Jin-Ming Yang

Overexpression of P-glycoprotein (P-gp), the MDR1 gene product, confers multidrug resistance (MDR) to cancer cells. Clinically, MDR is one of the major causes for chemotherapeutic treatment failure in cancer patients. To explore a new approach to circumventing MDR, we adopted RNA interference to target MDR1 gene expression. RNA interference is a conserved biological response to double-stranded RNA, which results in sequence-specific gene silencing [G. J. Hannon, Nature (Lond.), 418: 244-251, 2002]. We report that introduction of an MDR1-targeted small interfering RNA duplex into drug-resistant cancer cells markedly inhibited the expression of MDR1 mRNA and P-gp, as determined by reverse transcription-PCR and Western blot. Inhibition of P-gp expression by small interfering RNA enhanced the intracellular accumulation of and selectively restored sensitivity to drugs transported by P-gp. These studies indicate that RNA interference can modulate MDR in preclinical models.

Recent evidence suggests that the machinery of protein synthesis may provide novel targets for anticancer drugs. For example, aberrations in protein synthesis are commonly encountered in established cancers, and disruption by mutation or overexpression of translation factors can cause cellular transformation. We previously demonstrated that the activity of eukaryotic elongation factor 2 (eEF-2) kinase was markedly increased in several forms of malignancy and that nonspecific inhibitors of this enzyme promoted cell death. On the basis of the predicted amino acid sequence of eEF-2 kinase deduced from the cloned cDNA, we hypothesized that inhibitors of prokaryotic histidine kinases might also inhibit the activity of eEF-2 kinase. We describe herein the screening of a series of imidazolium histidine kinase inhibitors and the identification of an active lead compound, NH125. NH125 inhibited eEF-2 kinase activity (IC(50) = 60 nM) in vitro, blocked the phosphorylation of eEF-2 in intact cells, and showed relative selectivity over other protein kinases: protein kinase C (IC(50) = 7.5 microM), protein kinase A (IC(50) = 80 microM), and calmodulin-dependent kinase II (IC(50) > 100 microM). NH125 decreased the viability of 10 cancer cell lines with IC(50)s ranging from 0.7 to 4.7 microM. Forced overexpression of eEF-2 kinase in a glioma cell line produced 10-fold resistance to NH125. In conclusion, these results suggest that identification of potent inhibitors of eEF-2 kinase may lead to the development of new types of anticancer drugs.

Our laboratory discovered that p53 can regulate the sensitivity to cancer therapies by affecting three critical aspects of cancer pharmacology: 1). The expression of drug targets; 2). the access of drugs to intracellular targets; and the response to DNA damage. We review the effects of p53 on antimicrotubule drugs through transcriptional regulation of MAP4 and stathmin (Oncoprotein 18). These two p53-regulated proteins control microtubule dynamics, regulate the sensitivity to taxanes and vinca alkaloids by changing the polymerization dynamics of tubulin and affecting the binding of drugs to microtubules. We found that overexpression of MAP4 increased microtubule polymerization and increased taxane binding and sensitivity. Overexpression of stathmin, a microtubule destabilizer, virtually abolished cellular taxane binding and increased resistance by over 1000-fold. Yet, despite an increased binding of vinca alkaloids to stathmin transfectants, we did not observe increased drug sensitivity. This was explained, at least in part, by a delay in G2/M transit. We also discovered that p53 could regulate the expression of multidrug resistance protein-1 (MRP1), a member of the ABC family of transporters that mediates the sensitivity to vinca alkaloids and anthracyclines. We found that as prostate cancer progressed from low stage/low grade to high stage/high grade there was an increased expression of both MRP1 and staining for p53, a surrogate for p53 mutations. We went on to show that p53 regulated the expression of MRP1 and that this produced resistance to doxorubicin and vinblastine. We further demonstrated that MRP1 overexpression blocked the accumulation of flutamide and hydroxy-flutamide (the active metabolite) without affecting transport of dihydrotesterone, thereby blocking access of the anti-androgen but not the androgen to intracellular androgen receptors. Finally, we reviewed the effects of DNA damage on p53 expression and MAP4 repression as a means to increase the effectiveness of breast cancer treatment. These data demonstrated the possibility of individualizing treatment based on p53 status.

In order to survive under conditions of low oxygen, cancer cells can undergo a metabolic switch to glycolysis and suppress mitochondrial function in order to reduce oxygen consumption and prevent excessive generation of harmful reactive oxygen species (ROS). Nucleus accumbens-associated protein-1 (NAC1), a nuclear factor of the BTB/POZ gene family, has emerging roles in cancer. Here, we identified that NAC1-PDK3 pathway is required for suppression of mitochondrial mass, oxygen consumption, and ROS production and protects cancer cells from apoptosis in hypoxia. We show that NAC1 mediates suppression of mitochondrial function in hypoxia through inducing expression of pyruvate dehydrogenase kinase 3 (PDK3) by HIF-1α at the transcriptional level, thereby inactivating pyruvate dehydrogenase (PDH) and attenuating mitochondrial respiration. Re-expression of PDK3 in NAC1 absent cells rescued cells from hypoxia-induced metabolic stress and restored glycolysis in a mouse xenograft model, and demonstrated that knockdown of NAC1 expression can reinforce the antitumor efficacy of elesclomol, a pro-oxidative agent. Our findings define a novel mechanism by which NAC1 promotes stress resistance during cancer progression, and chemo-resistance in cancer therapy.