Estrogen Metabolism Role in Oncology Tom Archie, MD SLWRMC Tumor Board June 19, 2008

Early breast cancer – poorly differentiated 50yo Caucasian Female Early breast cancer – poorly differentiated MRI negative for contralateral tumor Receptor Status: Triple Negative (ER, PR, HER2/neu) Lumpectomy followed by Taxotere and Cytoxan

Estrogen Metabolism Testing Identify high risk patients for new breast cancer Others: prostate cancer, leukemia, olfactory tumors, and Parkinson’s Disease (probably more to come) Identify high risk of recurrence in breast cancer patients (and other cancers) Modify risk via modification of estrogen metabolism balance Future: “pre-mammogram” biomarker

Catechol Estrogen Quinones to DNA Adducts Endogenous Estrogens can become carcinogenic 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. Irregardless of ER status Mechanism: Sheer volume of DNA “apurinic” sites – DNA repair enzymes make mistakes, leading to single nucleotide polymorphisms (SNP)

Catechol Estrogen Quinones to DNA Adducts 4-OH-estrone induces DNA Adduct formation in normal breast epithelium (MCF-10F cells) Saeed M et al. Int J Cancer. 2007 Apr 15;120(8):1821-4.

Human Study of Urinary Estrogen Metabolites w/ and w/o Breast CA Human study comparing healthy controls, breast cancer patients, and “high risk” patients (as determined by oncologists in study) The levels of the ratios of depurinating DNA adducts to their respective estrogen metabolites/conjugates were significantly higher in high-risk women (p < 0.001) and women with breast cancer (p < 0.001) than in control subjects. This mechanism represents the best understood and documented initiation step in the formation of any cancer. Gaikwad NW et al. The molecular etiology of breast cancer: evidence from biomarkers of risk. Int J Cancer. 2008 May 1;122(9):1949-57.

COMT and CE (Catechol Estrogen) Quantitatively, the most active CE conjugative pathway is methylation. CE methylation is catalyzed by COMT Catechol-O-methyltransferase (COMT) a classical phase II enzyme, catalyzes the transfer of methyl groups from S-adenosyl methionine, the enzyme cofactor, to hydroxyl groups of a number of catechol substrates, including the CEs. Under normal circumstances, CEs are, for the most part, promptly O-methylated by COMT to form 2- and 4-O-methylethers, which are then excreted. While virtually all catechols are substrates for COMT, the highest affinities for the enzyme are exhibited by the CEs Journal of the National Cancer Institute Monographs No. 27, 2000

Low Functioning COMT and Breast Cancer Risk Genetic epidemiology studies have proposed a possible correlation between the low activity allele (COMTLL) and increased breast cancer risk Lavigne JA, et al. An association between the allele coding for a low activity variant of catechol-O-methyltransferase and the risk for breast cancer. Cancer Res 1997;57:5493–5497. Huang CS, et al. Breast cancer risk associated with genotype polymorphism of the estrogen metabolizing genes CYP17, CYP1A1, and COMT: A multigenic study on cancer susceptibility. Cancer Res 1999;59:4870–4875. Yim D-S, et al. Relationship between the val158met polymorphism of catechol O-methyl transferase and breast cancer. Pharmacogenetics 2001;11:1–8.

COMT and Breast Cancer COMT protects cells from the genotoxicity and cytotoxicity of catechol estrogens, by preventing their conversion to quinones Adds methyl group (-CH3) at the -OH site that would otherwise be oxidized by peroxidase enzymes Low activity of COMT leads to higher levels of depurinating estrogen-DNA adducts that can induce mutations and initiate cancer. MCF-10F (human breast epithelial cells that are ER neg) Estrogen-DNA adducts’ carcinogenicity independent of ER status Zahid M et al. Free Radic Biol Med. 2007 Dec 1;43(11):1534-40. Lu F et al.J Steroid Biochem Mol Biol. 2007 ; 105(1-5): 150–158.

Low Functioning COMT Common 25% of US Caucasians are homozygous for the val108/158met polymorphism in the COMT gene Lachman HM, et al. Pharmacogenetics 1996;6:243–250. Scanlon PD, et al. Science 1979;203:63–65. 27% Chinese Americans and 34% Japanese Americans Wu A et al. Cancer Res 2003;63: 7526–7529 Val108/158Met SNP associated with 3-4x reduction in functional enzymatic rate of COMT. Zhu BT. Curr Drug Metab 2002;3: 321–349

E2:E16 Ratio - Breast Cancer Risk Prospective Study 10,786 women aged 34-69 with 5 ½ yr followup Measured urinary estrogen metabolites 144 breast cancer pts w/ 4 matched controls for each cancer Highest quintile E2:E16 ratio Premenopausal: OR 0.58 (42% risk red) Postmenopausal: OR 1.29 (29% risk inc) Muti et al. Epidemiology. 2000 Nov;11(6):635-40

Broccoli increases E2:E16 ratio Increase E2:E16 ratio 29.5% with broccoli 500gr/day Cruciferous vegetables cause the upregulation of Cyp1A2 (19%) and Cyp1A1 and inhibit Cyp2E1 Indole 3 Carbinol (glucocinolate) Sulforaphane (isothiocyanate) Diindolylmethane (glucocinolate) Calcium D Glucarate

Interpretation Low 2-Hydroxyestrone/16α-Hydroxyestrone Ratio Premenopausal female Increased risk of ongoing carcinogensis leading to treatment failure

Interpretation Poor methylation capacity – she is a “slow methylator” 4-Methoxyestrone is undetectable 4-Hydroxyestrone is not being methylated adequately. 4-Hydroxyestrone level is high. This is associated with increased levels of 4-catechol estrogen DNA adducts, which are strongly associated with the initiation of breast and prostate cancer. COMT is likely genetically slow Principal agent for eliminating catechol estrogens

Interpretation Interestingly, the methylation of 2OHE is adequate, whereas methylation of 4OHE is not I find no literature citing methylation preferences for 2OHE vs. 4OHE Fact remains that additional methylation support is needed

Treatment Goals Enhance methylation Decrease Cyp1B1 activity Increase E2:E16 ratio Cruciferous vegetables

Improve Methylation Increase substrate for COMT (SAMe) Add methyl donors Folate, methylcobalamin (B12) Trimethylglycine (Betaine) Vit B6 (to discourage the accumulation of homocysteine and encourage the formation of glutathione via synthesis of cysteine) There is no physiological mechanism to suggest an adverse interaction between methylation and the metabolism of either taxotere or cytoxan

Improve Methylation COMT

Cyp1B1 inhibition (ie: reduction of DNA adducts) Reduce xenobiotic pollutant exposure N-acetyl Cysteine Sulforaphane (glucosinolate from broccoli) induces quinone reductase, which takes CEQs back to catechol estrogens, reducing the potential for the creating of DNA adducts. Hwang. J Med Food. 2005 Summer;8(2):198-203 Glutathione conjugates are not playing much of a role in protecting against DNA adducts.

Cyp 1B1 Inhibition to decrease DNA Adduct Formation Increased methylation of catechol estrogens leads to feedback inhibition of Cyp1B1 Dawling et al. Cancer Res. 2003 Jun 15;63 (12):3127-32.

Cyp 1B1 Inhibition to decrease DNA Adduct Formation Reduced Lipoic Acid N-acetyl Cysteine Resveratrol Melatonin (minimal but positive effect) Zahid M. et al. Inhibition of depurinating estrogen-DNA adduct formation by natural compounds. Chem Res Toxicol. 2007 Dec;20(12):1947-53. Epub 2007 Nov Chen et al. Resveratrol inhibits TCDD-induced expression of CYP1A1 and CYP1B1 and catechol estrogen-mediated oxidative DNA damage in cultured human mammary epithelial cells Carcinogenesis vol.25 no.10 pp.2005--2013, 2004 doi:10.1093/carcin/bgh183

Synergism b/t Paclitaxel and Broccoli Glucosinolate Diindolylmethane in combination with paclitaxel synergistically inhibits growth of Her2 / neu human breast cancer cells through G2M phase cell-cycle arrest and induction of apoptosis / necrosis McGuire KP, et al. J Surg Res. 2006 May 15;132(2):208-13. Epub 2006 Mar 31. 

Broccoli and Antitumor Effects Sulforaphane inhibits breast cancer growth and induces Quinone Reductase Hwang. J Med Food. 2005 Summer;8(2):198-203. I3C induces Br CA cell cycle arrest

Potential Risk Uncertain effect on Cyp3A4 (60% of drugs) Sulforaphane inhibtis Diindolylmethane has no effect Could affect concentration of these drugs and theoretically increase adverse drug events or decrease efficacy Taxotere metabolized by Cyp3A4 Consider avoiding near time of infusion

Prostate CA Small study of urine estrogen metabolites in men with prostate cancer vs. benign urological d/o vs. healthy controls 4-OHE1-DNA Adducts detected at higher levels in samples from subjects with prostate cancer and benign urological conditions compared to healthy males This is the first demonstration that CEQ-derived DNA adducts are present in urine samples from subjects with prostate cancer. Markushkin Y et al. Potential biomarker for early risk assessment of prostate cancer. Prostate. 2006 Oct 1;66(14):1565-71

Extension to Other Cancers This mechanism is also involved in Initiation of leukemia by benzene Rat olfactory tumors by naphthalene Neurodegenerative diseases such as Parkinson's disease by dopamine. Estrogens and Human Diseases. Volume 1089 published November 2006 Ann. N.Y. Acad. Sci. 1089: 286–301 (2006). doi: 10.1196/annals.1386.042

Conclusion No human intervention trials on manipulation of estrogen metabolism in patients w/ active breast cancer Epidemiologic studies support cruciferous vegetables and methyl donors to decrease breast cancer risk In vitro studies showing anticancer effects of brassica Risk of non-action vs. action? Enhance methylation now Increase E2:E16 ratio now but reduce likelihood of possible interaction w/ metabolism of taxotere by avoiding for 1 week prior and 2 days after administration of taxotere Inhibit Cyp1B1 now

In a word….

One Last Thought for Future Discussion Multifocal Angiostatic Therapy

Multifocal Angiostatic Therapy curcmin, artemsia, mistletoe, ginger scutellaria, resveratrol, grapeseed extract, green tea, gingko, squalamine, Vit D silymarin, glycine, Multifocal Angiostatic Therapy VEGFR EGCG, silymarin, quercetin, resveratrol, soy isoflavones, curcumin, EPA Cu antagonists curcmin, scutellaria, cartilege, silymarin, green tea ginger artemsia mistletoe curcumin scutellaria bFGFR and TNF-1: Cu antagonists Growth Factors NFkB COX-2 VEGF, AKs, bFGF, IL8, MMPs, TNF-1, heparinases, collagenases Silymarin, Glycine, Ginger Anti- NFkB: poria, coriolus, ginger, resveratrol, green tea, artemsia, quercetin, carnosol, panax ginseng, silymarin, salicylates, curcumin, picentannol, basil, Cu antagonists rosemary green tea quercetin magnolia resveratrol, soy, curcumin holy basil rosemary ganoderma licorice Vit E Anti-COX-2: quercetin, scutellaria, EPA/DHA, licorice, ginger, resveratrol, grapeseed extract, curcumin, salicylates, garlic, green tea, panax ginseng, silymarin, bilberry, antioxidants, boswellia, aloe