Our prior work shows malignant transformation occurs with continuous arsenic exposure of HaCaT cells to a low, environmentally relevant level of arsenic for ~28 weeks, as indicated by production of SCC in xenograft study, and MMP-9 hyper-secretion and multinuclear giant cell formation in vitro
(Pi et al., 2008
). MMPs degrade the extracellular matrix, aid in invasion and are often hyper-secreted by aggressive tumors (Liotta et al., 1980
). MMP-9 hyper-secretion is commonly observed after malignant transformation with arsenic (Pi et al., 2008
; Benbrahim-Tallaa et al., 2005
; Achanzar et al., 2002
). In the present study, HaCaT cells exposed to the same level of arsenic (Pi et al., 2008
) showed marked increases in colony formation, MMP-9 secretion and multinuclear giant cells at 20 weeks, indicating they had likely already acquired a malignant phenotype. In fact, secreted MMP-9 activity at 20 weeks was essentially the same as when these cells produce SCC upon inoculation (Pi et al., 2008
). Thus, it appears that the HaCaT cells exposed to arsenic in the present work have already acquired a malignant phenotype by 20 weeks of arsenic exposure. With this acquisition of malignant phenotype there was a dramatic and dynamic series of changes in CK expression in this study that were consistent with the process of epidermal carcinogenesis (Tseng et al., 1968
; Leigh et al., 1993
). This fortifies the use of these transformed cells as a valid in vitro
model of human skin cancer induced by arsenic.
Progressive alterations in keratin expressions are closely associated with the development of a variety of tumors including skin malignancies (Chu and Weiss, 2002
; Casanova et al., 2004
). Study of chronic arsenic poisoning in Taiwan indicates progressive alterations in CK expression occurs in various skin lesions, like hyperkeratosis and SCC (Yu et al., 1993
). However, an integrated investigation of CKs during chronic arsenic-induced acquired malignant phenotype in a specific target cell of concern is not available. The present study was performed to systematically assess the expression of CKs during arsenic-induced malignant transformation in a human skin keratinocyte line, and to help define the expression pattern of various CKs in arsenic-induced skin cancer in a relevant human model. Dramatic and dynamic alterations in CK expressions occurred with arsenic that are largely consistent with the process of epidermal carcinogenesis, and support and expand on the limited available human data with arsenic (Yu et al., 1993
Hyperkeratosis is one of the most common skin lesions with chronic arsenic poisoning (Pi et al., 2000
), and considered a sign of aberrant cell proliferation, and likely a precursor skin lesion of SCC (Alain et al., 1993
). The over-expression of CK1 and CK10 signal over-production of keratinocytes. Pathologically, keratinizing SCCs are consistently positive for CK1 and CK10, while nonkeratinizing SCCs are negative (Remotti et al., 2001
). In the present study, the time-dependent increases of CK1 and CK10 with arsenic exposure clearly indicate these two CKs are linked to arsenic-induced malignant transformation. This would be consistent with hyperkeratotic lesions and keratin positive SCC in arsenic-exposed patients, and the fact that arsenic-associated SCC typically arise in areas of arsenic-induced hyperkeratosis (Tseng et al., 1968
; Yu et al., 2001
). Furthermore, the SCC formed after inoculation of these arsenic-exposed cells are similarly keratin positive (Pi et al., 2008
Over-expression of CK13 appears to be a marker for skin tumor progression (Warren et al., 1993
; Slaga et al., 1995
), and occurs in HaCaT cells malignantly transformed with UV irradiation (He et al., 2006
). Although not normally expressed in the epidermis, CK13 is expressed in epidermal tumors (Caulin et al., 1993
). Again these findings suggest chronic arsenic exposure has initiated changes in CK expression in potential target cells consistent with similar CK expression seen in epidermal carcinogenesis.
Evolving theory in carcinogenesis proposes tumors originate from pluripotent stem cells with self-renewal capacity and conditional immortality, characteristics required for accumulating the genetic alterations needed for acquisition of cancer phenotype (Perez-Losada and Balmain, 2003
). CK5, 14 and 15 are all considered markers for epidermal stem cells (Gerdes and Yuspa, 2005
; Lyle et al., 1998
; Liu et al., 2003
). Recent evidence indicates that arsenic exposure in the fetal mouse facilitates cancer response in adulthood by distorting skin tumor stem cell signaling and population dynamics, creating an over-abundance of cancer stem cells in resulting SCC (Waalkes et al., 2008
). This implicates stem cells as a primary target of arsenic in the fetal basis of skin cancer in adulthood (Waalkes et al., 2008
). Other work (Patterson and Rice, 2007
) shows arsenic in vitro
delays exit of human epidermal stem cell into differentiation pathways, thus increasing stem cells and potentially increasing target cell number for carcinogenic insult. CK15 is expressed in various skin tumors (Misago and Narisawa, 2006
; Kanitakis et al., 1999
), while UV irradiation increases CK5 and CK14 expression of human keratinocytes (Kinouchi et al., 2002
). In the present study, arsenic increased CK5, CK14 and CK15. Together with the increased expression of additional stem cell markers in arsenic-treated HaCaT cells, such as p63 (~2-fold at 20 weeks; not shown), this indicates arsenic transformation might be involved at the level of stem cells. Additional study on chronic arsenic exposure and stem cells dynamics is required to elucidate this relationship, however.
CK8 and CK18 are often over-expressed in SCC, particularly when poorly differentiated (Markey et al., 1991
) or invasive (Schaafsma et al., 1993
). Again, the over-expression of CK8 and CK18 in arsenic-treated HaCaT cells in the present work is consistent with a molecular pathology of SCC.
Loricrin transcription can be up-regulated when keratinocytes differentiation is stimulated (Hohl et al., 1991
). Filaggrin is a filament-associated protein which binds to keratin fibers in epidermal cells. Involucrin expression is increased in cultured keratinocytes following treatment with TPA, a tumor promoter (Efimova et al., 1998
). The increased expressions of loricrin, filaggrin and involucrin in arsenic-treated HaCaT cells indicate that chronic arsenic exposure may also be involved in aberrant terminal differentiation of the epidermis.
Keratinocyte activation after wounding involves cellular changes at the wounds edge, which are accompanied by induction of stress response CKs 6, 16 and 17 (Mansbridge and Knapp, 1987
). Increases in CKs 6, 16, and 17 are considered a hallmark of keratinocyte activation and are seen in hyperproliferative skin disorders (Weiss et al., 1984
). CK6 and CK16 are found in SCC while CK17 is widely expressed in invasive SCC (Leigh et al., 1993
). CKs 6, 16 and 17 are also found in arsenic-induced SCC in human populations (Yu et al., 1993
). The expression of CK6/16 and 7/17 were increased by arsenic transformation in the present work, suggesting they are part of an adaptive response to chronic injury or stress. Again our in vitro
system appears to duplicate the responses seen in vivo
during epidermal oncogenesis with many of the specific changes seen with arsenic.
In conclusion, chronic arsenic exposure of human keratinocytes at environmentally relevant levels drives them towards malignant transformation. Multiple molecular events are likely associated with this arsenic-induced transformation. Dynamic and dramatic alterations in CK expression occur that frequently duplicate epidermal carcinogenesis in general and arsenic skin cancer in particular. This further establishes these transformed cells as an important model for in-depth molecular analysis of the events associated with arsenic-induced skin cancer.