vinblastine respectively) and synergy with CA-drug combinations. mRNA and protein expression studies showed that CA modulates expression of genes associated with apoptosis (Bax, Bcl-2, Bcl-X, BNIP3, caspase-3, mutant p53, and p21), cell proliferation (Ki67) and angiogenesis (VEGF), in vitro and in vivo and down regulation of AMPKα and AKT phosphorylation, HSP90 and inhibition of cell migration in a dose-dependent manner.

While direct targeting of tumor cells based on modulation of signaling pathways remains as we have demonstrated with CA in in vitro and in vivo studies remains an important and viable strategy for the development of cancer therapeutics, consideration of the cancer metabolism and the cancer microenvironment represents a potential and major paradigm shift. First, Cancer cells, unlike normal cells, in general display a preference for aerobic glycolysis and reduced mitochondrial oxidative phosphorylation for glucose metabolism, a phenomenon known as the Warburg effect. Cancer cells generally demonstrate high uptake of glucose and are more dependent on the aerobic glycolysis to produce ATP for growth and maintenance. Since therapeutic selectivity or preferential killing of cancer cells without significant toxicity to normal cells is one of the most important considerations in cancer chemotherapy, targeting metabolic pathway offers the potential for a selective approach to cancer treatment. Secondly, while cancer cells are considered as the primary drivers of tumor development, it is now clear that other cells such as stromal cells; fibroblasts, adipocytes, endothelial cells, immune cells, etc. also contribute to the tumorigenic microenvironments that enables cancer progression. For example, it has been well established that stromal fibroblasts infiltrate and are induced to differentiate into cancer associated fibroblasts (“CAFs”) and that they contribute to the energetics of tumor cells by fueling them with energy sources like lactate, ketones, glutamine, fatty acids or cysteine. Similarly, through lactate-induced extracellular acidification and secretion of inflammatory cytokines and matrix-degrading enzymes, CAFs enable metastasis and confer resistance to chemotherapeutic drugs. Therefore, more effective cancer treatment should also entail targeting of the tumor microenvironment including stromal cells such as cancer associated fibroblasts and adipocytes that fuel the cancer cells.

Recently our research has focused on these two areas of opportunity; cancer metabolism and the cancer microenvironment. We have previously shown strong synergistic effects of the combination of CA with two glycolysis inhibitors; 2-deoxy-D-glucose (“2-DG”) and sodium oxamate (“SO”) in the U-87MG human glioblastoma cell line. Specifically we confirmed that CA, 2-DG and SO and their combinations up regulate the ratio of Bax/Bcl-2, p21, TIMP1 and caspase 3 expression and down regulate mutant p53 and MMP2 expression that may increase apoptosis and inhibit cell proliferation as well as metastasis of tumor cells.

Another compound we have developed; GU-MCT810 has thoroughly documents effect on cellular energy metabolism which we hypothesize will also represent a potent adjuvant in disrupting cancer metabolism based on similar mechanism with metformin whose metabolic effects have been well established. Therefore, we wish to further explore the potential for the targeting of metabolism in brain tumor models that are relevant to pediatric oncology using CA, GU-MCT810 and glycolysis inhibitors.

The second are of the proposed research is the targeting of the cancer microenvironment. In previous research we have examined the role of CAFs, specialized fibroblasts considered to be the principal non-cancerous cell type within the tumor microenvironment (“TME”). This approach was suggested by recent research that addressed the role of CAFs in the progression and metastasis of cancer and the dynamic interaction between stroma and epithelium, while normal fibroblasts inhibit tumor progression, transformation of fibroblasts to a CAF phenotype and other alterations of the extratracellular matrix potentially produce a conducive environment for carcinogenesis. CAFs have been shown to promote proliferation and differentiation of tumor cells and other Hallmarks of cancer; angiogenesis and metastasis through remodeling of the extracellular matrix and epithelial to mesenchymal transition. It is also reported that stimulation of HIF-signaling is associated with increased glycolysis in CAFs, which in turn provide nutrients such as lactate to adjacent cancer cells.

Through our investigations of the metabolic coupling between carcinoma cell lines and CAF (and also cancer associated adipocytes) we demonstrated that CA treated MCF-7-CAF-05 co-culture systems substantially reduced lactate synthesis. Furthermore, CA treatment also down regulates other markers associated with Warburg and reverse Warburg effects such as HIF-1alpha, MCT1 and MCT4. Therefore, our results show that CA can target not only target cancer cells but also the tumor promoting CAFs and adipocytes in the tumor microenvironment.

In summary, we have compelling evidence that synergistic combinations of products that we’ve developed with glycolysis inhibitors and evaluated in different cancer models have direct impacts of cancer metabolism and indirectly through the targeting of metabolic coupling between cancer cells and stromal cells in the cancer microenvironment. Through the proposed research we wish to extend this research to the study of pediatric brain cancer models.

Description of Research Proposal

Budget

Collaborations and Conflicts of Interest