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

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 separation leading to chromosomal instability and aneuploidy

3/10/21 UKY 38 miR186 Overexpression in HaCaT

Jiguo Wu

3/10/21 UKY 39 hsa-miR-186 Expression in Transfected Keratinocytes

3/10/21 UKY Wu et al, TAAP 378, 114614,40 2019 Selected Clones

3/10/21 UKY Wu et al, TAAP 378, 114614,41 2019 hsa-miR-186 Expression in Selected Clones

Exposed to 0/100 nM NaAsO2 for 4/8 Weeks

3/10/21 UKY Wu et al, TAAP 378, 114614,42 2019 Chromosomal Abnormalities in HaCaT Over- Expressing hsa-miR-186

Jiguo Wu Sandra Wise

3/10/21 UKY Wu et al, TAAP 378, 114614,43 2019 hsa-miR-186 Overexpression Increases Mitoses with Supernumerary Chromosomes

3/10/21 UKY Wu et al, TAAP 378, 114614,44 2019 Evidence of Multiple Endomitoses

Tetraploid human cells shed chromosomes to

100 nM NaAsO2, 8 become ‘pseudo-tetraploid”. Second weeks endomitosis yields ‘pseudo-octoploidy’ >240 chromosomes Wu et al, TAAP 378, 114614, 2019 3/10/21 UKY 45 hsa-miR-186 Overexpression Increases Dicentric Chromosome Numbers

3/10/21 UKY Wu et al, TAAP 378, 114614,46 2019 Double Minutes Are Increased in Cells Overexpressing hsa-miR-186

DM’s are associated with gene amplification

3/10/21 UKY Wu et al, TAAP 378, 114614,47 2019 Ring Chromosomes Are Present Only in Cells Overexpressing hsa-miR-186

3/10/21 UKY Wu et al, TAAP 378, 114614,48 2019 Arsenite Exposure Enhances CIN in miR-186 Overexpressing Cells Ageliki Lykoudi miR186 miR186 + As

CIN

markers • 9q • 4p/18q • 3q/4p

3/10/21 UKY 49 hsa-miR-186 Induces Structural Chromosomal Instability

VC miR-186 0 AsnM

1: Chromosome 1 11: Chromosome 11 20: Chromosome 20 M: Marker chromosome D: Double minute chromosome 100 AsnM

3/10/21 UKY 50 15 metaphases were analyzed for the six HaCaT clones (exposed to 0 or 100 nM NaAsO2) for 9 weeks Clonal Variability in miR-186 Induced Structural Chromosomal Instability

40 15 Chromosome 1 Chromosome 11

30 10 15 metaphases were analyzed/ condition 20 5 after 9 weeks

10 0 Structural Abnormalities in Chromosome 11 Chromosome in Structural Abnormalities Structural Abnormalities in 1 Chromosome 0 -5 miR-186 - - + + miR-186 - - + + 3+ As3+ (100 nM) - + - + As (100 nM) - + - +

20 Chromosome 20 Double Minutes Data are expressed as mean

15 +/- SD. Data are analyzed with

10 two-way ANOVA and Tukey’s

5 multiple comparison post hoc test. The level of significance is 0 established at p value ≤ 0.05. Structural Abnormalities in Chromosome 20 -5 miR-186 - - + + 3/10/21 As3+ (100 nM) - + - + UKY 51 Summary

• Arsenite exposure enhances chromosome instability (CIN) • hsa-miR-186 overexpressed in SCC relative to premalignant HK • Overexpressing hsa-miR-186 in HaCaT induces CIN with increased chromosome numbers and increased structural abnormalities (aneuploidy) enhanced by arsenite exposure

3/10/21 UKY 52 KEGG Pathways Targeted by hsa-miR-186-5p

KEGG Pathway Pvalue # genes 1. Colorectal cancer 1.27e-06 11 Signaling pathways regulating pluripotency of stem 14 2. 0.0003 cells 3. Pathways in cancer 0.0003 23 4. Adherens junction 0.0008 8 5. Proteoglycans in cancer 0.0008 12 6. Cell cycle 0.0013 15 7. Endometrial cancer 0.005 7 8. TGF-beta signaling pathway 0.009 7

3/10/21 UKY 53 hsa-miR-186 Role in Cell Cycle/Mitosis

3/10/21 UKY 54 hsa-miR-186 Role in Cell Cycle/Mitosis

Spindle Assembly Checkpoint

3/10/21 UKY 55 hsa-miR-186 Role in Cell Cycle/Mitosis

Spindle Assembly Checkpoint

Anaphase Promoting Complex/ cyclosome components

3/10/21 UKY 56 BUB1 & CDC27 Responses

Ana Cardoso

Wu et al, TAAP, 2019

3/10/21 UKY 57 BUB1 Responds to hsa-miR-186 & NaAsO2

3/10/21 UKY Wu et al, TAAP 378, 114614,58 2019 BUB1 Suppressed by hsa-miR-186 Overexpression

3/10/21 UKY Wu et al, TAAP 378, 114614,59 2019 BUB1 Induced by Arsenite

3/10/21 UKY Wu et al, TAAP 378, 114614,60 2019 Arsenite Mitigates miR-186 BUB1 Suppression

3/10/21 UKY Wu et al, TAAP 378, 114614,61 2019 CDC27 Unchanged

3/10/21 UKY Wu et al, TAAP 378, 114614,62 2019 Spindle Assembly Checkpoint Summary

• BUB1 clearly targeted by hsa-miR-186, induced by NaAsO2 • CDC27 expression not suppressed by hsa-miR-186 overexpression or changed by NaAsO2 – Prediction incorrect? – Other factor(s) modulating expression? – CDC27 haploinsufficiency reported to suppress CIN (Sansregret et al, Cancer Discov. 2017 Feb;7(2):218-233)

3/10/21 UKY 63 hsa-miR-186 Embedded in ZRANB2

3/10/21 UKY 64 ZRANB2: Differential Splicing Factor with 2 Zn- Fingers

Structure of ZRANB2 zinc fingers (red circle=zinc) Plambeck et al., JBC 2003, 278, 22805-22811

3/10/21 UKY 65 Hypothesis

3/10/21 UKY 66 Arsenic Binds ZRANB2 Zinc Finger Peptides

_ j _ _ p ( ) ( ) 764.5182 8.45e5 729.1575 100 845202 1.16e6 100 1156041 728.9925 764.3493 1116497 790327 764.6873 728.8275 1059767 766464 729.3344 1037784

764.1924 704861

728.6625 930601

764.8563 611686 729.4995 814921 764.0234 573953

728.4975 636150

765.0253 416501 %

763.8545 % 394533 729.6646 ZNF1 533567 ZNF2

765.1823 262293 769.6814 253424 728.3207 Mayukh Banerjee 332161 729.8297 769.3423 769.8510 312958 222925 234753 733.9871 763.6856 285344 190887 767.0191 190158 731.4936 734.3302 253485 769.1849 772.5182 244448 167340 159217 731.8361 772.3484 772.8459 780.3403 227556 151316 149971 139550 734.4958 761.3468 731.9896 181862 773.0280;120461 780.8404 168617 119847 772.0208 777.8422 107335 108350 761.1782 761.8528 108593 783.0134 734.6615 750.3524 90121 93048 93072 151830 89630

0 m/z 738 740 742 744 746 748 750 752 754 756 758 760 762 764 766 768 770 772 774 776 778 780 782 784 786 788 790 792 794 796 798 800 802 804 806 808 810 812 814 816 818 820 822 0 m/z 702 704 706 708 710 712 714 716 718 720 722 724 726 728 730 732 734 736 738 740 742 744 746 748 750 752 754 756 758 760 762 764 766 768 770 _ j _ _ p ( ) ( ) 776.6737 5.51e5 741.3030 8.63e5 100 551262 776.8441 100 862935 535553

741.4813 763618

776.5156 741.1367 441871 687863 764.3613 428831

764.6873 401772 777.0145 396861

764.1924 741.6478 366610 575470

764.8563 319802 764.0234 +12 299366 777.1727 293422 % % 776.3453 255929 784.6757 249786 740.9703 370446 741.8024 784.8347 363005 765.0253 228021 ZNF1+As ZNF2+As 763.8545 222592 214405 777.3431 207149 784.5045 782.0120 200492 195698 785.0060 188275 781.8411 185453 782.1829 167838

781.6702 782.3417 785.1772 765.1944 145573 769.6935 140501 143014 137040 140354 777.5135 743.9799 131721 785.3362 769.3544 769.8510 787.3317 204534 123084 746.6378 763.6856 128666 130457 779.3406 117452 784.3333 728.9925 184472 111919 123766 744.1467 767.0312 770.0206 108825 175942 776.1871 779.6819 787.5032 179487 105690 100776 +12 746.8048 98503 99379 99895 729.1575 744.3134 165713 728.6625 160558 743.6465 772.6881 154139 136722 141258 774.0115 787.6748 746.9719 90993 790.1763 729.3226 750.3644 78407 87919 728.4857 120622 72716 111706 130687 73315 761.5275 740.8039 746.3277 67035 98822 94943 747.1389 750.8548 729.4877 87904 59430 95511

0 m/z 0 m/z 736 738 740 742 744 746 748 750 752 754 756 758 760 762 764 766 768 770 772 774 776 778 780 782 784 786 788 790 792 794 796 798 800 802 804 806 808 810 812 814 816 818 820 822 824 826 702 704 706 708 710 712 714 716 718 720 722 724 726 728 730 732 734 736 738 740 742 744 746 748 750 752 754 756 758 760 762 764 766 768 770

3/10/21 UKY Banerjee et al, CRT, 33, 1403–1417, 2020 67 Arsenite Exposure Increases ZRANB2 Levels But Decreases Function

3/10/21 UKY Banerjee et al, CRT, 33, 1403–1417, 2020 68 Summary

• Cells compensating for arsenite disruption of zinc fingers of ZRANB2 may lead to hsa-miR-186 overexpression • hsa-miR-186 overexpression and arsenite exposure acting together induce structural abnormalities • hsa-miR-186 overexpression and arsenite exposure both dysregulate BUB1 expression causing SAC imbalance • SAC imbalance may lead to CIN

3/10/21 UKY 69 Conclusions

• Arsenic exposure induced hsa-miR-186 likely contributes to chromosomal instability (aneuploidy) and carcinogenesis by inducing an imbalance in SAC components that can disrupt mitotic regulation and contribute to CIN • Confirmation in primary cells would validate this mechanism of carcinogenesis.

3/10/21 UKY 70 Acknowledgements

Jiguo Wu, Ph.D. Supported in part by R21-ES023627 Ana P. F. Cardoso, Ph.D. R01-ES027778 & P30ES030283, Mayukh Banerjee, Ph.D. University of Louisville EVPRI Laila Al-Eryani Competitive Enhancement Grant, Vanessa States Scholarship Fund of China Mark Doll Scholarship Council (No. Sandra S. Wise, Ph.D. 201408440133), and Wendell Cherry Ashok K. Giri, Ph.D. Chair in Clinical Trial Research. Susan Galandiuk, M.D. Shesh N. Rai, Ph.D.

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