Cancer Biomarkers and Drug Resistance
Associate Director / Section Leader
Ann M. Bode, Ph.D.
We continue to work with the National
Institutes of Health (NIH) to identify
biomarkers important in drug resistance to
cancer prevention and treatment. During
2014-2015, we published a number of papers in collaboration with NIH and the
University of Alabama.
1. Epidemiologic studies have shown that diabetics receiving the biguanide metformin, compared with sulfonylureas or insulin, have a lower incidence of breast cancer. Metformin reportedly increases the levels of activated AMPK (AMP-activated protein kinase) and decreases circulating insulin growth factor-1 (IGF-1), which has encouraged its potential use in both cancer prevention and therapeutic settings. In anticipation of clinical trials in nondiabetic women, we evaluated the efficacy of metformin in nondiabetic rat and mouse mammary cancer models. Metformin was administered by gavage or in the diet at a human equivalent dose in standard mammary cancer models: (i) methylnitrosourea (MNU)-induced estrogen receptor.positive (ER+) mammary cancers in rats, and (ii) MMTV-Neu/p53KO ER- (estrogen receptor.negative) mammary cancers in mice. In the MNU rat model, metformin dosing (150 or 50 mg/kg BW/d, by gavage) was ineffective in decreasing mammary cancer multiplicity, latency, or weight. Pharmacokinetic studies of metformin (150 mg/kg BW/d, by gavage) yielded plasma levels (Cmax and AUC) higher than humans taking 1.5 g/d. In rats bearing small palpable mammary cancers, shortterm metformin (150 mg/kg BW/d) treatment increased levels of phosphorylated AMPK and phosphorylated p53 (Ser20), but failed to reduce Ki-67 labeling or expression of proliferation-related genes. In the mouse model, dietary metformin (1,500 mg/kg diet) did not alter final cancer incidence, multiplicity, or weight. Metformin did not prevent mammary carcinogenesis in two mammary cancer models, raising questions about metformin efficacy in breast cancer in nondiabetic populations. Our immunohistochemistry results were a cover image for Cancer Prevention Research.
2. The COX inhibitors (NSAID/Coxibs) are a major focus for the chemoprevention of cancer. The COX-2-specific inhibitors have progressed to clinical trials and shown preventive efficacy in colon and skin cancers. They, however, have significant adverse cardiovascular effects. Certain NSAIDs (e.g., naproxen) have a good cardiac profile, but can cause gastric toxicity. The present study examined protocols to reduce naproxen.s toxicity. Female Fischer-344 rats were treated weekly with the urinary bladder-specific carcinogen hydroxybutyl(butyl)nitrosamine (OH-BBN) for eight weeks. Rats were dosed daily with naproxen (40 mg/kg body weight/day, gavage) or with the proton pump inhibitor omeprazole (4.0 mg/kg body weight/day) either singly or in combination beginning two weeks after the final OH-BBN. With the OH-BBNtreated rats, 96 percent developed urinary bladder cancers. While omeprazole alone was ineffective (97 percent cancers), naproxen alone or combined with omeprazole-prevented cancers, yielded 27 and 35 percent cancers, respectively. In a separate study, OH-BBN-treated rats were administered naproxen: (A) daily; (B) one week daily naproxen/1week vehicle; (C) three weeks daily naproxen/ three-week vehicle; or (D) daily vehicle beginning two weeks after last OH-BBN treatment. In the intermittent dosing study, protocol A, B, C, and D resulted in palpable cancers in 27 percent, 22 percent, 19 percent, and 96 percent of rats (p < 0.01). Short-term naproxen treatment increased apoptosis, but did not alter proliferation in the urinary bladder cancers. Two different protocols that should decrease the gastric toxicity of NSAIDs in humans did not alter chemopreventive efficacy. This should encourage the use of NSAIDs (e.g., naproxen) in clinical Cancer Biomarkers and Drug Resistance prevention trials. This study was a press release from the University of Michigan and the University of Minnesota.
3. Urinary bladder cancer prevention studies were performed with the nonsteroidal anti-inflammatory drugs (NSAID) naproxen (a standard NSAID with a good cardiovascular profile), sulindac, and their nitric oxide (NO) derivatives. In addition, we examined the effects of the ornithine decarboxylase inhibitor, difluoromethylornithine (DFMO), alone or combined with a suboptimal dose of naproxen or sulindac. Agents were evaluated at their human equivalent doses (HED) as well as at lower doses. In the hydroxybutyl(butyl) nitrosamine (OH-BBN) model of urinary bladder cancer, naproxen (400 or 75 ppm) and sulindac (400 ppm) reduced the incidence of large bladder cancers by 82 percent, 68 percent, and 44 percent, respectively, when the agents initially were given three months after the final dose of the carcinogen; microscopic cancers already existed. NO-naproxen was highly effective, whereas NO-sulindac was inactive. To further compare naproxen and NO-naproxen, we examined their effects on gene expression in rat livers following a seven-day exposure. Limited but similar gene expression changes in the liver were induced by both agents, implying that the primary effects of both are mediated by the parent NSAID. When agents were initiated two weeks after the last administration of OH-BBN, DFMO at 1,000 ppm had limited activity, and a low dose of naproxen (75 ppm) and sulindac (150 ppm) were highly and marginally effective. Combining DFMO with suboptimal doses of naproxen had minimal effects, whereas the combination of DMFO and sulindac was more active than either agent alone. Thus, naproxen and NO-naproxen were highly effective, whereas sulindac was moderately effective in the OH-BBN model at their HEDs.
“Our work funded by NIH has focused on biomarker
identification in breast and bladder cancer.“
Dr. Ann M. Bode
4. Naproxen [(S)-6-methoxy-alpha-methyl-2-naphthaleneacetic acid] is a potent nonsteroidal anti-inflammatory drug that inhibits both COX-1 and COX-2 and is widely used as an over-the-counter medication. Naproxen exhibits analgesic, antipyretic, and anti-inflammatory activities. Naproxen, as well as other nonsteroidal anti-inflammatory drugs, has been reported to be effective in the prevention of urinary bladder cancer in rodents. Potential targets other than the COX isozymes, however, have not been reported. We examined potential additional targets in urinary bladder cancer cells and rat bladder cancers. Computer kinase profiling results suggested that phosphoinositide 3-kinase (PI3K) is a potential target for naproxen. In vitro kinase assay data revealed that naproxen interacts with PI3K and inhibits its kinase activity. Pull-down binding assay data confirmed that PI3K directly binds with naproxen in vitro and ex vivo. Western blot data showed that naproxen decreased phosphorylation of Akt, and subsequently decreased Akt signaling in UM-UC-5 and UM-UC-14 urinary bladder cancer cells. Furthermore, naproxen suppressed anchorageindependent cell growth and decreased cell viability by targeting PI3K in both cell lines. Naproxen caused an accumulation of cells at the G1 phase mediated through cyclin-dependent kinase 4, cyclin D1, and p21. Moreover, naproxen induced significant apoptosis, accompanied with increased levels of cleaved caspase-3, caspase-7, and PARP in both cell types. Naproxen-induced cell death was mainly due to apoptosis that involved a prominent downregulation of Bcl-2 and up-regulation of Bax. Naproxen also caused apoptosis and inhibited Akt phosphorylation in rat urinary bladder cancers induced by N-butyl-N-(4- hydroxybutyl)-nitrosamine.