Non-canonical ALK7 pathways promote pancreatic cancer metastasis through β-catenin/MMP-mediated basement membrane breakdown and intravasation

Abstract

Breaching the vascular barrier is a critical step in pancreatic ductal adenocarcinoma (PDAC) metastasis, yet the mechanisms enabling this process remain incompletely understood. Transforming growth factor beta (TGFβ) receptors have been extensively studied in many cancer types. However, activin receptor-like kinase 7 (ALK7), one of the TGFβ receptors, is under-investigated, and its roles in PDAC metastasis have been unclear. This study identifies two distinct but interconnected ALK7-driven non-canonical pathways that promote PDAC dissemination. The ALK7-β-catenin-EMT axis enhances intrinsic tumor cell motility, driving epithelial-mesenchymal transition (EMT). In parallel, the ALK7-β-catenin-MMP axis facilitates metastatic invasion by upregulating MMP production, leading to ECM degradation and invadosome formation, which promote vascular barrier breakdown and intravasation. An orthotopic PDAC metastasis model reveals that both pharmacological and genetic ALK7 inhibition suppresses metastasis. 3D microfluidic vessel-on-chip platforms further demonstrate that ALK7 inhibition preserves basement membrane (BM) integrity, limiting intravasation. While MMP inhibition effectively blocks BM breakdown and intravasation, extravasation remains unaffected, highlighting distinct molecular requirements for different metastatic stages. These findings establish ALK7 as a dual-function pro-metastatic regulator that orchestrates both tumor cell plasticity and ECM remodeling, positioning ALK7 inhibition as a promising strategy to target early metastatic dissemination in PDAC.

Keywords: ALK7; Intravasation; Matrix metalloproteinases; Organ-on-a-chip; Pancreatic cancer.

Fig. 1

Pharmacological ALK7 blockade inhibits spontaneous PDAC metastasis in an orthotopic PDAC model in vivo. (A) A schematic of the spontaneous metastatic processes. (B) A schematic of the orthotopic PDAC spontaneous metastasis experiments with the SB431542 treatment in mice. Orthotopic PDAC tumor-bearing mice were prepared. After 1 week of tumor inoculation, DMSO or SB431542 (25 mg/kg/day, intraperitoneal (i.p.) injection) was treated for 2 weeks. (C) Representative IVIS images of PDAC tumor-bearing mice treated with DMSO or SB431542 (images at week 3). (D) Quantification of the bioluminescent signal from the luciferase-expressing tumors in PDAC tumor-bearing mice treated with DMSO or SB431542 at weeks 1, 2, and 3 after tumor inoculation (DMSO, n = 9; SB431542, n = 9). (E) Quantification of the weight of excised primary tumors obtained from the mice treated with DMSO or SB431542 (tumor excised at week 3). (F) Representative IVIS images of metastatic PDAC cells in the livers after treatment with SB431542 or DMSO (organs obtained at week 3). (G) Quantification of the bioluminescent signal from the luciferase-expressing PDAC cells metastasized to the livers of PDAC tumor-bearing mice treated with DMSO or SB431542. * p = 0.011, two-tailed unpaired Student t-test. (H) Representative IVIS images of metastatic PDAC cells in the lungs after treatment with SB431542 or DMSO (organs obtained at week 3). (I) Quantification of the bioluminescent signal from the luciferase-expressing PDAC cells metastasized to the lungs of PDAC tumor-bearing mice treated with DMSO or SB431542. * p = 0.0252, two-tailed unpaired Student t-test. Data were expressed as mean ± S.E.M. Individual data points are presented on the graphs. ‘ns’ means not significant. Representative data from one of three independent experiments is shown

Fig. 2

Genetic ALK7 inhibition suppresses spontaneous PDAC metastasis in an orthotopic PDAC model in vivo. (A) A schematic of the orthotopic PDAC spontaneous metastasis model with or without ALK7 knockdown (KD). PDAC cells were treated with shALK7#2, a shRNA targeting ALK7 (shALK7KD), or with a scrambled control shRNA (shControl). shALK7KD and shControl PDAC cells were injected into the tail of the pancreas of NSG mice (NOD-scid IL2Rgammanull) (n = 10), and tumor progression was observed for 3 weeks. (B) Primary tumor growth monitored with an IVIS imager (photons/s) at week 2. (C) IVIS images of PDAC tumor-bearing mice at week 3. (D) Quantification of bioluminescence signal from all primary tumors in shALK7KD and shControl mice at week 3 (n = 10). (E) Quantification of the weight of excised primary tumors obtained from the shALK7KD and shControl mice (n = 10). (F) Quantification of bioluminescence signal from the lungs (n = 10). IVIS images of metastatic PDAC cells in the lungs obtained from shALK7KD and shControl mice (organs obtained at week 3). (G) IVIS images of metastatic PDAC cells in the lungs obtained from shALK7KD and shControl mice (organs obtained at week 3). (H) Quantification of bioluminescence signal from the livers (n = 10). (I) IVIS images of metastatic PDAC cells in the livers obtained from shALK7KD and shControl mice (organs obtained at week 3). (J) IVIS images and total flux from CTCs ex vivo obtained from shControl- vs. shALK7KD PDAC tumor-bearing mice (n = 10). (K) The Cancer Genome Atlas (TCGA) survival analysis of human PDAC patients with upregulated ALK7 signaling compared to those with normal ALK7 signaling, Kaplan-Meier analysis with log-rank test in the regular ALK7 expression (n = 158, overall survival = 21.71 months) or ALK7 up-regulation (n = 19, overall survival = 15.11 months). O.S. indicates Overall Survival. * (p < 0.05) indicates statistical significance. ‘ns’ indicates not significant

Fig. 3

A non-canonical ALK7/β-catenin pathway promotes EMT and aggressiveness in PDAC. (A) Western blotting to evaluate the activation status of canonical and non-canonical TGFβ receptor downstream pathways. Smad2/3 is associated with the canonical pathway, while RhoA, MLC2, AKT, ERK (MAPK), JNK, and p38 are associated with the non-canonical pathway. (B) Western blotting to assess the GSK3β/β-catenin axis. (C) Immunofluorescence to examine nuclear translocation of β-catenin. (D) Western blotting of β-catenin after cell fractionation: total, cytosolic, and nuclear fractions of cell lysates. (E) Western blotting to evaluate the expression of epithelial-mesenchymal transition (EMT) markers (ZEB1, SLUG, SNAIL, and TWIST1). (F) Western blotting to assess the expression of EMT markers in ALK7 KD PDAC cells, with or without transfection of β-catenin expressing plasmids. (G) Western blotting to examine known β-catenin target genes involved in cell proliferation (c-Myc and CyclinD1), stemness (CD44, SOX2, and OCT4), and cell migration (N-cadherin and Fibronectin) in ALK7 KD PDAC cells, with or without transfection of β-catenin expressing plasmids. (H) A summary of the findings. Scale bars (C) = 10 μm. * (p < 0.05), ** (p < 0.01), *** (p < 0.001) indicate statistical significance. ‘ns’ indicates not significant.Immunoblotting experiments were independently performed in triplicate (n = 3)

Fig. 4

ALK7/β-catenin signals upregulate MMP7/9 expression, enhancing invasiveness in PDAC cells. (A) A bulk RNA-sequencing (RNA-seq) and gene set enrichment analyses in ALK7-knockdown or control PDAC cells. The count means gene numbers categorized in a specific pathway identified. (B) Western blotting to assess MMP7 and MMP9 expression in wild-type PDAC or ALK7-silenced PDAC cells with or without β-catenin induction. (C) RT-qPCR to evaluate MMP7 and MMP9 mRNA transcription in wild-type PDAC or ALK7-silenced PDAC cells with or without β-catenin induction (n = 3). One-way ANOVA with Tukey’s multiple comparisons (D) Representative immunofluorescent (IF) images of PDAC cells (anti-GFP, green; phalloidin, red; and merged images with DAPI, blue), embedded in 2.5 mg/mL collagen with or without Batimastat (20 μM) treatment (24 h). (E) Number of invadosome-like protrusions in PDAC cells with or without Batimastat treatment (n = 4). (F) Protrusion lengths in PDAC cells with or without Batimastat treatment (n = 4). (G) Percent of circular cells with or without Batimastat treatment (n = 4). Scale bars (D) = 50 μm; ** (p < 0.01), *** (p < 0.001) **** (p < 0.0001) indicate statistical significance. ‘ns’ indicates not significant. Immunoblotting experiments were independently performed in triplicate (n = 3).

Fig. 5

MMP inhibition minimally affects PDAC extravasation. (A) A schematic of the bioengineered blood vessel (BV)-on-chip. (B) HUVECs in monoculture stained with anti-CD31 (red), anti-Collagen IV (yellow) antibodies, and DAPI (blue). Collagen IV, a vascular basement membrane (BM) protein, is secreted by HUVECs and deposited in the basal side of the engineered BV (side view, left: apical side, right: basal side). (C) A schematic of the extravasation-on-chip workflow. After 3 days of HUVEC seeding, HUVECs form an engineered BV, fully depositing BM. Then, PDAC cells were introduced into the BV lumens through the media reservoirs. Batimastat (20 µM) or DMSO (a vehicle) was treated on Day 3 when PDAC cells were introduced. (D) Representative immunofluorescent (IF) images of PDAC extravasation, staining for PDAC (green, GFP+), Collagen IV (yellow), CD31 (red), and merged images. (E) Percent PDAC invasion in the BV (n = 6). (F) Extravasated PDAC cells (n = 6). (G) Percent CD31 + area out of the total area of the BVs (n = 6). (H) Percent Col IV + area out of the total area of the BVs (N = 6). Scale bars (B) = 50 μm; (D) = 250 μm; ‘ns’ indicates not significant

Fig. 6

MMP inhibition effectively limits PDAC intravasation by preserving BM integrity. (A) A schematic of the PDAC intravasation-on-chip. PDAC cells were seeded in the Col I bulk at 1.5 × 10⁶ cells/mL; immediately after, HUVECs were seeded into microchannels. Devices were treated with Batimastat or DMSO for 1 or 2 days. (B) Representative images for PDAC (green), CD31 (red), and Col IV (yellow) at 24 h. (C) Percent Col IV + area out of the total area of the BVs with or without Batimastat at 24 h (n = 4–5). (D) Percent CD31 + area out of the total area of the BVs with or without Batimastat at 24 h (n = 4–5). (E) Vessel permeability in normal and PDAC conditions with or without Batimastat at 24 h (n = 7–9). (F) Representative images for PDAC (green), CD31 (red), and Collagen IV (yellow) at 48 h. (G) Percent Col IV + area out of the total area of the BVs with or without Batimastat at 48 h (n = 5). (H) Percent CD31 + area out of the total area of the BVs with or without Batimastat at 48 h (n = 5). Scale bars (A) = 75 μm, (B, F) = 100 μm; * (p < 0.05), ** (p < 0.01), **** (p < 0.0001) indicate statistical significance. ‘ns’ indicates not significant

Fig. 7

Genetic ALK7 inhibition preserves basement membrane (BM) integrity and endothelial barrier, limiting PDAC intravasation. (A) Representative images for PDAC (green), CD31 (red), and Col IV (yellow) in the intravasation-on-chip at 24 h. Control vs. ALK7 KD PDAC cells were seeded in Col I bulk at 0.7 × 10⁶ cells/mL. (B) Percent CD31 + area out of the total area of the BVs with PDAC control and ALK7 KD cells at 24 h (n = 5). (C) Percent Col IV + area out of the total area with PDAC control and ALK7 KD cells at 24 h (n = 5). (D) Quantification of PDAC cells on the image at 24 h of co-culture. (E) Correlation between Col IV and CD31 expression at 24 h. (F) Quantification of protrusion numbers and length of control and ALK7 KD PDAC cells. (G) Representative images for PDAC (green), CD31 (red), and Col IV (yellow) at 48 h. (H) Percent CD31 + area out of the total area of the BVs with PDAC control and ALK7 KD cells at 48 h (n = 5). (I) Percent Col IV + area out of the total area with PDAC control and ALK7 KD cells at 48 h (n = 5). (J) Correlation between Col IV and CD31 expression at 48 h (K) Quantification of PDAC cells on the image at 48 h of co-culture (n = 5). (L) Summary of our findings on the mechanisms of ALK7 in PDAC metastasis. PDAC cells that express ALK7 receptor invade into ECM, break BM, and intravasate into blood vessels via GSK3β/β-catenin signaling that mediates MMP secretion and EMT transition. Scale bars (A, F) = 50 μm; * (p < 0.05), ** (p < 0.01), indicate statistical significance. ‘ns’ indicates not significant