ZIP7 Maintains Colon Cancer Radioresistance by Regulating Zinc-dependent Epithelial-mesenchymal Transition


 Background: Irradiation-induced radioresistance often leads to the therapeutic failure of colon cancer. By regulating the redistribution of intracellular zinc, ZIP7 plays a dominating role in the activation of many critical signaling pathways in the progression of tumors. However, the relationship between ZIP7 and radioresistance is still unclear. Methods: ZIP7 expression of colon cancer was evaluated by analyzing public data from the GEPIA and CPTAC databases and validated based on immunohistochemistry (IHC) staining. Clonogenic survival assay was employed to examine the influence of intracellular zinc interference and ZIP7 knockdown on radioresistance of colon cancer, based on radioresistant colon cancer cells. At last, Western blot was performed to preliminarily explore the potential mechanisms. Results: ZIP7 was significantly upregulated in human colon cancers compared with adjacent normal tissues. ZIP7 knockdown could significantly reduce the radioresistance of colon cancer cells, while transmembrane ionophore of zinc could partially reverse this effect. In terms of mechanisms, ZIP7 knockdown significantly reversed the radiation-induced expression of elevated ZEB1 and down-regulated E-cadherin through regulating zinc. Conclusion: ZIP7 is crucial to maintain radioresistance of colon cancer cells through regulation of zinc distribution, and at least partially by maintenance of epithelial-mesenchymal transition (EMT).


Introduction
Colon cancer is one of the most commonly diagnosed malignancies, which severely threatens public health. [1] According to the latest data of the United States, the annual age-standardized colon cancer incidence rate was 37.6 per 100,000 persons, and about one third of which died from this disease. [2] Radiotherapy is an irreplaceable part of systemic therapy for solid tumors, including colon cancer. [3,4] However, the intrinsic and irradiation-induced radioresistance often lead to the failure of treatment, while the mechanisms and coping strategies need to be further studied. [3,5] Zinc is the second most abundant indispensable trace nutrient for life, and is an important signal mediator of cells, including cancer cells.
[6] ZIP (ZRT, IRT-like protein) family, the channels facilitating the in ux of zinc into the cytosol, are the critical proteins to initiate the role of zinc to act as the second messenger.
[6] Zinc dysregulation and ZIPs abnormal expressions are related to the malignant biological behaviors of various tumors, and are considered as potential therapeutic targets. [7] For esophageal cancer, high expression of ZIP5 or ZIP6 were highly responsible for poor prognosis, and down-regulation of these gene expressions signi cantly inhibited the progression of the disease. [8,9] ZIP4 was signi cantly overexpressed in pancreatic and nasopharyngeal cancer tissue, which was implicated in chemoresistance and radioresistance. [10,11] ZIP7, regulating the redistribution of intracellular zinc, is a special member of the ZIPs family and just localized inside the cells.
[6] Analysis of the information of the Oncomine database showed that, ZIP7 is one of the 10% of genes overexpressed in breast cancer patients with poor prognosis. [12] Taylor, et al. found that ZIP7 was essential for the activation of growth factor receptors, and removal of ZIP7 may be an effective strategy to prevented anti-hormone resistance in breast cancer. [13] In cervical and gastric cancer, downregulated the expression of ZIP7 signi cantly inhibited the proliferation, migration of tumor cells, but promoted apoptosis. [14,15] However, the relationship between ZIP7 and colon cancer, especially in regard to radiotherapy, is still unclear.
In this study, the role of ZIP7 in colon cancer radioresistance and the preliminarily relevant mechanisms were explored. We found that ZIP7 was signi cant to maintain radioresistance of colon cancer cells through regulation of zinc distribution, and at least partially by maintenance of epithelial-mesenchymal transition (EMT).
[16] UALCAN (http://ualcan.path.uab.edu/) is an integrated platform based on multi-omics data and clinic-pathological pro les. [17] In the present study, the GEPIA website and the CPTAC database, a sub-database of UALCAN, were respectively employed to explore the mRNA and proteomic levels of ZIP7 in colon cancer and adjacent normal tissues.

Tissue samples
Fifty pairs of colon cancer tissues and the paired para-tumor normal colon tissues were obtained from surgical procedures from the Wuxi People's Hospital A liated to Nanjing Medical University (Wuxi, China) between 2017 and 2018 with the consent of all patients. This study was approved by the Ethics Committee of Wuxi People's Hospital A liated to Nanjing Medical University.

Radioresistant HCT116 cells establishment
HCT116 cells were plated in 10-cm culture dishes with medium supplemented with 10% FBS (HyClone, UT, USA). After 24h culture, the cells were exposed to a signal dose of 6 Gy X-rays at a dose rate of 500 cGy/min. After 2 weeks of incubation, the colonies were digested and replated, and then the above radiotherapy process was repeated. The cells undergone two cycles of radiotherapy were named P2 HCT116.

Clonogenic survival assay
The cells were plated into six-well plates at a density of 300-3,000 cells/well depending on the dose of radiation. The application concentration of TPEN and pyrithione (Py) were 3 µmol/L and 500 nmol/L, respectively. And then, the cells were irradiated with 0, 1, 2, or 4 Gy X-ray. After the irradiation, the cells were incubated for 10-14 days. And then crystal violet staining was carried out. Colonies consisting of 50 or more cells were counted as a clone. Survival fraction was calculated as: (number of colonies / number of cells plated) irradiated / (number of colonies/number of cells plated) control .

Lentivirus construction and transfection
Lentiviruses carrying shRNA gene sequence targeting ZIP7 and green uorescent protein gene sequence were designed and constructed by Hanbio Biotechnology Co., Ltd. (Shanghai, China). The targeting sequence were 5'-CCACAATGACTGTCCTGCTACATGA-3' for the No. 1 lentivirus (Sh -ZIP7 #1), 5'-GCCTTTCTTGTCGTGGAGAAA-3' for the No. 2 lentivirus (Sh -ZIP7 #2), and 5'-TTCTCCGAACGTGTCACGT-3' for the control lentivirus (Sh-Con). HCT116 cells were plated into six-well plates at a density of 10 4 cells/well, and were transfected with the above lentiviruses at a multiplicity of infection (MOI) value of 20. The polybrene at the concentration of 6 ug/ml was used during lentivirus transfection. At last, Western blot was carried out to verify the effectiveness of gene knockdown.

Western blot analysis
According to the instructions of the BCA kit (Beyotime Biotechnology, Shanghai, China), the total cell proteins were collected. Proteins were separated by SDS-PAGE and blotted onto a nitrocellulose membrane, following incubation with the speci c primary antibodies, and then peroxidase-conjugating corresponding secondary antibodies. At last, the bands were visualized by chemiluminescence and were analyzed by Image J. Anti-ZIP7 (1:1000 dilution, Cat. ab117560, Abcam), ZEB1 (1:500 dilution, Cat. ab203829, Abcam), E-cadherin (1: 1000 dilution, Cat. 3195S, CST), GAPDH (1:1000 dilution, Cat. 2118S, CST) were applied as primary antibodies. For Py intervention experiment, the cells were treated with Py or control medium for 48h before protein collection.

Statistical analysis
The data were exhibited as mean ± standard deviation (SD) and analyzed by SPSS19.0 software (V19.0 for Windows; SPSS, Inc., Chicago, IL, USA). Statistical signi cance was determined via a one-way analysis of the variance (ANOVA), and the Student's t-test was used when there were two groups. The sensitizer enhancement ratios (SER) were measured according to the multi-target single hit model. P < 0.05 was considered statistically signi cant, and P < 0.01 and P < 0.001 were considered highly signi cant.

ZIP7 was upregulated in human colon cancers
To explore the correlation between ZIP7 and colon cancer, the expression levels of ZIP7 in colon cancer and control normal tissues were explored using public databases and IHC staining. The mRNA expression analysis of the GEPIA database showed that ZIP7 was overexpressed in colon cancer compared with adjacent normal colon tissues (P < 0.001, Fig. 1A). Moreover, the analysis of the CPTAC database suggested that ZIP7 protein expression was also upregulated in colon cancer tissues (P < 0.001, Fig. 1B). The result of IHC staining was consistent with the GEPIA database, and the immunoreactivity score (IRS) of ZIP7 in colon cancer tissues was 5.57 ± 2.75, signi cantly higher than 2.31 ± 1.40 in paired normal tissues (P < 0.001, Fig. 1C-D).

Colon cancer radioresistance was related to intracellular zinc
In order to identify the in uencing factors of colon cancer radioresistance, the radioresistant HCT116 cells were constructed based on the method of desensitization by repeated radiotherapy (Fig. 2A). The results of clonogenic survival assay showed that, the survival fractions of all cells decreased in a dosedependent manner, and the radioresistance of P2 HCT116 was signi cantly higher than that of parental cells (Fig. 2B). The mean lethal doses (D 0 ) for the parent cell and P2 HCT116 were 1.34 and 1.88 Gy, and the quasithreshold doses (D q ) were 0.39 and 0.10 Gy, respectively. The SER was 0.71 for desensitization radiotherapy.
Then, the relationship between radioresistance of colon cancer and zinc was studied by application of TPEN, a transmembrane zinc chelator. As shown in Fig. 2C, TPEN

ZIP7 maintainscolon cancer radioresistance through regulation of zinc
To investigate the impacts of ZIP7 on colon cancer radioresistance, ZIP7 knockdown colon cancer cells were established by lentivirus mediated RNA interference technology based on P2 HCT116. As shown in Fig. 3A, the infection e ciencies were more than 90% in both targeting and control groups. The following results of western blot showed that, lentivirus with ZIP7 shRNA (sh-ZIP7) sequence could signi cantly reduce the expression of ZIP7 (P < 0.01, Fig. 3B-C). Further results of clonogenic survival assay showed that, ZIP7 knockdown signi cantly reduced the radioresistance of P2 HCT116, which effect could be partially, but signi cantly reversed by Py, a transmembrane ionophore of zinc (Fig. 3D). Meanwhile, we found that the Py, at the corresponding concentration, had no effect on the radioresistance of the control P2 HCT116 cells (Fig. 3B). The D 0 of sh-Con, sh-Con + Py, sh-ZIP7 and sh-ZIP7 + Py group were 1.86, 1.78, 1.23 and 1.49 Gy, while the D q of each group were 1.04, 1.23, 0.41, and 0.57, respectively. The D 0 and D q of sh-ZIP7 group were the lowest. The SER was 1.51 for ZIP7 knockdown P2 HCT116 to sh-Con cells, and was 0.83 for Py intervention in ZIP7 knockdown P2 HCT116 cells.

ZIP7 maintains colon cancer EMT through regulation of zinc
The occurrence of EMT plays an important role in tumor radioresistance. In order to preliminarily explore the mechanisms of colon cancer acquired radioresistance regulated by ZIP7, the expressions of relating proteins were detected by Western blot. As Fig. 4A showed that, desensitization radiotherapy signi cantly upregulated the expression of ZIP7 and ZEB1, and the gray values of ZIP7 and ZEB1 in P2 HCT116 were about 1.85 and 2.08 times of that in parental cells, respectively (P < 0.01). Contrarily, the expression of Ecadherin was signi cantly downregulated in P2 HCT116, and the relative gray value was just about 71% of that in parental cells (P < 0.01). Further study showed that, ZIP7 knockdown signi cantly downregulated the expression of ZEB1 by 86% in P2 HCT116, while upregulated the expression of Ecadherin 1.57 times (P < 0.01, Fig. 4B). More meaningfully, Py treatment partially reversed the expression changes of the above protein causing by ZIP7 knockdown (P < 0.01, Fig. 3B). The relative gray value of ZEB1 for ZIP7 knockdown alone group was just about 20% of that for ZIP7 knockdown plus Py treatment group, while E-cadherin expression was 1.73 times higher than that for ZIP7 knockdown plus Py treatment group.

Discussion
Radioresistance is still a huge obstacle of colon cancer treatment, although the effectiveness of some new radiotherapy technologies, such as selective internal radiotherapy and stereotactic body radiotherapy, have been con rmed. [4,18,19] The exploration of radioresistance mechanisms could provide strategies for improving the e cacy of radiotherapy. [5] The results of information analyses of databases and IHC staining showed that ZIP7 was highly expressed in colon cancer tissues, indicating it may be a therapeutic target.
Zinc is an important signal mediator in the progression of various tumors, and plays key roles in many malignant biological behaviors. Cheng et al. con rmed that zinc supplementation could signi cantly increase the metastasis and invasion abilities of esophageal cancer cells, which effects were effectively reversed by zinc chelator TPEN. [20] And these phenomena may attribute to zinc activated PI3K/AKT and MAPK/ERK signaling pathway. [20] In addition, it has been reported that TPEN could cause cell death in a dose-dependent manner by inducing oxidative stress and inhibiting autophagy, and zinc application completely reversed this kind of death. [21] For tumor radiotherapy, autophagy plays a cytoprotective role. In glioma cells, the chelation of intracellular zinc blocked the lysosomal degradation of autophagic vesicles, and further reduced the survival fraction of cells in radiotherapy. However, the equivalent dose of TPEN has no effect on glioma cells without radiotherapy. [22] In this study, we found that low concentration TPEN signi cantly reversed the radioresistance of progeny colon cancer cells, which provides a theoretical basis for the application of TPEN in colon cancer radiotherapy.
The impact of ZIP7 on some malignant biological behaviors of cancer has been reported, including breast cancer, cervical cancer and gastric cancer. [13][14][15] Sheng et al. found that downregulation of ZIP7 signi cantly inhibited cell growth and induced apoptosis in colon cancer cells. [23] However, little is known about the role of this molecule in cancer radioresistance. In this study, knockdown ZIP7 was found could signi cantly reduce the survival ability of radioresistant colon cancer cells after irradiation, while transmembrane ionophore of zinc partially reversed this effect. This result indicated that ZIP7 kept colon cancer radioresistance through regulation of zinc.
ZIP7 has been considered to be "a hub for tyrosine kinase activation". [12] In the progression of cancer, some major pathways, such as MAPK, PI3K-AKT and mTOR, could be actived by ZIP7-mediated zinc release from intracellular stores. [24] Other members of ZIPs family have also been con rmed to promote tumor development through the above signaling pathways. [7,20] In addition, ZIP7 plays an important role in maintenance the function state of endoplasmic reticulum (ER). Knockdown or knockout the expression of ZIP7 could induce ER stress and impact protein transport. [25][26][27] Therefore, the in uence of ZIP7 on colon cancer radioresistance, as veri ed in this study, may be completed through a complex network.
For basic mechanisms, EMT acting as an essential process involved in acquired radioresistance has been widely accepted.
[28] Irradiation can induce the occurrence of EMT through many signaling pathways. [28] During EMT, not only the interactions of cell-cell and cell-extracellular matrix are remodeled, but also the cancer stem cell properties, enhanced DNA damage response and antioxidant activity abilities are obtained. [29,30] In this study, we found that irradiation induction promoted ZIP7 and ZEB1 expression, and the occurrence of EMT. Knockdown of ZIP7 could downregulate ZEB1 expression, but upregulate Ecadherin expression, and both of which could be partially reversed by Py treatment. These results indicated that ZIP7, through regulation of zinc, kept colon cancer radioresistance at least partially by maintenance of EMT. Therefore, ZIP7 may be a novel druggable node in radiosensitization treatment of colon cancer, and this could be possible because a small molecule inhibitor of ZIP7 has been developed. [31]

Conclusions
To sum up, ZIP7 was upregulated in human colon cancers compared with adjacent normal tissues. Irradiation induction increased the radioresistance of colon cancer, which could be reversed by chelation of intracellular zinc and ZIP7 knockdown. In terms of mechanisms, the keeping role of ZIP7 on colon cancer radioresistance at least partially attributing to maintenance of EMT. Consequently, ZIP7 may be a novel target in radiosensitization treatment of colon cancer.     Western blot analyses of ZIP7, ZEB1 and E-cadherin protein levels in sh-ZIP7 and sh-Con P2 HCT116 cells. n = 3, sh-ZIP7 vs sh-Con * P < 0.01, * * P < 0.001, sh-ZIP7 vs sh-ZIP7+Py #P < 0.01.