Figure 3: Indicated by the pro-apoptosis factors Bax and Caspases 3, 9, and 10 cell death was very high in the first week of exposure to Cd. The return back to control levels at six weeks likely plays a role in the recovery and adaptation of the cells.

Background: Cadmium is a multisite human carcinogen that possibly targets the prostate. Cadmium is present in cigarette smoke, food, drinking water, and air from industrial and agricultural sources2. It has previously been shown that chronic exposure to cadmium can malignantly transform the human prostate epithelial cell line, RWPE-1.Objective: Here, we hypothesize that chronic exposure to cadmium can similarly transform the isogenic human prostate progenitor/stem cells, WPE-stem, which were isolated from RWPE-1. Typically, progenitor/stem cells are resistant to toxins compared to their differentiated counterparts. Methods: To determine if cadmium exposure induced a cancer cell phenotype in WPE-stem cells, several characteristics commonly associated with cancer cells were analyzed, including secreted MMP-9 levels, epithelial-mesenchymal transition (EMT), and colony formation in soft agar.Results: Unexpectedly the WPE-stem cells were highly sensitive to cadmium after one week of exposure compared with RWPE-1 (90% cell loss vs 15%, respectively). However, the WPE-stem cells quickly recovered back to control levels in as early as four weeks of exposure, which appears to be related to several apoptotic (Bax; Casp-3,9,10) and cadmium adaptation-associated (MT-1/2; NRF2, ZIP8, ZNT1) factors. In as early as four weeks of cadmium exposure, MMP-9 levels increased by 1500% and colony formation increased by approximately 100% compared with untreated controls. Decreased expression of E-cadherin, a hallmark of EMT, and of the PTEN tumor suppressor gene was seen starting at six weeks.Conclusions: These data suggest that while initially hypersensitive to cadmium, WPE-stem cells quickly adapt and acquire a cancer cell phenotype during chronic exposure.

Figure 2: MT-1, MT-2, NRF2, ZNT1 and ZIP8 are all factors known to play a role in cadmium resistance and adaptation. The changes of expression in these factors appear to play a role in initial sensitivity but then apparent adaptation to Cd during chronic exposure.

Abstract

Factors Involved in Cd Adaptation• Cadmium (Cd) and Cd exposure:- Occupational and environmental concern1

• Fertilizers, fossil fuel combustion, tobacco, metal industries- Classified as a known human carcinogen by IARC1

• Prostate is a target

• Stem Cells (SCs) and cancer SCs (CSCs)3: - Typically possess an intrinsic survival selection advantage compared to their mature differentiated counterparts- Believed to play key roles in cancer initiation, tumorigenesis, and relapse

• Many factors involved in general toxicant and/or Cd resistance4,5,6:- Apoptotic factors

• BAX, Caspases- Uptake/efflux factors for metals

• ZnT1, ZIP8- Direct Cd resistance

• Metallothionein (MT), NRF2

• Common cancer associated factors- Increased MMP-9 levels and colony formation- Epithelial-mesenchymal transition (EMT)- Depletion of PTEN tumor suppressor gene

Introduction

Apoptosis Factors During Chronic Exposure

• SCs showed an early hypersensitivity to sub-chronic Cd exposure.• SCs quickly adapt to Cd exposure becoming transformed in

6-8wks

• Factors favoring early Cd sensitivity in SCs:• Increased pro-apoptotic BAX and caspases 3, 9 and 10• Large increases in Cd/Zn transporters ZnT1 and ZIP8• Factors associated with Cd resistance

• Smaller increases in MT-1/2 at week one• Decreased NRF2 expression

• Factors involved in adaptation to Cd• BAX and caspase levels decreased to control levels or below• Decreased levels of Cd/Zn transporters• Further increases in MT-1/2 and NRF2

• Chronic Cd exposure induced an acquired cancer phenotype• Decreased PTEN tumor suppressor gene• Induction of EMT• Increased MMP-9 activity • Apparent increase in colony formation and clear increase in

colony size

Summary and Discussion

Authors would like thank the members of the Stem Cell Toxicology Group in the NTPL for their help with this project and the National Toxicology Program at the National Institute of Environmental Health Sciences.

Acknowledgments

1. IARC: IARC Monographs on the Evaluation of Carcinogenic Risksk to Humans. Cadmium and Cadmium Compounds, vol 100c. Lyon, France: IARC, 2011.

2. ATSDR: ATSDR, ToxGuide for Cadmium Cd. CDC, Oct. 2012.3. Reya T et al. Stem cells, cancer, and cancer stem cells. Nature

414:105-111, 2001.4. Moulis JM. Cellular mechanisms of cadmium toxicity related to

the homeostasis of essential metals. Biometals 23:877-896, 2010.

5. Joseph P. Mechanisms of cadmium carcinogenesis. Toxicol Appl Pharmacol 238:272-279, 2009.

6. Chen J, Shaikh ZA. Activation of Nrf2 by cadmium and its role in protection against cadmium-induced apoptosis in rat kidney cells. Toxicol Appl Pharmacol 241:81-89, 2009.

References

Cells and Cell Culture: WPE-stem is a stem/progenitor cell line isolated from RWPE-16. All cell lines were maintained in Keratinocyte Serum Free Medium (KSFM) with EGF and BPE supplements and 1% antibiotics. The treated cells were in medium with 10mM Cd concentration.

Real Time RT-PCR: Gene expression levels were measured by real time RT-PCR, normalized to β-actin and GAPDH levels and expressed as percent control.

Soft Agar: Size and number of colonies was measured by plating cells in soft agar and growing them for two weeks. Colonies were stained, fixed, and counted with a colony counter.

Zymography: SDS-PAGE zymography to measure MMP-9 activity

Western Blot: Protein was collected with M-PER reagent and separated in 4-12% gradient gels. Protein bands were visualized with chemiluminescent reagent.

Statistical Analysis: Data represent mean ± SEM (n ≥ 3) and are considered significant if P < 0.05 as signified by an asterisk (*).

Materials and Methods

Figure 5: SNAIL1 showed a significant increase and E-Cadherin showed a significant decrease starting at six weeks. These results suggest chronic Cd exposure induces EMT, a common characteristic of cancer cells.

EMT Associated Factors

Figure 4: Unexpectedly, there was a drop by almost 50% in the KRAS oncogene that typically increases in cancer cells. PTEN, a tumor suppressor gene, goes down in Cd treated cells, indicating that the treated cells have acquired a cancer phenotype.

Cancer Associated Genes

Although initially hypersensitive to Cadmium, the WPE-stem cells quickly recover, adapting to the metal and acquiring a cancer phenotype during chronic exposure.

Conclusion

Figure 1: Chronic exposure to 10 mM Cd in parental cell line (PCL) and normal stem cells (NSCs) viability. Picture insets taken of each cell line after 1 week of exposure.Cd was clearly more toxic to SCs initially but they showed a rapid rebound back to control numbers.

Early Sensitivity of Stem Cells to Cadmium

Chronic Cadmium Exposure Causes Prostate Stem/Progenitor Cells to Acquire a Cancer PhenotypeSarah R. Sugarman, Matthew Bell, Olive Ngalame, Erik J. Tokar

Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709

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Figure 6: After chronic exposure to Cadmium, MMP-9 levels increased by approximately 900% starting at 6 weeks. The dramatic increase in MMP-9 levels of treated cells could indicate that cancer caused by Cd is prone to metastasis.Colony formation increased in size and number.

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• The KRAS oncogene typically increases in expression in cancer cells, including the Cd transformed RWPE-1 cells. Further study is needed to elucidate the reason for the unexpected decrease in KRAS expression in the Cd treated stem cells.

• Initial sensitivity of stem cells to Cd and the mechanisms involved are currently being explored by the Stem Cell Toxicology Group.

Future Directions

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