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Microrna Dysregulation and Aneuploidy in Arsenic-Induced Carcinogenesis

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Microrna Dysregulation and Aneuploidy in Arsenic-Induced Carcinogenesis MicroRNA Dysregulation and Aneuploidy in Arsenic-induced Carcinogenesis J. Christopher States, Ph.D. Professor, Distinguished University Scholar Vice Chair for Research Dept. Pharmacology & Toxicology Associate Dean for Research Director, Center for Integrative Environmental Health Sciences March 10, 2021 Conflict of Interest • No commercial conflicts of interest • Work supported by U.S. National Institute of Environmental Health Science grants R21ES023627, R01ES027778 & P30ES030283, University of Louisville EVPRI Competitive Enhancement Grant, the Scholarship Fund of China Scholarship Council (No. 201408440133) and Wendell Cherry Chair in Clinical Trial Research 3/10/21 UKY 2 Outline • Background – Environmental arsenic exposure – Chronic arsenic exposure & cancer – miRNA function • Studies on miRNA dysregulation • Impact of hsa-miR-186 overexpression on chromosome stability • Potential mechanism of hsa-miR-186 overexpression • Future Directions & Conclusions 3/10/21 UKY 3 Key Points • Arsenite transformation of keratinocytes induces chromosomal instability • hsa-miR-186 elevated in As-induced SCC • hsa-miR-186 induces chromosome instability that is enhanced by arsenite exposure • hsa-miR-186 may be induced as consequence of arsenite disruption of ZRANB2 function 3/10/21 UKY 4 “King of Poisons” • Long history of use for homicide – Tasteless, odorless – Famous examples: • Napoleon Bonaparte (victim?) • Cesare Borgia (perpetrator?) • Chronic ingestion in drinking water – largest mass poisoning: >30M in Bangladesh 3/10/21 UKY 5 Arsenic Is a Metaloid 3/10/21 UKY 6 http://periodictable.com/ Arsenic Is Widely Distributed & Causes Multiple Diseases • 20th most common element in earth’s crust • >200 million people exposed worldwide to arsenic in drinking water • #1 on ATSDR National Priority List of hazardous ⇒Known human carcinogen substances > 20 years ⇒Known human atherogen • Variable disease outcome ⇒Probable human teratogen 3/10/21 UKY 7 U.S. counties with arsenic concentrations exceeding 10 ppb in 10% or more of ground-water samples 3/10/21 UKY 8 Private Well Water Arsenic Exposure in U.S. • EPA limits arsenic in public water supplies - not private wells • 2.1 million people in U.S. exposed to arsenic in private well water https://www.usgs.gov/media/images/estimated-population-arsenic-greater-10-micrograms-liter 3/10/21 UKY 9 Arsenicism in Michigan 3/10/21 UKY 10 Arsenic in Kentucky Groundwater Fisher & Goodman, 2002 NWQMC 3/10/21 UKY 11 Burning Coal Contaminates Air and Water with Arsenic and Heavy Metals Estimated sources of total arsenic exposure in Coal ash piles at Kentucky Utilities’ E.W. Southwest Guizhou are contaminated food (50- Brown plant site in Harrodsburg, KY are still 80%), air (10-20%), water (1-5%), and direct contact leaking pollution – water pollution often in coal-mining workers (1%) (Liu et al., EHP continues after conversion to natural gas 110:119, 2002) EarthJustice.org 3/10/21 UKY 12 Coal Ash Uncovered: Polluted Groundwater Found At 14 Kentucky Sites Ryan Van Velzer: https://wfpl.org/coal-ash-uncovered-polluted-groundwater-found-at-14-kentucky-sites/ 3/10/21 UKY 13 Arsenic in Food: Rice & Fruit Juice “At one point during the reign of King Cotton, farmers in the south central United States controlled boll weevils with arsenic- based pesticides, and residual arsenic still contaminates the soil. Today, rice paddies cover fields where cotton once grew” Apple & Grape Juices https://www.consumerreports.org/food-safety/arsenic-and-lead-are-in-your-fruit-juice-what-you-need-to-know/ 3/10/21 UKY 14 Arsenic in Treated Lumber A 12-foot section of pressure- "In less than two weeks, an average treated lumber contains about five-year-old playing on an arsenic- an ounce of arsenic, or treated playset would exceed the lifetime enough to kill 250 people cancer risk considered acceptable" 3/10/21 UKY 15 Chronic Arsenicism • Chronic ingestion causes: – Peripheral neuropathy – Hyperkeratosis – Bowen’s Lesions – Diffuse melanosis – Arteriosclerosis – Wasting 3/10/21 UKY 16 Arsenic Induced Arteriolosclerosis • Peripheral vascular disease Blackfoot disease in Gangrene examples endemic area of Taiwan from Bangladesh 3/10/21 UKY 17 Arsenic Induced Arteriolosclerosis • Peripheral vascular disease Blackfoot disease in Gangrene examples endemic area of Taiwan from Bangladesh Also increased MI, stroke, kidney disease… 3/10/21 UKY 18 Arsenic Is a Human IARC Class 1 Carcinogen Squamous Cell Carcinoma Basal Cell Carcinoma mybiopsy.org Bladder Cancer missinglink.ucsf.edu http://www.greenmagz.info/arsenic/ Lung Cancer medicalpictures.net quit-smoking-stop.com o Hepatocellular Carcinoma o Prostate Carcinoma academics.skidmore.edu Kidney Cancer o seerpress.com o Ovarian Cancer healthnewstrack.com 3/10/21 UKY 19 Proposed Mechanisms of Arsenic Induced Carcinogenesis • DNA repair inhibition / Co-mutagenicity • Epigenetics: DNA methylation / miRNA expression • Oxidative stress • Cell proliferation/stem cell expansion • Differential alternative mRNA splicing • Aneuploidy, chromosomal instability (numerical and structural chromosomal abnormalities ) 3/10/21 UKY 20 Arsenic Mode of Action “The most accepted explanation for the mode of action for arsenic carcinogenicity is that it induces chromosomal abnormalities without interacting directly with DNA.” Subcommittee on Arsenic in Drinking Water National Research Council Arsenic in Drinking Water National Academies Press, 1999, p. 4 3/10/21 UKY 21 Proposed Mechanisms of Arsenic Induced Carcinogenesis • DNA repair inhibition / Co-mutagenicity Epigenetics: DNA methylation / miRNA expression • Oxidative stress • Cell proliferation/stem cell expansion • Differential alternative mRNA splicing Aneuploidy, chromosomal instability (numerical and structural chromosomal abnormalities ) 3/10/21 UKY 22 Arsenite Disrupts Mitosis, Induces Aneuploidy • Disrupts cell division, aneuploidogenic in PBLs in vitro (Vega et al, Mutat. Res. 334:365, 1995) • Disrupts spindle retraction, delays mitotic exit, aneuploidogenic in HDFs in vitro (Yih et al, Cancer Research 57:5051, 1997) • Chromosome & chromatid deletions in PBLs, micronuclei in oral and urinary epithelial cells of arsenic-exposed people (Gonsebatt et al, Mutat. Res. 386:219, 1997) • Chromosomal aberrations in PBLs arsenic-exposed people (Mäki‐Paakkanen et al, Environ. Mol. Mutagen. 32:301, 1998) 3/10/21 UKY 23 Chronic Arsenite Exposure Increases Aneuploidy in Human Keratinocytes (HaCaT) Laila Al-Eryani 4 cultures incubated with 0 & 4 with 100 nM NaAsO2 for 28 weeks 3/10/21 UKY Wu et al, TAAP 378, 114614,24 2019 miRNA Structure & Function • Short non-coding RNAs that regulate gene expression post-transcriptionally • Processed from large RNAs transcribed by RNA pol II as ‘miRNA only’ genes or embedded in introns in protein coding genes 3/10/21 UKY 25 Overview of miRNA Biogenesis & Effector Pathways Chiara Alberti, and Luisa Cochella Development 2017;144:2548-2559 © 2017. Published by The Company of Biologists Ltd 3/10/21 UKY 26 Many miRNAs Play Role in Multiple Cancers Ahmad J, Hasnain SE, Siddiqui MA, Ahamed M, Musarrat J, Al-Khedhairy AA. MicroRNA in carcinogenesis & cancer diagnostics: A new paradigm. Indian J Med Res 2013;137:680-94 3/10/21 UKY 27 Role of miRNAs in Cancer • Association between miRNA expression and tumor development, progression and response to therapy • Potential biomarkers for disease diagnosis, prognosis and therapy (already entering the clinic) Paranjape T, Slack FJ, Weidhaas JB MicroRNAs: tools for cancer diagnostics Gut 2009;58:1546-1554. 3/10/21 UKY 28 Ganges Delta Region World’s Largest Mass Poisoning 3/10/21 UKY 29 Arsenic-Induced Skin Cancer Ashok Giri, IICB 3/10/21 UKY 30 Arsenic-Induced Skin Cancer Ashok Giri, IICB 3/10/21 UKY 31 miRNA Expression Analysis Assay miRNA on RT- Purify RNA qPCR arrays H & E LCM-procured Vanessa States cells HK BD/SCC 3/10/21 UKY 32 miRNAs Differentially Expressed between Hyperkeratoses & SCC mean Fold Target Name change (HK-SCC) Compared two squamous cell carcinomas hsa-miR-186 1351.2 (SCC) vs two hyperkeratosis (HK) hsa-miR-454 274.4 hsa-miR-331-5p 207.9 hsa-let-7f 128.0 hsa-miR-375 119.4 mmu-miR-134 90.5 hsa-miR-301 68.6 hsa-miR-628 59.7 hsa-miR-31 55.7 hsa-miR-520f 42.2 hsa-miR-342 42.2 hsa-miR-28 42.2 hsa-miR-345 42.2 hsa-miR-21 34.3 hsa-miR-636 4.81E-03 hsa-miR-208 3.01E-04 3/10/21 UKY 33 miRNAs Differentially Expressed between Hyperkeratoses & SCC mean Fold hsa-miR-186 targets securin (aka PTTG1) Target Name change (HK-SCC) hsa-miR-186 1351.2 Securin validated as miR-186 target: hsa-miR-454 274.4 hsa-miR-331-5p 207.9 Li et al, Carcinogenesis 34:2145 (2013) hsa-let-7f 128.0 hsa-miR-375 119.4 mmu-miR-134 90.5 Predicted binding: hsa-miR-301 68.6 microRNA.org: hsa-miR-628 59.7 3' ucgGGUUUUCCUCUUAAGAAAc 5' hsa-miR-186 mirSVR score: -0.9169 hsa-miR-31 55.7 ::|| | || ||||||| hsa-miR-520f 42.2 PhastCons score: 0.6066 33:5' guaUUAAUA-AAGCAUUCUUUa 3' PTTG1 hsa-miR-342 42.2 hsa-miR-28 42.2 hsa-miR-345 42.2 hsa-miR-21 34.3 hsa-miR-636 4.81E-03 hsa-miR-208 3.01E-04 3/10/21 UKY 34 Arsenic Inhibition of Mitosis: Cyclin B & Securin Stabilization States, Biol. Trace Elem. Res. 166:34-40 (2015) 3/10/21 UKY 35 CDKN1A Inhibits CDK1 States, Biol. Trace Elem. Res. 166:34-40 (2015) 3/10/21 UKY 36 Potential hsa-miR-186 Role in Mitosis States, Biol. Trace Elem. Res. 166:34-40 (2015) 3/10/21 UKY 37 Impacts of Aberrant Securin Levels • Too much securin (e.g. APC/C inhibition) – Inhibit chromatid separation leading to lagging chromosomes and aneuploidy • Too little securin (e.g. excess miR-186) – Premature chromatid
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