LRH1 as a driving factor in pancreatic cancer growth

Abstract

Liver receptor homolog 1 (LRH1), directs the development and differentiation of embryonic pancreas, and is overexpressed in pancreatic cancer (PC). We hypothesized that LRH1 promotes PC growth. Cell proliferation and tumorigenicity in nude mice were compared between empty vector-transfected (control) and stable LRH1-overexpressed PC cell lines. The subsequent tumor burden, vasculature development, and histologic features were evaluated. LRH1 overexpression enhanced the expression of downstream target genes (cyclin D1/E1) and stimulated cell proliferation in PC cell lines. LRH1 upregulated cyclin E1 truncated T1/T2 isoforms expression which may occur through ERα-calpain1 signaling. Compared with the control, LRH1 overexpressing stable cells generated tumors with increased weight, proliferation index and enhanced angiogenesis. Cyclin D1/E1 and calpain1 were overexpressed in human PC tumors compared to adjacent normal pancreas. These observations demonstrate that LRH1 promotes PC growth and angiogenesis, suggesting that LRH1 is a driving factor in tumorigenesis and may serve as a potential therapeutic target.

Keywords: Cyclin D1; Cyclin E1; Liver receptor homolog 1; Pancreatic cancer.

Fig. 1

LRH1 upregulated downstream target genes in PC. (A) Endogenous expression of LRH1 in PC cell lines. LRH1 was highly expressed in MIA PaCa-2 and Hs766T, compared with HPDE (negative control), as well as HepG2 and HEK-293 cells transfected with pcDNA3-LRH1 (positive controls). (B) Expression levels of cyclin D1 and cyclin E1 truncated T1/T2 isoforms were significantly upregulated in LRH1 overexpressing stable Capan-1 and AsPC-1 cells than in controls. Cyclin E1 (FL), full length cyclin E1. (C) The mRNA levels of cyclin D1/E1 were upregulated in PC cell line.

Fig. 2

Growth curves of stable PC cells with LRH1 overexpression. Accelerated cell growth was observed in LRH1 stable transfected Capan-1 and AsPC-1 cells than in controls (Student t test, 2-tailed, *p<0.01).

Fig. 3

LRH1 promoted PC tumor growth in nude mice. (A) Representative established subcutaneous (s.c.) tumors by inoculation of 1×10⁷ Capan-1 cells transfected with LRH1 or vector control. Right shoulder tumors were derived from LRH1-transfected cells, left shoulder tumors were derived from vector control cells. Tumor weights were analyzed within 3 weeks after inoculation of Capan-1 cells stably transfected with LRH1 or vectors (n=7). Compared to vectors, LRH1 expressing stable Capan-1 cells produced much larger tumors (paired t test, 2-tailed, **p<0.01). Vertical bars, standard error. (B) H&E staining of two subcutaneous tumors. (C) Proliferation Index as measured by positive Ki67 staining of tumor nuclei (Student t test, 2-tailed, ***p<0.0001). (D) Examples of enhanced CD34 expression in endothelial indicating increased vascularity in two LRH1 induced tumors.

Fig. 4

Representative examples of LRH1 related genes expression in PC. Weak-negative staining for LRH1 (a1), cyclin D1 (b1), cyclin E1 (c1), and calpain1 (d1) was observed for all components of normal exocrine pancreas. In human PC tumor cells, elevated levels of LRH1 (a2), cyclin D1 (b2), cyclin E1 (c2), and calpain1 (d2) were detected. The original magnification (×40) is specified.

Fig. 5

Hypothesized oncogenic role of LRH1 in PC. We suggest that LRH1 promotes PC tumor growth and angiogenesis by upregulating cyclin D1/E1. In combination with estrogen (E) and estrogen receptor (ER), LRH1 acts to upregulate caplain1. Calapin1 is known to convert full-length cyclin E1 into truncated T1/T2 isoforms through post-translational processing [2, 6]. These isoforms may exhibit enhanced oncogenic and angiogenic properties [2, 6].