Apoptotic resistance,cell cycle dysregulation and autophagy activation contribute to arsenic carcinogenesis

Background: Arsenic is a common environmental pollutant, chronic arsenic exposure causes multiple cancers including skin cancer. However, the underlying mechanisms of arsenic-driven skin carcinogenesis remains unclear. Methods: HaCaT cells were exposed to a low level of arsenic (100nM) for 28 weeks. Then, xenograft mouse model and H&E staining were used to test their ability of tumors formation. The effect of arsenic on apoptosis demonstrated by TUNEL assay. XIAP and cIAP1 were determined by immunocytochemistry. MMP9, p16, Cyclin D1, CDK4 and Rb were detected by Real-time PCR. The protein expression level of Cyclin D1 was detected by Western blot . The formation of autophagosome was examined by a transmission electron microscope. Results: After HaCaT cells were chronic exposed to arsenic for 28 weeks, malignant transformation occurred as evidenced by the formation of highly aggressive squamous cell carcinoma after inoculation into nude mice. In addition to increased secretion of MMP9, apoptotic resistance generalized and members of the inhibitor of apoptosis (IAP), including XIAP and cIAP1, were significantly elevated in arsenic-transformed cells (termed AS-TM). Furthermore, p16, Cyclin D1, CDK4, and Rb were significantly increased in AS-TM. The protein expression level of Cyclin D1 in AS-TM cells was significantly higher than that in control. More interesting, arsenic was found to induce autophagy after chronic arsenic exposure, heightened autophagosome release was observed in AS-TM. Conclusion: Apoptotic resistance, cell cycle dysregulation and activation of autophagy are the underlying mechanisms of arsenic-driven skin carcinogenesis, which provide new insight on the pathogenesis of arsenic-induced skin cancer.

exposure on skin have been documented. Arsenic was demonstrated that the exposure of arsenic in humans is associated with a marked increase in skin cancer, such as squamous cell carcinoma and basal cell carcinoma [7,8]. Studies have shown that human keratinocytes (HaCaT) can be converted into acquired malignant phenotype cells by continuous exposure to low concentrations of arsenic,which was evidenced by the formation of highly aggressive squamous cell carcinoma after inoculation into nude mice [9]. It has been recognized as a cell research model for arsenic-induced skin cancer. Due to the complex role of arsenic, although scholars have proposed some hypothesis mechanisms of arsenic carcinogenesis, the exact mechanism of arsenic carcinogenesis is still unclear.
Apoptosis, a closely regulated programmed cell death mechanism, is an essential process to maintain tissue homeostasis. Resistance to apoptotic stimuli is one of the hallmarks of cancer [10], and can be achieved by overexpression of anti-apoptotic proteins. There are several factors have been found correlated to the carcinoma cell apoptotic resistance, including members of the inhibitor of apoptosis (IAP). X-linked inhibitor of apoptosis protein(XIAP) and cellular inhibitor of apoptosis protein 1(cIAP1)were the most potent caspase inhibitor in the IAP family, they play a critical role in the anti-apoptotic and pro-survival signaling pathways. XIAP and cIAP1 are highly expressed in almost all of a series of 60 human cancer cell lines studied [11]. It remains unclear how IAPs regulate the cell cycle. The typical example is survivin, an IAP that connects antiapoptotic pathways and the cell cycle [12]. XIAP and its E3 ligase also played a crucial role in regulation of Cyclin D1 expression in cancer cells [13]. Cell cycle dysregulation is an important mechanism of tumorigenesis, the abnormal changes of the regulatory factors in the G1/S phase of the cell cycle are particularly closely related to cell carcinogenesis. The role and significance of the p16-cyclin D1/CDK4-pRb pathway in various tumors has been confirmed [14]. In recent years, Eshkoor et al [14] found that Cyclin D1, P16 are highly expressed in squamous cell carcinoma of the skin.
Autophagy is considered as an evolutionarily cell survival process, which is responsible for degradation of long-lived proteins and removal of dysfunctional organelles [15]. It seems that autophagy and apoptosis function appositely. While apoptosis is a cellular suicidal program, autophagy can either be a cell survival mechanism as a homeostatic process or a stress-induced cell death pathway depending on variable context [16,17]. High levels of XIAP and cIAP1 expression can induce the formation of autophagosomes by up-regulating Beclin 1 expression via the activation of the NFκB pathway [18]. Cyclin D1 activity can regulate autophagy and senescence in the mammary epithelium [19].
Although apoptosis, cell cycle and autophagy have been well documented, the role of apoptosis,cell cycle and autophagy in arsenic carcinogenesis and the interaction between them have not been well understood in human Keratinocytes.
Therefore, in search for crosstalk between apoptosis,cell cycle and autophagy pathways and to see how arsenic affects these processes, will help to better understand the mechanism of arsenic carcinogenesis and find new targets for clinical prevention and treatment of skin cancer.

Cell culture and arsenic exposure
The human keratinocyte cell line HaCaT (maintained in our lab) cells were cultured in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% FBS (Fetal bovine serum), 100 U of penicillin/ml, and 100 mg of streptomycin/ml.
Cultures were maintained at 37 ℃ in a humidified 5% CO2 atmosphere. For chronic arsenic exposure, cells were maintained continuously in medium containing 100 nM of arsenic trioxide (Sigma, St. Louis, MO) for 28 weeks [20,21]. All the materials for cell culture were purchased from Thermo Scientific HyClone (Logan,UT, USA).

Xenograft mouse model
To test their ability to form tumors, female BALB/c nude mice (4-6 weeks old) were randomly divided into 2 groups: those that received 1) passage-matched control

Western blotting
Western blotting was performed as described previously [20]. Cells were lysed in RIPA buffer, and protein concentrations were determined by a BCA Protein Assay Kit (Santa Cruz, CA, USA). Cyclin D1 antibody (Santa Cruz, sc-4074 ) was diluted at 1:500. Blots were normalized to β-actin (Cell Signaling Technology, 3700S).
Quantification of bands was performed using the ImageJ software (v4.18).

Transmission electron microscopy (TEM)
HaCaT and AS-TM cells were fixed in 2.5% glutaraldehyde in 0. uranyl acetate and lead citrate [22]. The formation of autophagosome was examined by a transmission electron microscope (JEM1230, JEOL, Japan).

Statistical analysis
All statistical analyses were performed using SPSS 17.0 software (SPSS Inc.,Chicago,IL, USA). Data were expressed as mean ± SD. Student's t test was used to determine the statistical differences. P<0.05 was considered statistically significant.

Low dosage of arsenic induces HaCaT cells malignant transformation
To achieve oncogenic transformation, HaCaT cells were continuously exposed to a low level (100 nM) of arsenic trioxide for 28 weeks. Our previous research [20] has confirmed that after 28 weeks of continuous arsenic exposure, the arsenic-treated cells  (Fig.1B). In addition, a marked increase in secretion of matrix metalloproteinase-9 (MMP9) from AS-TM cells occurred (Fig.   1C). Extracellular MMP9 activity was 11.5-fold higher in AS-TM cells compared to control. Thus, based on variety of criteria, it is clear that the AS-TM cells have acquired a highly malignant phenotype.

Arsenic exposure causes cell apoptotic resistance
To demonstrate the effect of arsenic on apoptosis, a TUNEL assay was applied.
we found that there were some cells with positive signals in the HaCaT control group.
However, after exposure to arsenic (100 nM) for 28 weeks, numbers of cells with positive signals obviously decreased ( Fig.2A-B). This indicated that arsenic promoted cell proliferation through apoptosis resistance.

Arsenic treatment induces high expression of anti-apoptotic proteins
To dissect arsenic's action mechanism, we further tested the expression level of XIAP and cIAP1 by immunocytochemistry. The results showed that both two anti-apoptotic proteins (XIAP and cIAP1) were highly expressed in AS-TM cells, of which XIAP was mainly expressed in cytoplasm and cIAP1 was mainly expressed in cytoplasm and nucleus (Fig.3A). The positive expression rates of XIAP and cIAP1 in AS-TM cells were significantly higher than those in HaCaT control cells (Fig.3B).
These results indicated that arsenic may induce apoptotic resistance through anti-apoptotic proteins expression.

Arsenic treatment causes cell cycle dysregulation through upregulation of p16,Rb,CDK4 and Cyclin D1
Since XIAP played a crucial role in regulation of cyclin D1 expression in cancer cells [13]. Overexpression of cIAP1 increased the phosphorylation of Rb [23]. Cyclin D1, p16 are highly expressed in squamous cell carcinoma of the skin [14]. We suspect that arsenic treatment causes cell cycle dysregulation. Consistent with the assumption, Real-time PCR results shown that compared with the control HaCaT cells (0 weeks), the mRNA expression levels of p16, Rb, CDK4 and Cyclin D1 in the arsenic treatment group at 6 weeks, 15 weeks and 28 weeks were significantly increased. The highest mRNA expression levels of P16, Rb and Cyclin D1 appeared at 6 weeks, and fell at 15 weeks and 28 weeks, but they were still significantly higher than the control group (Fig.4A). Cyclin D1, a key cell cycle regulator, is required for completion of the G 1/S transition in normal mammalian cells. It has been verified that high expression of Cyclin D1 is associated with invasion and metastasis of squamous cell carcinoma [24].

Arsenic activated autophagy in HaCaT cells
Given high levels of XIAP and cIAP1 expression induce the formation of autophagosomes [18], the central role of Cyclin D1 as an integrator of growth-promoting signals that can also have an impact on autophagy [19]. We speculate that autophagy may play a role in arsenic carcinogenesis. To test this hypothesis, we studied AS-TM cells at the ultrastructural level using transmission electron microscopy (TEM). As shown in Fig.5A, many autophagosomes, which are intracellular double-membraned vesicles characteristic of autophagy, were identified in AS-TM cells. The number of autophagosomes in the AS-TM group was significantly higher than control (Fig.5B).

Discussion
Arsenic is a common environmental pollutant, which has been studied for its toxic effects for many years. It has been well documented that chronic low-dose arsenic exposure increases proliferation and transformation of human and rodent cells [25]. However, the underlying mechanism of arsenic carcinogenicity is poorly understood. This study aimed to investigate a possible interaction between apoptosis, cell cycle, and autophagy and how the interaction relates to arsenic carcinogenesis.
After 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 (Fig.1A-B), a common form of skin cancer in arsenic-exposed humans [26], after inoculation of nude mice. Importantly, by studying this cell model system the important biomarker MMP9 for arsenic-induced malignant transformation was increased (Fig.1C). 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 [27]. Elevated expression levels of MMP9 are strongly correlated with malignant phenotype in SCC [28] and are characteristic of malignant transformation of cells [29]. Thus, the dramatically increased MMP9 observed in AS-TM cells might be an important biomarker for arsenic-induced malignant transformation in human keratinocytes.
Resistance to apoptotic stimuli is a hallmark feature of various cancers. One of the mechanisms through which tumor cells are believed to acquire resistance to apoptosis is by overexpression of inhibitor of apoptosis proteins(IAPs) [30]. XIAP and cIAP1 were the most common members of IAP, XIAP is one of the best characterized member of the IAP family in terms of its potent caspase inhibitory mechanisms and is considered as the prototype of the IAP protein family [31]. cIAP1 can directly inhibit the activity of caspase-3. It has been reported that high levels of XIAP and cIAP1 expression could induce chemo-resistance and radio-resistance of human cancers [32,33]. Thus, XIAP and cIAP1 have been postulated to contribute to the development of some tumors [34]. Our study shown that both two anti-apoptotic proteins (XIAP and cIAP1) were highly expressed in AS-TM cells (Fig.3),which may contribute to the arsenic-induced malignant transformation of HaCaT cells and the apoptotic resistance (Fig.2).
Cell cycle dysregulation is an important mechanism of tumorigenesis. During tumor progression, changes in cell damage are achieved by regulating genes involved in the G1-S phase pathway in the cell cycle [35]. The regulation involves two pathways: one is the p16-cyclin D1/CDK4-pRb pathway and the other is the p53 pathway. Studies have confirmed that the p16-cyclin D1/CDK4-pRb pathway has been shown to be abnormal in many human cancers [36]. Cyclin D1 belongs to one of the G1 phase cyclins. Cyclin D1 binds to CDK4 and CDK6, phosphorylates the downstream protein retinoblastoma protein (Rb), which releases the transcription factor E2F, promotes transcription of genes involved in DNA replication, promotes cell transition from G1 to S phase, and lead to cell proliferation [37][38][39][40]. Therefore, Cyclin D1 is an important family member of cyclins involved in cell cycle regulation, and its high expression is also thought to promote cell proliferation and promote tumor progression [41]. Eshkoor et al [14] showed that cyclin D1, p16 expression increased in squamous cell carcinoma tissues. In our study, in addition to the significant increase in XIAP expression long-term treatment of HaCaT cells with arsenic significantly increased cyclin D1 mRNA and protein expression levels ( Fig.4 A-B). The expression of CyclinD1, a gene related to cell cycle regulation, was changed during the formation of HaCaT malignant phenotype cells induced by arsenic.
The study also found that the level of Cyclin D1 mRNA expression in HaCaT cells at Autophagy is a key mechanism for cancer cells adapted to a strict environment, which is involved in cancer progression and drug resistance [42]. Apoptosis, cell cycle and autophagy are interconnected cellular processes sharing regulatory components.
Anti-apoptotic protein XIAP played a crucial role in regulation of cyclin D1 expression in cancer cells [13].Overexpression of cIAP1 increased the phosphorylation of Rb [23]. High levels of XIAP and cIAP1 expression induce the formation of autophagosomes [18], and the central role of cyclin D1 as an integrator of growth-promoting signals that can also have an impact on autophagy [19]. Therefore, the crosstalk between these pathways plays an important role in regulating physiological and pathophysiological processes. Our results indicated increases in autophagosomes caused by arsenic exposure in HaCaT cells (Fig.5 A-B), which suggest the enhancement of autophagy activity under the action of arsenic exposure.

Conclusion
In conclusion, the data from present study pointed out that arsenic-driven skin carcinogenesis involving intricate interactions between autophagy, cell cycle and apoptosis. While a low dose of arsenic can activate both processes, arsenic induce apoptotic resistance through anti-apoptotic proteins XIAP and cIAP1 expression may be an initial event in HaCaT cells and then cause cell cycle dysregulation and autophagy activation, leading to tumorigenesis (Fig.6). Therefore, apoptotic resistance, cell cycle dysregulation and autophagy activation may contribute to arsenic-induced carcinogenesis in the skin.

Acknowledgements
Not applicable.

Funding
The present study was supported by the National Natural Science Foundation (grant no. 81350023). This work was also supported by the China Scholarship Council(201806370114).

Availability of data and materials
All data generated or analyzed during this study are included in this published article.

Authors'contributions
MYG designed and supervised the present study. YYZ, XLW and YHLperformed the majority of the experiments. YYZ written the manuscript. WMZ analyzed the data.
YFW and GDP provided technical and material support. All authors have read and approved the final version of the manuscript.

Ethics approval and consent to participate
Not applicable.

Patient consent for publication
Not applicable.      Fig.6 The schematic diagram elucidates the mechanism whereby apoptotic resistance,cell cycle dysregulation and autophagy activation contribute to arsenic carcinogenesis. A low dose of arsenic treatment induce apoptotic resistance by increasing anti-apoptotic proteins XIAP and cIAP1 expression, XIAP and cIAP1 activate autophagy by regulating the p16-cyclin D1/CDK4-pRb pathway. At the same time, XIAP and cIAP1 can also directly activate autophagy, both of them ultimately leading to tumorigenesis.