The introduction of enzalutamide and abiraterone has led to improvement in the treatment of metastatic castration-resistant prostate cancer (mCRPC). However, acquired resistance to enzalutamide and abiraterone therapies frequently develops within a short period in many patients. In the present study, we developed enzalutamide resistant prostate cancer cells in an effort to understand the mechanisms of resistance. Global gene expression analysis showed that steroid biosynthesis pathway is activated in enzalutamide resistant prostate cancer cells. One of the crucial steroidogenic enzymes, AKR1C3, was significantly elevated in enzalutamide resistant cells. In addition, AKR1C3 is highly expressed in metastatic and recurrent prostate cancer and in enzalutamide resistant prostate xenograft tumors. Liquid Chromatography-Mass Spectrometry (LC-MS) analysis of the steroid metabolites revealed that androgen precursors such as cholesterol, DHEA and progesterone, as well as androgens are highly up regulated in enzalutamide resistant prostate cancer cells compared to the parental cells. Knock down of AKR1C3 expression by shRNA or inhibition of AKR1C3 enzymatic activity by indomethacin resensitized enzalutamide resistant prostate cancer cells to enzalutamide treatment both in vitro and in vivo. In contrast, overexpression of AKR1C3 confers resistance to enzalutamide. Furthermore, the combination of indomethacin and enzalutamide resulted in significant inhibition of enzalutamide-resistant tumor growth. These results suggest that AKR1C3 activation is a critical resistance mechanism associated with enzalutamide resistance, targeting intracrine androgens and AKR1C3 will overcome enzalutamide resistance and improve survival of advanced prostate cancer patients.

Estrogens can become endogenous carcinogens via formation of catechol estrogen quinones, which react with DNA to form specific depurinating estrogen-DNA adducts. The mutations resulting from these adducts can lead to cell transformation and the initiation of breast cancer. Estrogen metabolites, conjugates and depurinating DNA adducts in urine samples from 46 healthy control women, 12 high-risk women and 17 women with breast cancer were analyzed. The estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified by using ultraperformance liquid chromatography/tandem mass spectrometry. The levels of the ratios of depurinating DNA adducts to their respective estrogen metabolites and conjugates were significantly higher in high-risk women (p < 0.001) and women with breast cancer (p < 0.001) than in control subjects. The high-risk and breast cancer groups were not significantly different (p 5 0.62). After adjusting for patient characteristics, these ratios were still significantly associated with health status. Thus, the depurinating estrogen-DNA adducts are possible biomarkers for early detection of breast cancer risk and response to preventive treatment. ' 2007 Wiley-Liss, Inc.Key words: breast cancer risk; depurinating estrogen-DNA adducts; estrogen biomarkers; balance in estrogen metabolism Development of noninvasive tests of breast cancer risk has been a major goal for more than 30 years. In this article we present biomarkers of risk that are related to the hypothesized first critical step in the initiation of breast cancer, namely, the reaction of catechol estrogen quinone metabolites with DNA.1 Prevention of cancer can be achieved by blocking this DNA damage, which generates the mutations leading to the initiation, promotion and progression of cancer. 2Exposure to estrogens is a known risk factor for breast cancer.3,4 The discovery that specific oxidative metabolites of estrogens, namely, catechol estrogen quinones, can react with DNA [5][6][7][8][9] led to and supports the hypothesis that these metabolites can become endogenous chemical carcinogens. Some of the mutations generated by this specific DNA damage can result in the initiation of cancer. 1,5 This paradigm suggests that specific, critical mutations generate abnormal cell proliferation leading to cancer. 1,[10][11][12][13] As illustrated in Figure 1, in the metabolism of catechol estrogens there are activating pathways 14 that lead to the formation of the estrogen quinones, estrone (estradiol) quinones [E 1 (E 2 )-Q], which can react with DNA. There are also deactivating pathways that limit formation of the quinones and/or prevent their reaction with DNA. These are methylation of catechol estrogens, 15 conjugation of the E 1 (E 2 )-Q with glutathione (GSH) 16 and reduction of the quinones to catechols 17 (Fig. 1). When E 1 (E 2 )-3,4-Q react with DNA, they form predominantly the depurinating adducts 4-hydroxyestrone(estradiol)-1-N3Ade-nine [4-OHE 1 (E 2 )-1-N3Ade] and 4-hydroxyestrone(estradiol)-1-N7Guanine [4-OHE 1 (E 2...

Endogenous estrogens can be bio-activated to endogenous carcinogens via formation of estrogen quinones. Estrogen-3,4-quinones react with DNA to form mutagenic depurinating estrogen-DNA adducts. The carcinogenicity of endogenous estrogens is related to unbalanced estrogen metabolism leading to excess estrogen quinones and formation of depurinating DNA adducts. The present studies were initiated to confirm that relatively high levels of depurinating estrogen-DNA adducts are present in women at high risk for breast cancer or diagnosed with the disease. These adducts may be biomarkers for early detection of breast cancer risk. The estrogen metabolites, conjugates and depurinating DNA adducts were identified and quantified by using ultraperformance liquid chromatography/tandem mass spectrometry to analyze urine samples from 40 healthy control women, 40 high-risk women and 40 women with newly diagnosed breast cancer. Estrogen metabolism was shifted from protective methoxylation and conjugation pathways in healthy control women towards activating pathways leading to formation of depurinating DNA adducts in women at high risk or with breast cancer. These results support the hypothesis that breast cancer is initiated by mutations derived from depurination of estrogen-DNA adducts. Therefore, relative levels of depurinating estrogen-DNA adducts could become biomarkers for early detection of breast cancer risk and aid in determining preventive strategies.

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