Pancreatic Cancer-Derived Exosomes Cause Paraneoplastic β-cell Dysfunction

Abstract

Purpose: Pancreatic cancer frequently causes diabetes. We recently proposed adrenomedullin as a candidate mediator of pancreatic β-cell dysfunction in pancreatic cancer. How pancreatic cancer-derived adrenomedullin reaches β cells remote from the cancer to induce β-cell dysfunction is unknown. We tested a novel hypothesis that pancreatic cancer sheds adrenomedullin-containing exosomes into circulation, which are transported to β cells and impair insulin secretion.

Experimental methods: We characterized exosomes from conditioned media of pancreatic cancer cell lines (n = 5) and portal/peripheral venous blood of patients with pancreatic cancer (n = 20). Western blot analysis showed the presence of adrenomedullin in pancreatic cancer-exosomes. We determined the effect of adrenomedullin-containing pancreatic cancer exosomes on insulin secretion from INS-1 β cells and human islets, and demonstrated the mechanism of exosome internalization into β cells. We studied the interaction between β-cell adrenomedullin receptors and adrenomedullin present in pancreatic cancer-exosomes. In addition, the effect of adrenomedullin on endoplasmic reticulum (ER) stress response genes and reactive oxygen/nitrogen species generation in β cells was shown.

Results: Exosomes were found to be the predominant extracellular vesicles secreted by pancreatic cancer into culture media and patient plasma. Pancreatic cancer-exosomes contained adrenomedullin and CA19-9, readily entered β cells through caveolin-mediated endocytosis or macropinocytosis, and inhibited insulin secretion. Adrenomedullin in pancreatic cancer exosomes interacted with its receptor on β cells. Adrenomedullin receptor blockade abrogated the inhibitory effect of exosomes on insulin secretion. β cells exposed to adrenomedullin or pancreatic cancer exosomes showed upregulation of ER stress genes and increased reactive oxygen/nitrogen species.

Conclusions: Pancreatic cancer causes paraneoplastic β-cell dysfunction by shedding adrenomedullin(+)/CA19-9(+) exosomes into circulation that inhibit insulin secretion, likely through adrenomedullin-induced ER stress and failure of the unfolded protein response.

Figure 1. PC sheds CA19-9 and AM-containing exosomes that readily enter β-cells

(A) NanoTracker analysis (average of 5, 60 second movies) of size distribution of microparticles isolated from conditioned medium of a PC patient–derived cell line (left), PANC-1 cell line (center), and plasma from a PC patient (right). Red bars indicate ±1 standard error of the mean. (B) Western blot for CA19-9 in 15 µg of exosomes from HUVEC and HPDE (controls), and PC patient plasma (PC1-5). (C–D) Western blot probing for AM in 20 µg of exosomes. (C) PC-Exo isolated from PANC-1 and HPDE cell lines and PC1-PC4 (PC patient–derived xenograft cell lines). (D) PC-Exo isolated from peripheral venous blood (pev) or portal venous blood (pov) of PC patients. (E) Electron microscopy of exosomes isolated from PANC-1 cell line. Scale bar, 200 µm. (F) Confocal microscopy after co-incubation for 48 hours of PKH67-dyed exosomes from the PANC-1 cell with human islet cells. Scale bar, 50 µm.

Figure 2. PC-Exosomes decrease insulin secretion with the effect abrogated by ADMR blockade

Glucose-stimulated insulin secretion in INS-1 cells (A, C–E) and human islets (B) co-incubated with PC-Exo (+Exo) compared to control culture medium (-Exo), PC-Exo with ADMR inhibitor (+Exo + AM inhib) and PC-conditioned medium depleted of exosomes (Exo-free medium). Exosomes were isolated from a PC patient–derived cell line (A–B), PANC-1 (C), and plasma from 2 PC patients (D–E). (F) Insulin secretion dose response curve with increasing amounts of PC-Exo in INS-1 cells. * P<.05; ** P<.001; *** P<.0001.

Figure 3. PC-Exosomes deliver AM to INS-1 cells and increase AM/ADMR interaction with the effect abrogated by ADMR blockade

(A–B) The Duolink Assay System was used to assess the interaction of AM/ADMR in INS-1 cells treated with increasing amounts (0, 5, 10, or 15 µl) of PKH67-dyed PC-Exo in the absence (A) and presence (B) of AM 22–52, an ADMR antagonist. Scale bar, 10 µm.

Figure 4. Pharmacological inhibition of the endocytic pathway blocks exosome uptake into INS-1 cells

(A) INS-1 cells were treated with varying concentrations of Amiloride (25, 50, and 100 µM) for 15 minutes. PANC-1 exosomes were dyed with PKH67 and incubated with the cells after Amiloride treatment for 5 hours to allow exosome uptake. Scale bars represent 10 µm. (B) Zoom images (10.5X) of exosome internalization into INS-1 cells in the presence or absence of 100 µM Amiloride. Scale bar represents 5 µm. (C) Quantification of green fluorescence/cell in all treatment groups. (D) INS-1 cells were treated with 25 µg/ml or 50 µg/ml Nystatin for 30 minutes. PKH67-dyed exosomes were incubated with the cells for 5 hours to allow internalization. Scale bars represent 10 µm. (E) Zoom images (10.5X) of cells treated with 50 µg/ml Nystatin compared to control (untreated) cells show decreased PKH67-dyed exosome internalization. Scale bar represents 5 µm. (F) Green fluorescence quantification of internalized exosomes in the presence of absence of Nystatin. ***, P<.0001.

Figure 5. AM and PC-Exosomes increase expression of ER stress markers

(A–C) mRNA expression of ER stress markers Bip (A), Chop (B), and Xbp-1s (C) in INS-1 cells after treatment with varying concentrations of AM peptide, or peptide + AM inhibitor (AM 22–52). (D–E) mRNA expression of ER stress markers Bip (D) and Chop (E) in INS-1 cells after treatment with 50 µg of PC-Exo (+Exo) from a PC patient–derived cell line compared with control culture medium (-Exo). (F) Percentage of apoptotic cells determined by annexin V staining of INS-1 cells treated with 50 µg of PC-Exo (+Exo) compared to control (untreated) cells (-Exo). (G) Western blot for pro-insulin protein levels with the addition of AM peptide (1, 20, or 40 pM AM) vs. untreated INS-1 cells. * P<.05; ** P<.001; *** P<.0001.

Figure 6. AM-containing PC-Exosomes can increase Bip/pro-insulin interactions in β-cells

(A)The Duolink Assay System was used to assess Bip/pro-insulin interactions with increasing amounts (0, 5, 10, or 15 µl) of PKH67-dyed PC-Exo. Scale bar represents 10 µm. (B) The potential mechanism for exosomal AM release and signaling differs from the conventional mechanism shown in 1) in which AM binds to cell surface AM receptors (ADMRs) and activates the cAMP-dependent pathway. Instead PC-Exo internalize through either caveolin-mediated endocytosis or macropinocytosis thus fusing to the early endosome which is also the site of endocytosed ADMRs (2). The early endosome/multivesicular body could be another site of AM/ADMR interaction. Once the pathway is activated adenyl cyclase activation of cAMP releases the catalytic subunits of PKA, which translocate to the nucleus and subsequently activates insulin gene transcription. Excessive exosomal AM activates ER stress proteins that regulate degradation of misfolded or unfolded ER proteins. However, overproduction of insulin due to excessive AM pathway activation eventually leads to failure of the UPR marked by increase Bip/proinsulin coupling in the ER, increased ROS/RNS production, and an increase in Chop, an inducer of apoptosis, leading to a decrease in insulin secretion due to β-cell death.