Low-level, chronic arsenic exposure can induce malignant transformation in various human and rodent cells [28
]. In the present study when HaCaT cells were continuously exposed to environmentally relevant levels of arsenic for a protracted period, malignant transformation occurred as evidenced by the formation of highly aggressive SCC, a common form of skin cancer in arsenic-exposed humans [1
], after inoculation of nude mice. Importantly, by studying this cell model system several important biomarkers for arsenic-induced malignant transformation, including increased secretion of MMP-9, expression profile of Nrf2-target genes and cytokeratins, acquired apoptotic resistance and decreased Nrf2-mediated antioxidant response, were indentified.
MMPs are a family of matrix-degrading enzymes that play a critical role in invasion and metastasis of malignant tumors and hypersecretion of MMPs is common in highly aggressive tumors [33
]. MMP-9 specifically targets type IV collagen, a major component of basement membrane, and appears to play a crucial role in tumor invasion across the basement membrane [34
]. Elevated expression levels of MMP-9 are strongly correlated with malignant phenotype in SCC [34
] and are characteristic of malignant transformation of cells [29
]. In human keratinocyes constitutive production and secretion of MMP-9 correlate with the degree of tumorigenicity in malignantly transformed clones [33
]. Thus, the dramatically increased secretion of MMP-9 observed in As-TM cells might be an important biomarker for arsenic-induced malignant transformation in human keratinocytes. It is noteworthy that the promoter region of MMP-9 gene contains multiple highly conserved AREs [35
], suggesting Nrf2 may act as a direct transcriptional regulator of MMP-9.
Skin hyperkeratosis is common in chronic arsenosis [22
]. Importantly, SCC can arise from arsenic-induced hyperkeratotic lesions [36
], suggesting dysfunctional keratinization may be critical in arsenic-induced skin carcinogenesis. In the current study, As-TM cells were observed to exhibit dramatically increased expression levels of keratin-1, keratin-10, involucrin, and loricrin, major markers of squamous differentiation that play important roles in skin keratinization [37
]. Since aberrant over-expressions of these markers have been observed in skin hyperkeratosis [38
], skin SCC [40
], bladder SCC [41
] and lung cancer [42
], the enhanced expressions observed in As-TM cells may represent additional indicators for arsenic-induced malignant transformation. Interestingly, emerging data suggests that Nrf2 may act as a direct transcriptional regulator of certain squamous differentiation genes through their respective AREs [43
]. Thus, the enhanced expression of these genes and marked increase in secretion of active MMP-9 observed in As-TM cells might be a consequence of chronic Nrf2 activation. It should be noted that the expression of keratin-10 in As-TM cells is different from what was observed in higher concentrations of arsenite-induced malignant transformation of keratinocyte [30
CK2 is a ubiquitous and highly conserved pleiotropic serine/threonine protein kinase, and it appears to function predominantly in regulating the activities of nuclear proteins, including the regulation of cell growth and proliferation, as well as cell survival [25
]. Dysregulation of CK2 expression imparts a potent oncogenic potential to cells and overexpression of CK2 is often a key factor in carcinogenic transformation [45
]. A consistent elevation of CK2 has been observed in rapidly proliferating tissues and a wide variety of tumors [46
]. In the present study, increased expression and activity of CK2 was observed in As-TM cells, suggesting increase in CK2 might be a critical event during the process of arsenic-induced malignant transformation of human keratinocytes.
Apoptosis normally functions to control the integrity of cell populations by eliminating aberrant clones, whereas failure of apoptosis likely is a key contributor to tumor initiation and progression as well as drug resistance in skin cancer and cancer in general [47
]. Thus, the acquired, generalized apoptotic resistance in As-TM cells may be an important event in the process of arsenic-induced malignant transformation. Our previous studies have indicated that arsenic-induced apoptotic resistance is potentially associated with PI3K/PKB-mediated cellular survival signaling pathways [10
]. In the present study, increased levels of CK2, which participates in inhibition of apoptosis and negatively regulates caspase activity in tumor cells [49
], was observed. This finding suggests the enhanced CK2 expression of As-TM cells might be involved in their acquired apoptotic resistance.
In contrast to increased expression of Nrf2 and its target genes, including GCLC as well as elevated levels of intracellular GSH, a weakened Nrf2-mediated antioxidant response was observed in As-TM cells. The decrease of Nrf2 activation in response to high levels of oxidative stressors observed in As-TM cells, coupled with their acquired apoptotic resistance might increase the likelihood to acquire heritable oxidative DNA damage [7
]. This may help explain the remarkable co-carcinogenic effects of arsenic and UV irradiation observed in mouse models of dermal carcinogenesis but the absence of activity for arsenic alone [3
]. Our previous study indicated that exogenous GSH might markedly suppress hypochlorous acid-induced Nrf2 activation in mouse macrophages [50
], suggesting the enhanced GSH levels in As-TM cells might be a critical factor for the weakened Nrf2 activation. In addition, increased activities of Nrf2 downstream phase II enzymes in As-TM cells might detoxify both arsenic and tBHQ, and thus results in decreased Nrf2 activation. Furthermore, our recent studies revealed that CK2 is an important protein kinase for Nrf2 phosphorylation, which is involved in Nrf2 activation and degradation [24
]. The enhanced activity of CK2 and subsequent Nrf2 phosphorylation of As-TM cells might also contribute to their decreased Nrf2-medicated antioxidant response.
In summary, long-term arsenic exposure at levels in the range of human exposure causes malignant transformation of human keratinocytes in vitro. This occurs concurrently with acquired apoptotic resistance, increased expression of CK2, elevated basal Nrf2 activity, and decreased Nrf2-mediated antioxidant response. Arsenic-induced apoptotic resistance and weakened antioxidant response may be critical steps in development of dermal cancer after exposure to the metalloid.