ZIP4 Increases Expression of Transcription Factor ZEB1 to Promote Integrin α3β1 Signaling and Inhibit Expression of the Gemcitabine Transporter ENT1 in Pancreatic Cancer Cells

Abstract

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 non-tumor 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 small 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 PCR, 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 associated with shorter survival times. MIA PaCa-2 cells that overexpressed ZIP4 had increased resistance to gemcitabine, 5-FU, 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 of ITGA3 and ITGB1 was 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 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.

Screening of Methylation Gene Sites as Prognostic Signature in Lung Adenocarcinoma

“…In mice and pancreatic cancer cell lines, the expression of ZEB1, which activated the expression of ITGA3 and ITGB1, also increased, affecting the resistance of pancreatic cancer to chemotherapy drugs. 40 ZEB1 uniquely promotes disease progression in NSCLC. In many epithelial tumors, dysregulation of ZEB1 expression is associated with poor clinical prognosis and significantly drives EMT in the pathogenesis of LC.…”

Intraductal pancreatic cancer is less responsive than cancer in the stroma to neoadjuvant chemotherapy

“…One possible explanation is that molecular changes unique to COD cells drive their lack of response to chemotherapy. While multiple possible molecular mechanisms for therapeutic resistance have been proposed (for example, loss of TP53 function activating JAK2-STAT3 signaling [ 27 ], expression of the gemcitabine transporter ENT1 and the solute carrier protein ZIP4 [ 28 ]), there is no evidence to date that these are differentially present in intraductal cancer cells. For example, similar patterns of TP53 and SMAD4 loss are seen in intraductal cancer cells and cancer in the stroma [ 10 ].…”

Transmembrane 163 (TMEM163) Protein: A New Member of the Zinc Efflux Transporter Family

“…Very recently, using 743 nonconsanguineous Chinese patients with early onset PD, multiple novel TMEM163 variants demonstrated association with PD. Notably, one variant in particular, c.98C>G, was found to be significantly associated with PD (Li et al, 2020). There were a few limitations of this study, such as the lack of age and sex-matched controls and opting rather for gnomAD v2.1.1 data from East Asians.…”