Characterization of Niemann-Pick Type C2 protein expression in multiple cancers using a novel NPC2 monoclonal antibody

Abstract

Niemann-Pick Type C2 (NPC2) plays an important role in the regulation of intracellular cholesterol homeostasis via direct binding with free cholesterol. However, little is known about the significance of NPC2 in cancer. In this study, we have pinpointed the impact of various different cancers on NPC2 expression. A series of anti-NPC2 monoclonal antibodies (mAbs) with the IgG2a isotype were generated and peptide screening demonstrated that the reactive epitope were amino acid residues 31-40 of the human NPC2 protein. The specificity of these mAbs was confirmed by Western blotting using shRNA mediated knock-down of NPC2 in human SK-Hep1 cells. By immunohistochemical staining, NPC2 is expressed in normal kidney, liver, breast, colon, lung, esophageal, uterine cervical, pancreatic and stomach tissue. Strong expression of NPC2 was found in the distal and proximal convoluted tubule of kidney and the hepatocytes of liver. Normal esophageal, uterine cervical, pancreatic, stomach, breast, colon and lung tissue stained moderately to weakly. When compared to their normal tissue equivalents, NPC2 overexpression was observed in cancers of the breast, colon and lung. Regarding to breast cancer, NPC2 up-regulation is associated with estrogen receptor (-), progesterone receptor (-) and human epidermal growth factor receptor (+). On the other hand, NPC2 was found to be down-regulated in renal cell carcinoma, liver cirrhosis and hepatoma tissues. By antigen-capture enzyme immunoassay ELISA, the serum NPC2 is increased in patients with cirrhosis and liver cancer. According to western blot data, the change of glycosylated pattern of NPC2 in serum is associated with cirrhosis and liver cancer. To the best of our knowledge, this is the first comprehensive immunohistochemical and serological study investigating the expression of NPC2 in a variety of different human cancers. These novel monoclonal antibodies should help with elucidating the roles of NPC2 in tumor development, especially in liver and breast cancers.

Figure 1. Development of NPC2 monoclonal antibodies.

(A) Construction of the expression plasmids of GST-NPC2 and His-NPC2. (B) Bacterial NPC2 fusion proteins were expressed in BL21 cells and then induced using 1 mM IPTG. SDS-PAGE and Coomassie blue staining were used to show the molecular weights of the two proteins were approximately 43.3 kD for GST-NPC2 and 17 kD for His-NPC2. (C) Western blot analysis using the mAbs (14-8D, 5-5B, 5-4B, 4-12C, 3-6B, 2-7D, 1-5B and 1-2G) against GST-NPC2 and His-NPC2. (D) Western blot analysis using the mAbs against mouse epididymis.

Figure 2. Characterization of NPC2 monoclonal antibodies.

(A) Epitope region mapping of the monoclonal NPC2 antibodies. Different length of pNPC2-HA including full-length pNPC2-HA, N-terminal half (1-80 a.a.), C-terminal half (81-151 a.a.) and 41-105 a.a. were transfected into 293T cells and 24 hrs later were harvested for Western blot analysis. Lane 1, 1-2G; Land 2, 1-5B; Lane 3, 2-7B; Lane 4, 3-6B; Lane 5, mAb-HA; Lane 6, 4-12C; Lane 7, 5-4B, Lane 8, 5-5B; Lane 9, 14-8D. (B) An ELISA plate coated with four peptides covering the 1-40 a.a. region of NPC2 protein was used for epitope mapping. Representative results from the mAbs (14-8D, 5-4B, 3-6B, 2-7D, 1-5B and 1-2G) are shown. (C) The knockdown effect of shNPC2 on SK-Hep1 cells was detected using anti-NPC2 mAbs (3-6B, 14-8D, 5-4B and 2-7B).

Figure 3. IHC staining of NPC2 protein expression in paired human tumor tissue samples and their normal counterparts.

Magnification: 200x. (A) NPC2 was up-regulated in breast, colon and lung tumor samples. (B) NPC2 was down-regulated in kidney and liver tumor samples. (C) The expression of NPC2 remained unchanged in the pancreatic, esophageal, uterine cervical and stomach tissue sample pairs.

Figure 4. Dysregulation of NPC2 is associated with cirrhosis and HCC.

(A) ELISA analysis of serum NPC2 levels in 42 healthy controls, 20 patients with chronic HBV infection, 2 patients with chronic HCV infection, 27 patients with fatty liver, 28 cirrhosis and 46 HCC patients. (B) IHC staining of NPC2 protein expression in normal, chronic hepatitis and cirrhosis tissues. (C) IHC analysis of hepatic NPC2 expression in 44 normal, 21 chronic hepatitis and 60 cirrhosis tissue samples (*, p<0.05, Mann-Whitney U test). (D) Human sera (60 μg) were incubated with peptide N-glycosidase F (PNGase F) and subjected to Western blot analysis. (E) Western blot analysis of serum NPC2 expression in 14 healthy controls, 7 fatty liver, 4 cirrhosis and 4 HCC patients. Each line was loaded 100 µg protein.

Figure 5. The effects of NPC2 on MAPK signaling.

Total and phosphorylated ERK1/2, p38, JNK were detected by Western blotting from the SK-hep1 cells. Knockdown of NPC2 results in activation of ERK1/2.