Our Science – Waalkes Website

Michael P. Waalkes, Ph.D.

Laboratory of Comparative Carcinogenesis Head, Inorganic Carcinogenesis Section Senior Investigator Building 101SC, Room F095 National Institute of Environmental Health Sciences 111 Alexander Drive Research Triangle Park, NC 27709 Phone:   919-541-2328 Fax:   919-541-3970 E-Mail:   waalkes@niehs.nih.gov

Biography

Dr. Waalkes received his Ph.D. degree in pharmacology and toxicology in 1981 from West Virginia University where he studied perinatal toxicology of cadmium. He then completed postdoctoral work at the University of Kansas where his studies focused on the cellular and molecular mechanisms of acquired tolerance to metal toxicity. He has been with the NCI since 1983.

Research

Molecular Mechanisms of Inorganic Carcinogenesis

Some of the highest priority hazardous substances are inorganics. The EPA annually lists agents posing the greatest hazard to the U.S. population and for many years inorganics, such as arsenic, and cadmium have consistently topped this list. These agents are particularly hazardous as they cannot be metabolized into less toxic subunits in organisms. Inorganics are also an important class of human carcinogens, but their modes of action are as yet undefined. Our work has focused on defining the molecular mechanisms of inorganic carcinogenesis.

Arsenic has been a suspected human carcinogenic for over 100 years. Despite this, the mode of carcinogenic action for this important environmental contaminant is still unknown. Arsenic undergoes mono- and di-methylation, consuming cellular methyl groups in the process. Defining the carcinogenic mechanisms of inorganic arsenic has been hampered by the lack of animal models for research. We recently found inorganic arsenic to be a very effective transplacental (TPL) carcinogen or co-carcinogen in several mouse strains, as brief exposure to arsenic in the drinking water of pregnant mice initiates or induces tumors of the urinary bladder (UB), lung, skin, liver, kidney, ovary, adrenal, and uterus in the offspring in adulthood and long after all arsenic exposure has ended. These findings have clear human significance because the UB, lung, skin, liver, and kidney are recognized or probable target sites of arsenic carcinogenesis in humans. Additionally, our mouse TPL studies are increasingly relevant to human risk analysis because recent studies have now shown that arsenic has TPL/early life carcinogenic activity in humans. We have now greatly lowered dose by using "whole life" exposure (fetal, translactational, weanling, adult), which more precisely duplicates human exposure. Tumor sites are unaltered but incidence often increases, suggesting fetal exposure dictates target site while exposure in other periods promotes these lesions.

One possible mechanism for TPL arsenic carcinogenesis is attack on fetal stem cells (SCs). We recently found that fetal arsenic exposure caused a striking increase in cancer SCs (CSCs) in squamous cell carcinomas and distorted SC signaling in these tumors and earlier in fetal skin. To further elucidate possible SC-associated mechanisms and the co-carcinogenic capability of arsenic in the skin, we plan to give Tg.AC mice TPL arsenic then ultraviolet irradiation, a human relevant carcinogen, in adulthood. The arsenic-driven aberrant accumulation of SCs/CSCs could be due to a survival selection advantage that favors accumulation and transformation. To examine this hypothesis, we are studying the role of SCs in arsenic carcinogenesis in in vitro models. We find superior intrinsic and acquired arsenic resistance in vitro in human prostate and rodent kidney SC lines, involving apoptotic-, stress-, and arsenic-specific adaptation genes. During malignant transformation of a mature heterogeneous prostate line, we find that a stunning over-production of CSCs is unique to arsenic but not the other well-known carcinogens, including the inorganic carcinogen, cadmium. Given this initial SC selection, we will test if arsenic directly targets SCs by determining if it transforms enriched SC lines (urogenital, renal) more rapidly when isolated from their mature counterparts. Together, these studies provide clear evidence that arsenic can target and may malignantly transform fetal SCs, greatly impacting oncogenesis in adulthood and forming a toxicological mechanism for the fetal basis of adult disease.

We tested the role of arsenic biomethylation (As-BML) in oxidative DNA damage (ODD) and transformation using the recently developed immuno-spin trapping method, which minimizes isolation artifacts. When a BML-capable liver line and a BML-deficient prostate line were exposed to transforming levels of arsenic, a delayed increase in ODD occurred only in the BML-capable cells prior to transformation. The BML-deficient cells showed no ODD despite exposure past transformation. The human bladder cell line, UROtsa, which poorly methylates arsenic, and its stable arsenic methyltransferase transductant, UROtsa/F35, also shows that arsenic-induced ODD occurs only in As-BML-capable cells. Thus, As-BML is obligatory for ODD and can correlate with acquired cancer phenotype, but some cells acquired a cancer phenotype without ODD. This clearly indicates arsenic has multiple carcinogenic mechanisms. We are currently testing methylated arsenicals for ODD and linkage to transformation and possible mutations. Also, we will look at potential epigenetic mechanisms in cells that do not show ODD yet still undergo arsenic-induced transformation.

We have elucidated a potential mechanism of skin co-carcinogenesis with arsenic and UV irradiation. We malignantly transformed a human skin keratinocyte line with arsenic, which adapts via diminished oxidative stress response and apoptotic resistance. Once adapted, they are cross-adapted to UV irradiation. Consequently, they show UV-induced ODD at a much higher level than control due to adaptive apoptotic by-pass. This demonstrates that adaptation to arsenic is not always beneficial. We plan to test this co-carcinogenesis mechanism in an in vivo model (see above). As-BML requires S-adenosylmethionine (SAM). The arsenic-transformed and -adapted human CAsE-PE cells show signs of methyl depletion (DNA hypomethylation) even though they only very poorly methylate arsenic. During early adaptation, homocysteine (Hcy) increases, and SAM decreases along with inter-converting enzymes, indicating reduced conversion of Hcy to SAM. SAM loss is directly related to DNA hypomethylation. Additionally, the transulfuration pathway is activated to increase glutathione (GSH) production for arsenic efflux. Thus, adaptation preserves cells but pre-disposes to a potential epigenetic mode of carcinogenesis. Further study will focus on other metabolic factors potentially altered by arsenic that could drain SAM, such as polyamine synthesis and cobalamin transport.

The prostate is a target of cadmium. We have found that cadmium, similar to arsenic, can malignantly transform human prostate epithelial cells. However, in contrast to arsenic, cadmium selectively kills prostate SCs early in exposure and at a dramatically higher percentage when compared to the heterogeneous parental mature cells. Though depleted, remaining SCs show signs of rapid malignant transformation. We plan to determine if cadmium has malignantly transformed these SCs and compare them to the mature line during transformation for selective "hyper-resistant" SC targeting. The pancreas is another potential human target of cadmium. Human pancreatic epithelial cells were transformed by cadmium, with markedly increased floating "pancreaspheres" highly enriched in viable CSC-like cells. Thus, cadmium over-produces CSCs while transforming pancreatic cells. We are comparing cadmium and other carcinogens for CSC formation and plan to define if an early "bottleneck" event, like with prostate SCs, occurs with cadmium killing most of the SCs in this cell system. Human breast cancer has recently been linked to cadmium with some feeling it acts as a metalloestrogen via estrogen receptor (ER) stimulation. We malignantly transformed the ER-negative MCF10A normal human breast epithelial cell line with chronic cadmium. The transformed cells showed basal-like breast carcinoma (BLBC) characteristics as well as over-expression of SC markers after cadmium exposure. Thus, cadmium transforms breast epithelial cells via a non-ER mechanism producing a BLBC phenotype, noteworthy for its poor prognosis. We will determine more precisely the mode of cadmium transformation in these cells, looking at potential non-ER-based mechanisms.

This page was last updated on 11/25/2009.