Interaction and Collaboration of SP1, HIF-1, and MYC in Regulating the Expression of Cancer-Related Genes to Further Enhance Anticancer Drug Development.

Specificity protein 1 (SP1), hypoxia-inducible factor 1 (HIF-1), and MYC are important transcription factors (TFs). SP1, a constitutively expressed housekeeping gene, regulates diverse yet distinct biological activities; MYC is a master regulator of all key cellular activities including cell metabolism and proliferation; and HIF-1, whose protein level is rapidly increased when the local tissue oxygen concentration decreases, functions as a mediator of hypoxic signals. Systems analyses of the regulatory networks in cancer have shown that SP1, HIF-1, and MYC belong to a group of TFs that function as master regulators of cancer. Therefore, the contributions of these TFs are crucial to the development of cancer. SP1, HIF-1, and MYC are often overexpressed in tumors, which indicates the importance of their roles in the development of cancer. Thus, proper manipulation of SP1, HIF-1, and MYC by appropriate agents could have a strong negative impact on cancer development. Under these circumstances, these TFs have naturally become major targets for anticancer drug development. Accordingly, there are currently many SP1 or HIF-1 inhibitors available; however, designing efficient MYC inhibitors has been extremely difficult. Studies have shown that SP1, HIF-1, and MYC modulate the expression of each other and collaborate to regulate the expression of numerous genes. In this review, we provide an overview of the interactions and collaborations of SP1, HIF1A, and MYC in the regulation of various cancer-related genes, and their potential implications in the development of anticancer therapy.

Tetra-O-methyl-nordihydroguaiaretic acid inhibits energy metabolism and synergistically induces anticancer effects with temozolomide on LN229 glioblastoma tumors implanted in mice while preventing obesity in normal mice that consume high-fat diets.

Tetra-O-methyl-nordihydroguaiaretic acid (terameprocol; M4N), a global transcription inhibitor, in combination with a second anticancer drug induces strong tumoricidal activity and has the ability to suppress energy metabolism in cultured cancer cells. In this study, we showed that after continuous oral consumption of high-fat (HF) diets containing M4N, the M4N concentration in most of the organs in mice reached ~1 µM (the M4N concentration in intestines and fat pads was as high as 20-40 µM) and treatment with the combination of M4N with temozolomide (TMZ) suppressed glycolysis and the tricarboxylic acid cycle in LN229 human glioblastoma implanted in xenograft mice. Combination treatment of M4N with TMZ also reduced the levels of lactate dehydrogenase A (LDHA), a key enzyme for glycolysis; lactate, a product of LDHA-mediated enzymatic activity; nicotinamide phosphoribosyltransferase, a rate-limiting enzyme for nicotinamide adenine dinucleotide plus hydrogen (NADH)/NAD+ salvage pathway; and NAD+, a redox electron carrier essential for energy metabolism. It was also shown that M4N suppressed oxygen consumption in cultured LN229 cells, indicating that M4N inhibited oxidative phosphorylation. Treatment with M4N and TMZ also decreased the level of hypoxia-inducible factor 1A, a major regulator of LDHA, under hypoxic conditions. The ability of M4N to suppress energy metabolism resulted in induction of the stress-related proteins activating transcription factor 4 and cation transport regulator-like protein 1, and an increase in reactive oxygen species production. In addition, the combination treatment of M4N with TMZ reduced the levels of oncometabolites such as 2-hydroxyglutarate as well as the aforementioned lactate. M4N also induced methylidenesuccinic acid (itaconate), a macrophage-specific metabolite with anti-inflammatory activity, in tumor microenvironments. Meanwhile, the ability of M4N to suppress energy metabolism prevented obesity in mice consuming HF diets, indicating that M4N has beneficial effects on normal tissues. The dual ability of combination treatment with M4N to suppress both energy metabolism and oncometabolites shows that it is potentially an effective therapy for cancer.

Tetra-O-Methyl Nordihydroguaiaretic Acid Broadly Suppresses Cancer Metabolism and Synergistically Induces Strong Anticancer Activity in Combination with Etoposide, Rapamycin and UCN-01.

The ability of Tetra-O-methyl nordihydroguaiaretic acid (M4N) to induce rapid cell death in combination with Etoposide, Rapamycin, or UCN-01 was examined in LNCaP cells, both in cell culture and animal experiments. Mice treated with M4N drug combinations with either Etoposide or Rapamycin showed no evidence of tumor and had a 100% survival rate 100 days after tumor implantation. By comparison all other vehicles or single drug treated mice failed to survive longer than 30 days after implantation. This synergistic improvement of anticancer effect was also confirmed in more than 20 cancer cell lines. In LNCaP cells, M4N was found to reduce cellular ATP content, and suppress NDUFS1 expression while inducing hyperpolarization of mitochondrial membrane potential. M4N-treated cells lacked autophagy with reduced expression of BNIP3 and ATG5. To understand the mechanisms of this anticancer activity of M4N, the effect of this drug on three cancer cell lines (LNCaP, AsPC-1, and L428 cells) was further examined via transcriptome and metabolomics analyses. Metabolomic results showed that there were reductions of 26 metabolites essential for energy generation and/or production of cellular components in common with these three cell lines following 8 hours of M4N treatment. Deep RNA sequencing analysis demonstrated that there were sixteen genes whose expressions were found to be modulated following 6 hours of M4N treatment similarly in these three cell lines. Six out of these 16 genes were functionally related to the 26 metabolites described above. One of these up-regulated genes encodes for CHAC1, a key enzyme affecting the stress pathways through its degradation of glutathione. In fact M4N was found to suppress glutathione content and induce reactive oxygen species production. The data overall indicate that M4N has profound specific negative impacts on a wide range of cancer metabolisms supporting the use of M4N combination for cancer treatments.