CLIC1 and CLIC4 complement CA125 as a diagnostic biomarker panel for all subtypes of epithelial ovarian cancer

Abstract

New plasma and tissue biomarkers of epithelial ovarian cancer (EOC) could improve early diagnosis and post-diagnosis clinical management. Here we investigated tissue staining and tissue secretion of CLIC1 and CLIC4 across EOC subtypes. CLIC1 and CLIC4 are two promising biomarkers we previously showed were elevated in EOC patient sera. Individually, CLIC1 or CLIC4 stained larger percentages of malignant tumors across all EOC subtypes compared with CA125, particularly early stage and mucinous tumors. CLIC4 also stained benign tumors but staining was limited to nuclei; whereas malignant tumors showed diffuse cellular staining of stromal and tumor cells. Both proteins were shed by all EOC subtypes tumors in short term organ culture at more consistent levels than CA125, supporting their potential as pan-subtype serum and tissue biomarkers. Elevated CLIC4 expression, but not CLIC1 expression, was a negative indicator of patient survival, and CLIC4 knockdown in cultured cells decreased cell proliferation and migration indicating a potential role in tumor progression. These results suggest CLIC1 and CLIC4 are promising serum and tissue biomarkers as well as potential therapeutic targets for all EOC subtypes. This justifies development of high throughput serum/plasma biomarker assays to evaluate utility of a biomarker panel consisting of CLIC1, CLIC4 and CA125.

Figure 1

CLIC1 staining in normal, benign and EOC tissues. Representative images of CLIC1 staining of normal ovary, fallopian tubes, benign tumors, and serous (stage IV) and mucinous (stage I) EOC tissue. Red arrows = stroma, black arrows = normal epithelial cells and yellow arrow = tumor cells. Locations of high magnification insert are indicated by the black box.

Figure 2

CLIC4 staining in normal, benign and EOC tissues. Representative images of CLIC4 staining of normal ovary, fallopian tubes, benign tumors, and serous and mucinous EOC tissue. Tissue cores are identical to the ones used in Fig. 1. Red arrows = stroma, black arrows = normal epithelial cells and yellow arrow = tumor cells. Locations of high magnification insert are indicated by the black box.

Figure 3

Changes in CLIC4 staining at different EOC stages. (a) Representative images of CLIC4 staining in stage I, II, III and IV serous EOC tumors. Red arrows = staining in the stroma and yellow arrow = staining in tumor cells. (b) Percentage of tumor nests (T) and stroma (S) that show the indicated distribution of nuclear, cytoplasmic, nuclear + cytoplasmic or negative staining for CLIC4 across different EOC stages and subtypes.

Figure 4

CA125 staining in normal tissues, benign tissues and EOC tissues. Representative images of CA125 staining in the normal ovary, fallopian tubes, benign tumors, and serous and mucinous EOC tissue. Tissue cores are identical to the ones used in Fig. 1. Red arrows = stroma, black arrows = normal epithelial cells and yellow arrow = tumor cells. Locations of high magnification insert are indicated by the black box.

Figure 5

Sensitivity of CLIC1 and CLIC4 as EOC biomarkers. Intensity of CLIC1, CLIC4 and CA125 staining across all tumors (a) and tumors of each EOC subtype (b). (c) Complementarity among CLIC1, CLIC4, and CA125 staining. (d) Quantitation of CLIC1 and CLIC4 shed from benign and malignant ovarian tumors expressed as protein intensity; B = Benign (n = 3), CC = Clear cell (n = 3), S = Serous (n = 8), M = mucinous (n = 3), E = Endometrioid (n = 6), M/R = Mixed/Rare (n = 1). The red (1.8E + 9) and blue (1.4E + 9) horizontal lines indicate the average secretion of CLIC1 and CLIC4, respectively, from the benign tumors. (e) Quantitation of CA125 from the same secretomes as in panel d. Black horizontal line (1.5E + 8) indicates the average CA125 shed from the benign tumors.

Figure 6

Elevated CLIC4, but not CLIC1, is a negative indicator of patient survival. Upper panels – CLIC1 mRNA levels correlated with overall and progression free survival in all EOC patients and patients with low CA125 (bottom quartile). Lower panels – CLIC4 mRNA levels correlated with overall and progression free survival in all EOC patients and patients with low CA125 (bottom quartile).

Figure 7

Knockdown of CLIC1 or CLIC4 decreases EOC cell proliferation and cell migration. TOV21G, OV90 and TOV112D cells were transduced with control, CLIC1 or CLIC4 shRNA. (a) Monoplast colony formation reflecting cell proliferation. Cells were stained with hematoxylin. (b) Wound healing assay to assess cell migration. Values represent the mean +/− SD of three independent experiments. Statistical significance was determined by performing ANOVA multiple comparisons, using GraphPad Prism 6 software (GraphPad Software, La Jolla, CA). ns = not significant. *p < 0.05; **p < 0.01; ***p < 0.001.