LRH1 promotes pancreatic cancer metastasis

Abstract

The transcriptional factor liver receptor homolog 1 (LRH1) regulates pancreatic development, and may participate in pancreatic oncogenesis through activation of growth factor signaling transduction cascades. We measured transcriptional activity of β-catenin in response to LRH1 stimulation by a Topflash reporter assay. The pancreatic cancer (PC) phenotype was then characterized by cell migration, wound healing, invasion, and sphere formation in vitro, as well as tumor formation and distant metastatic spread in vivo. We compared results between vector control and LRH1-overexpressing stable PC cell lines. In addition, tumor burden, angiogenesis, histologic characteristics, and hepatic spread were assessed in orthotopic and experimental liver metastatic murine models. Expression of downstream LRH1 related genes was evaluated by Western blot and immunohistochemistry in PC cell lines and human tumor specimens. Specific inhibition of LRH1 expression and function was accomplished by shRNAs "knockdown" experiments. It was found that LRH1 enhanced transcriptional activity of β-catenin and the expression of downstream target genes (c-Myc, MMP2/9), as well as promoted migration, wound healing, invasion, and sphere formation of PC cell lines. Specific inhibition of LRH1 by shRNAs reduced cell migration, invasion, sphere formation and expression of c-Myc and MMP2/9 target genes. Mice injected with LRH1 overexpressing stable PC cells developed tumors with increased size and exhibited striking hepatic metastatic spread. More important, LRH1 was overexpressed in PC tumors compared to adjacent normal pancreas. Our findings demonstrate that LRH1 overexpression is associated with increased PC growth and metastatic spread, indicating that LRH1-targeted therapy could inhibit tumor progression.

Keywords: Liver receptor homolog 1; MMP2; MMP9; Pancreatic cancer; c-Myc.

Fig. 1.

Synergistic effect of LRH1 on transcriptional activity of β-catenin was reversed by quercetin. LRH1 potentiates β-catenin/Tcf4-mediated transactivation in PC cell lines (A) Capan-1, (B) AsPC-1, (C) MIA PaCa-2, as well as (D) HEK 293 cells. Reversal of LRH1-induced effect was obtained by quercetin. The above LRH1–β-catenin/Tcf4 synergy was reversed by quercetin in (E) Capan-1 and (F) AsPC-1 cells.

Fig. 2.

LRH1 upregulated downstream target genes in PC. (A) Expression levels of c-Myc, MMP2 and MMP9 (in Capan-1) were significantly enhanced in LRH1 overexpressed stable Capan-1 and AsPC-1 cells than vector control and (B) LRH1 expression in stable MIA PaCa2 cells with constitutive expression of specific shRNAs was significantly reduced, and the expression of c-Myc, MMP2 and MMP9 was downregulated in response to LRH1 knockdown.

Fig. 3.

LRH1 promoted PC cell migration and invasion. (A) Pro-migration of LRH1 on Capan-1 and AsPC-1 cells. LRH1 promoted Capan-1 and AsPC-1 cell Migration using BD Bioscience Chamber. Compared to vectors, LRH1 expressing stable Capan-1 and AsPC-1 cells showed enhanced cell migration at 24 h in Boyden chamber assay. A substantial increase in migration was present in LRH1 stable transfected Capan-1 and AsPC-1 cells compared to vector control (**p < 0.01). (B) LRH1 overexpression promoted Capan-1 and AsPC-1 cell invasion. Compared to vector control, LRH1 stable overexpressing Capan-1 and AsPC-1 cells conferred enhanced cell invasion at 24 h. Enhanced invasion was present in LRH1 stable transfected Capan-1 and AsPC-1 cells compared to vector control (**p < 0.01). Significant attenuation of cell migration (C) and invasion (D) was observed at 36 h in three stable MIA PaCa2 cell lines expressing LRH1-shRNAs (**p < 0.01).

Fig. 4.

LRH1 promoted wound healing and sphere formation. (A) LRH1 accelerated wound healing of PC cell lines Capan-1 and AsPC-1. Compared to vector control, LRH1 stable expressing Capan-1 and AsPC-1 cells exhibited accelerated wound healing at 24 h. An increase in relative migration distance was present in LRH1 stable cells compared to vector control (**p < 0.01), (B) LRH1 stable overexpression promoted sphere formation in Capan-1 and AsPC-1 cells. Morphologic changes were observed in LRH1 stable overexpressing Capan-1 and AsPC-1 cells. Compared to vector control, LRH1 stable overexpressing Capan-1 and AsPC-1 cells produced spheres with increased size (**p < 0.01) and (C) Significant reduction in the size of spheres was detected in three MIA PaCa2 cells expressing specific LRH1-shRNAs (**p < 0.05).

Fig. 5.

LRH1 promoted PC tumor growth and distant metastasis in nude mice. (A) Orthotopic PC tumor growth. Representative established orthotopic tumors produced by inoculation of 3 × 10⁶ Capan-1 cells transfected with LRH1 or vector control cells into the body of the pancreas. Primary tumors were removed and three representative pictures from each group are shown. Tumor weights were analyzed at 3 weeks after inoculation of Capan-1 cells stably transfected with LRH1 or vector (n = 5). Compared to vector control, LRH1 expressing stable Capan-1 cells produced much larger tumors (paired t test, 2-tailed, **p < 0.01). Vertical bars, standard error, (B) metastatic rate of orthotopic tumors. Liver metastatic rate of Capan-1–LRH1 and Capan-1–Vector mice was 100% vs. 20%, respectively (*p < 0.05), (C) IHC for Ki67 and the calculation of the proliferation Index as measured by positive Ki67 staining of tumor nuclei (***p < 0.0001), (D) H&E staining of two orthotopic tumors, (E) PC liver metastatic model produced by injection of Capan-1 cells transfected with vector or LRH1 into the spleen (upper and bottom, respectively). H&E staining of representative sections of the metastatic liver foci derived from vector cells and LRH1 expressing cells (upper and bottom, respectively). Magnification was 40 × (left) and 200 × (right), respectively. Black arrows indicate the tip of infiltrated tumor derived from the LRH1 expressing cells and (F) compared to vector control cells, LRH1 expressing stable Capan-1 cells aggressively invade through the portal vein into the liver parenchyma. The yellow bar is 1 cm in length. LRH1 expressing stable Capan-1 cells produced much larger tumors in the liver compared to vector-transfected control cells (paired t test, 2-tailed, *p < 0.01).

Fig. 6.

Expression of LRH1 and related genes in PC. LRH1 was overexpressed in tumor tissues (a2) compared with adjacent normal pancreas (a1) of patients with PC. Negative staining was observed for all components of normal exocrine pancreas. In tumor cells, an elevated level of LRH1 was detected either in the nuclei (insert A) or in the cytoplasm (insert B) or both. The original magnification (40×) is specified. The expression of β-catenin (b1–b2) was comparable between the tumorous and adjacent normal pancreas. Weak-negative staining for c-Myc (c1) was observed for all components of normal exocrine pancreas. In tumor cells, an elevated level of c-Myc (c2) was detected. The original magnification (40×) is specified.

Fig. 7.

Diagram illustrating the proposed oncogenic role of LRH1 in PC. We hypothesize that LRH1 promotes pancreatic oncogenesis (cell migration, invasion, tumor formation, angiogenesis and liver metastasis) by upregulating c-Myc and MMP2/9.