ZIP4 Promotes Muscle Wasting and Cachexia in Mice With Orthotopic Pancreatic Tumors by Stimulating RAB27B-Regulated Release of Extracellular Vesicles From Cancer Cells

Abstract

Background & aims: Cachexia, which includes muscle wasting, is a frequent complication of pancreatic cancer. There are no therapies that reduce cachexia and increase patient survival, so it is important to learn more about its mechanisms. The zinc transporter ZIP4 promotes growth and metastasis of pancreatic tumors. We investigated its effects on muscle catabolism via extracellular vesicle (EV)-mediated stimulation of mitogen-activated protein kinase 14 (p38 MAPK).

Methods: We studied nude mice with orthotopic tumors grown from human pancreatic cancer cell lines (AsPC-1 and BxPC-3); tumors were removed 8 days after cell injection and analyzed by histology. Mouse survival was analyzed by Kaplan-Meier curves. ZIP4 was knocked down in AsPC-1 and BxPC-3 cells with small hairpin RNAs; cells with empty vectors were used as controls. Muscle tissues were collected from mice and analyzed by histology and immunohistochemistry. Conditioned media from cell lines and 3-dimensional spheroid/organoid cultures of cancer cells were applied to C2C12 myotubes. The myotubes and the media were analyzed by immunoblots, enzyme-linked immunosorbent assays, and immunofluorescence microscopy. EVs were isolated from conditioned media and analyzed by immunoblots.

Results: Mice with orthotopic tumors grown from pancreatic cancer cells with knockdown of ZIP4 survived longer and lost less body weight and muscle mass than mice with control tumors. Conditioned media from cancer cells activated p38 MAPK, induced expression of F-box protein 32 and UBR2 in C2C12 myotubes, and also led to loss of myofibrillar protein myosin heavy chain and myotube thinning. Knockdown of ZIP4 in cancer cells reduced these effects. ZIP4 knockdown also reduced pancreatic cancer cell release of heat shock protein (HSP) 70 and HSP90, which are associated with EVs, by decreasing CREB-regulated expression of RAB27B.

Conclusions: ZIP4 promotes growth of orthotopic pancreatic tumors in mice and loss of muscle mass by activating CREB-regulated expression of RAB27B, required for release of EVs from pancreatic cancer cells. These EVs activate p38 MAPK and induce expression of F-box protein 32 and UBR2 in myotubes, leading to loss of myofibrillar myosin heavy chain and myotube thinning. Strategies to disrupt these pathways might be developed to reduce pancreatic cancer progression and accompanying cachexia.

Keywords: Mouse Model; Muscle Loss; Pancreas; Signal Transduction.

Figure 1.. ZIP4 knockdown in pancreatic cancer cells reduced body weight loss in mice underwent tumor resection.

(A) AsPC-shV/shZIP4 and (B) BxPC-shV/shZIP4 cells (3 × 10⁶) were orthotopically inoculated into the nude mice. The primary tumors were removed on day 8 post injection by distal pancreatectomy/splenectomy, and five mice from each group were shown. Survival of the mice was shown in the Kaplan-Meier survival curve for (C) AsPC-shV/shZIP4 groups and (D) BxPC-shV/shZIP4 groups.

Figure 2.. Knockdown of ZIP4 reduced muscle wasting in vivo.

Mice with orthotopic xenograft tumor of AsPC-shV/shZIP4 were subjected to distal pancreatectomy/splenectomy on day 8. On day 18, mice were euthanized and muscle samples were collected for analyses. (A) The body weight data of mice in AsPC-shV/shZIP4 groups. Log-rank (Mantel-Cox) test showed significant loss of body weight in the AsPC-shV group vs. AsPC-shZIP4 group (P<0.05). (B) TA muscle mass. (C) Body weight. (D) TA muscle mass relative to mice body weight. (E) Tyrosine release from EDL. (F) H&E stained TA muscle sections. The scale bar represents 200 μm. (G) Fiber cross-sectional area (CSA). CSA of TA cross-sections was measured as described in materials and methods.

Figure 3.. ZIP4 mediates pancreatic cancer-induced muscle catabolism by activating p38MAPK in vitro.

Conditioned media were collected to test their capacity in inducing myotube catabolism. (A) ZIP4 mediates p38MAPK activation induced by conditioned media treatment with C2C12 myotubes for 1 hr. (B) ZIP4 mediates ATROGIN1 upregulation induced by conditioned media treatment with C2C12 myotubes for 8 hrs. (C) ZIP4 mediates myofibrillar protein myosin heavy chain (MHC) loss induced by conditioned media treatment with C2C12 myotubes for 72 hrs. (D) ZIP4 mediates myotube atrophy induced by conditioned media. Immunofluorescence staining of MHC was performed (bar = 100 μm), and diameter of myotubes was determined. (E) ZIP4 mediates p38MAPK activation induced by BxPC-3 cell-conditioned media in C2C12 myotubes.. (F) C2C12 myotubes were treated with BxPC-3 cell-conditioned media for 8 hrs.

Figure 4.. ZIP4 mediates pancreatic cancer-induced muscle catabolism by activating p38MAPK in vivo.

(A) Conditioned media was collected from methylcellulose (MC) based hanging drop 3D cancer spheroids and the classical matrigel based 3D organoid culture from AsPC-shV/shZIP4 cells, then added onto C2C12 myotube cells for 1 hr to check the activation of p38. (B) C2C12 myotubes were treated with the 3D organoid culture media for 8 hrs to check the induction of UBR2. (C) Mice with orthotopic xenograft of AsPC-shV/shZIP4 cells were subjected to distal pancreatectomy/splenectomy on day 8. On day 18, TA lysate was analyzed by western blot analysis for p38MAPKactivity and ATROGIN1 expression. Data are expressed as means ± SE and analyzed by ANOVA. * denotes a difference (P<0.05) from control and # denotes a difference from AsPC-shV (P<0.05)

Figure 5.. ZIP4 promotes p38MAPK-mediated muscle catabolism through increasing pancreatic cancer cell release of HSP70 and HSP90-positive EVs.

(A) AsPC-1 cells release higher levels of HSP70 and HSP90 in a ZIP4-dependent manner. HSP70 and HSP90 levels in cell conditioned media of indicated cells were measured by ELISA. (B) AsPC-1 cells release higher levels of EVs in a ZIP4-dependent manner as measured by AchE activity. (C) EVs isolated from conditioned media of AsPC-1 cells activates p38MAPK-mediated catabolic response in C2C12 myotubes in a ZIP4-dependent manner. C2C12 myotubes were treated with EVs isolated from AsPC-1 cells and analyzed by western blotting in 1 hr (p38MAPK) or 8 hrs (UBR2 and ATROGIN1). (D) Serum HSP90 levels in nude mice with orthotopic xenograft of AsPC-1 cells are reduced by ZIP4 knockdown. Serum HSP90α was analyzed by ELISA from nude mice grafted with AsPC-shV/shZIP4 cells on day 27 (n = 8 and 7, respectively). * denotes P<0.05.

Figure 6.. ZIP4 is critical to CREB-regulated RAB27B expression and EV release by pancreatic cancer cells.

(A) ZIP4 knockdown suppresses RAB27B expression in AsPC-1 cells. RAB27B expression was analyzed by both western blotting and qPCR. (B) ZIP4 knockdown suppresses RAB27B expression in BxPC-3 cells. (C) ZIP4 knockdown decreases HSP70 and HSP90-positive EV release. EVs were isolated from conditioned media of AsPC-shV/shZIP4 cells and analyzed by western blotting for multiple EV markers. Ponseau S staining was used as control for equal loading. (D) ZIP4 knockdown attenuates CREB binding to the RAB27B promoter. ChIP assay was conducted to analyze CREB binding in AsPC-shV/shZIP4 cells to two potential CREB binding motifs (bold letters) identified in the human RAB27B promoter by database search at JASPAR and ConSite. Homologous nucleotides in the binding motifs in mouse RAB27B promoter are shown in red.

Figure 7.. RAB27B mediates ZIP4 stimulation of HSP70 and HSP90-positive EV release by pancreatic cancer cells.

AsPC-shV/shZIP4 cells were transduced with lentivirus encoding RAB27B as described in Materials and Methods. (A) Overexpression of RAB27B restores high-level HSP70 and HSP90 release by ZIP4-knockdown AsPC-shZIP4 cells. RAB27B overexpression was verified by western blotting (left panel). HSP70 and HSP90 release was measured by ELISA (right panel). (B) Overexpression of RAB27B restores high-level EV release by AsPC-shZIP4 cells. EV release levels are shown in left panel. EV markers, HSP70, HSP90 and RAB27B content in isolated EVs were examined by western blotting (middle and right panel). (C) Schematic diagram of the mechanism for ZIP4 promoting pancreatic cancer-associated muscle wasting by stimulating RAB27B-mediated release of HSP70 and HSP90-positive EVs.