BACKGROUND & AIMS Pancreatic tumors undergo rapid growth and progression, become resistant to chemotherapy, and recur after surgery. We studied the functions of the solute carrier family 39 member 4 (SLC39A4, also called ZIP4), which regulates concentrations of intracellular zinc and is increased in pancreatic cancer cells, in cell lines and mice. METHODS We obtained 93 pancreatic cancer specimens (tumor and adjacent nontumor tissues) from patients who underwent surgery and gemcitabine chemotherapy and analyzed them by immunohistochemistry. ZIP4 and/or ITGA3 or ITGB1 were overexpressed or knocked down with short hairpin RNAs in AsPC-1 and MIA PaCa-2 pancreatic cancer cells lines, and in pancreatic cells from KPC and KPC-ZEB1-knockout mice, and pancreatic spheroids were established; cells and spheroids were analyzed by immunoblots, reverse transcription polymerase chain reaction, and liquid chromatography tandem mass spectrometry. We studied transcriptional regulation of ZEB1, ITGA3, ITGB1, JNK, and ENT1 by ZIP4 using chromatin precipitation and luciferase reporter assays. Nude mice were given injections of genetically manipulated AsPC-1 and MIA PaCa-2 cells, and growth of xenograft tumors and metastases was measured. RESULTS In pancreatic cancer specimens from patients, increased levels of ZIP4 were associated with shorter survival times. MIA PaCa-2 cells that overexpressed ZIP4 had increased resistance to gemcitabine, 5-fluorouracil, and cisplatin, whereas AsPC-1 cells with ZIP4 knockdown had increased sensitivity to these drugs. In mice, xenograft tumors grown from AsPC-1 cells with ZIP4 knockdown were smaller and more sensitive to gemcitabine. ZIP4 overexpression significantly reduced accumulation of gemcitabine in pancreatic cancer cells, increased growth of xenograft tumors in mice, and increased expression of the integrin subunits ITGA3 and ITGB1; expression levels of ITGA3 and ITGB1 were reduced in cells with ZIP4 knockdown. Pancreatic cancer cells with ITGA3 or ITGB1 knockdown had reduced proliferation and formed smaller tumors in mice, despite overexpression of ZIP4; spheroids established from these cells had increased sensitivity to gemcitabine. We found ZIP4 to activate STAT3 to induce expression of ZEB1, which induced expression of ITGA3 and ITGB1 in KPC cells. Increased ITGA3 and ITGB1 expression and subsequent integrin α3β1 signaling, via c-Jun-N-terminal kinase (JNK), inhibited expression of the gemcitabine transporter ENT1, which reduced gemcitabine uptake by pancreatic cancer cells. ZEB1-knockdown cells had increased sensitivity to gemcitabine. CONCLUSIONS In studies of pancreatic cancer cell lines and mice, we found that ZIP4 increases expression of the transcription factor ZEB1, which activates expression of ITGA3 and ITGB1. The subsequent increase in integrin α3β1 signaling, via JNK, inhibits expression of the gemcitabine transporter ENT1, so that cells take up smaller amounts of the drug. Activation of this pathway might help mediate resistance of pancreatic tumors to chemotherapeutic agents.

中文翻译：

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ZIP4增加转录因子ZEB1的表达，以促进整合素α3β1信号传导，并抑制胰腺癌细胞中吉西他滨转运蛋白ENT1的表达。

背景与目的胰腺肿瘤经历快速的生长和发展，对化学疗法产生抗药性，并在手术后复发。我们研究了溶质载体家族39成员4（SLC39A4，也称为ZIP4）的功能，该成员调节细胞内锌的浓度并在胰腺癌细胞，细胞系和小鼠中增加。方法我们从接受手术和吉西他滨化疗的患者中获得了93份胰腺癌标本（肿瘤和邻近的非肿瘤组织），并通过免疫组织化学对其进行了分析。ZIP4和/或ITGA3或ITGB1在AsPC-1和MIA PaCa-2胰腺癌细胞系中以及在KPC和KPC-ZEB1基因敲除小鼠的胰腺细胞中被短发夹RNA过度表达或敲低，并建立了胰腺球体。通过免疫印迹分析细胞和球状体，逆转录聚合酶链反应，以及液相色谱串联质谱。我们使用染色质沉淀和荧光素酶报告基因检测技术研究了ZIP4对ZEB1，ITGA3，ITGB1，JNK和ENT1的转录调控。给裸鼠注射基因操纵的AsPC-1和MIA PaCa-2细胞，并测量异种移植肿瘤和转移的生长。结果在患者的胰腺癌标本中，ZIP4水平升高与生存时间缩短有关。过度表达ZIP4的MIA PaCa-2细胞对吉西他滨，5-氟尿嘧啶和顺铂的耐药性增加，而具有ZIP4抑制作用的AsPC-1细胞对这些药物的敏感性增加。在小鼠中，由具有ZIP4抑制作用的AsPC-1细胞生长的异种移植肿瘤较小，对吉西他滨更敏感。ZIP4的过表达显着减少了吉西他滨在胰腺癌细胞中的积累，增加了小鼠异种移植肿瘤的生长，并增加了整联蛋白亚基ITGA3和ITGB1的表达。ZIP4敲低的细胞中ITGA3和ITGB1的表达水平降低。尽管ZIP4过表达，但具有ITGA3或ITGB1抑制作用的胰腺癌细胞在小鼠中的增殖减少并形成了较小的肿瘤。由这些细胞建立的球体对吉西他滨的敏感性增加。我们发现ZIP4激活STAT3以诱导ZEB1的表达，从而诱导KPC细胞中ITGA3和ITGB1的表达。ITGA3和ITGB1表达的增加以及随后的整联蛋白α3β1信号通过c-Jun-N-末端激酶（JNK）抑制了吉西他滨转运蛋白ENT1的表达，减少了胰腺癌细胞对吉西他滨的摄取。ZEB1基因敲低细胞对吉西他滨的敏感性增加。结论在胰腺癌细胞系和小鼠的研究中，我们发现ZIP4增加了转录因子ZEB1的表达，而转录因子ZEB1激活了ITGA3和ITGB1的表达。随后通过JNK整合素α3β1信号转导的增加抑制了吉西他滨转运蛋白ENT1的表达，因此细胞吸收的药物量较小。该途径的激活可能有助于介导胰腺肿瘤对化学治疗剂的抗性。随后通过JNK整合素α3β1信号转导的增加抑制了吉西他滨转运蛋白ENT1的表达，因此细胞吸收的药物量较小。该途径的激活可能有助于介导胰腺肿瘤对化学治疗剂的抗性。随后通过JNK整合素α3β1信号转导的增加抑制了吉西他滨转运蛋白ENT1的表达，因此细胞吸收的药物量较小。该途径的激活可能有助于介导胰腺肿瘤对化学治疗剂的抗性。