Integrin alpha2 mediates selective metastasis to the liver

Abstract

Cancers display distinct patterns of organ-specific metastasis. Comparative analysis of a broad array of cell membrane molecules on a liver-metastasizing subline of B16 melanoma versus the parental B16-F0 revealed unique up-regulation of integrin alpha2. The direct role of integrin alpha2 in hepatic metastasis was shown by comparison of high versus low-expressing populations, antibody blockade, and ectopic expression. Integrin alpha2-mediated binding to collagen type IV (highly exposed in the liver sinusoids) and collagen type IV-dependent activation of focal adhesion kinase are both known to be important in the metastatic process. Analysis of primary colorectal cancers as well as coexisting liver and lung metastases from individual patients suggests that integrin alpha2 expression contributes to liver metastasis in human colorectal cancer. These findings define integrin alpha2 as a molecule conferring selective potential for formation of hepatic metastasis, as well as a possible target to prevent their formation.

Figure 1

Establishment and characterization of B16-KY8. A, scheme for establishment of B16-KY8. B16-F0 was subjected to eight rounds of sequential splenic administration followed by extraction and culture of liver nodules. With each passage, tumors were selected from the liver until the eighth passage produced a cell line with high propensity to form liver metastases. B, patterns of hepatic and i.p. metastases at death or when moribund. C, appearance of liver disease in mice challenged with the various B16 cell lines and necropisied on day 17. D, weight of livers of mice from C.

Figure 2

Integrin α₂ expression by B16-KY8 and effect of integrin α2 blockade on patterns of metastasis. A, FACS analysis of the two subunits of VLA2 on all B16 sublines. B, 10 of 10 mice (100%) challenged with integrin α2 high cells developed liver metastases, and 3 of 10 (30%) mice had limited peritoneal disease (P = 0.001). Eleven of 11 mice (100%) challenged with integrin α2 low cells developed peritoneal carcinomatosis, as well as hepatic metastases. Numbers of nodules in the liver and peritoneum for integrin α2 high versus integrin α2 low cell lines, and fewer peritoneal nodules compared with the integrin α2 low cell line.

Figure 3

Integrin α2 blockade inhibits formation of hepatic metastases. Forced expression of integrin α2 in a B16 cell line that normally has low expression alters the pattern of metastatic spread. A, number of liver nodules after challenge of B16-F0, B16-KY8, and B16-SKY via hemispleen was counted. Effect of anti–integrin α2 blockade on B16-SKY metastasis patterns to liver, lung, and peritoneum after hemispleen injection was shown as percent of mice with metastases in each of the organ sites (10 mice/group). Effect of anti–integrin α2 blockade on B16-SKY liver metastases was shown as number of nodules per liver (B16-SKY isotype antibody, 30 mice/group; B16-SKY anti–integrin α2 blockade, 19 mice/group). Effect of anti–integrin α2 blockade on B16-SKY peritoneal metastases was shown as number of nodules per peritoneum (10 mice/group). B, effect of anti–integrin α2 blockade on B16-SKY metastasis patterns to liver, lung, and peritoneum after i.v. injection was shown as percent of mice with metastases in each of the organ sites (10 mice/group). C, comparison of B16-SKY and B16-SKYP and effect of anti–integrin α2 blockade on B16-SKY and B16-SKYP liver metastases was shown as number of nodules per liver.

Figure 4

Blocking integrin α2 expression on CT26 and 4T-1 alters their patterns of metastases. A and B, effect of blocking integrin α2 expression on CT26 on the pattern of metastases in mice challenged via splenic injection route. The number of nodules within the liver and peritoneum in the CT26 unblocked (isotype control) versus CT26 integrin α2 blocked is shown. C, effect of blocking integrin α2 expression on 4T-1 on the pattern of metastases in mice challenged via splenic injection route. The number of nodules within the liver in the 4T-1 unblocked versus 4T-1 blocked is shown. Ab, antibody.

Figure 5

Effect of collagen type IV, I/III, fibronectin, and lamin in attachment of the various B16 cell lines. A and B, to study the intensity of attachment of the B16 cell lines to collagen type IV–coated plates, an attachment assay was performed using B16-F0 and B16-KY8 (8 HPF) per group. C, when either B16-KY8 or B16-SKYP was blocked with anti–α2 integrin blocking antibody, both cell lines significantly lost their ability to attach to collagen type IV–coated plates. When the B16-SKYP and B16-KY8 cell lines were used in these assays comparing to B16-F0, they also attached strongly to the collagen IV–coated plates (4 HPF per group). When these cells were incubated on collagen type I/III or fibronectin-coated plates, there was very little attachment and no enhancement by integrin α2 expression. Integrin α2 expression marginally enhanced binding to laminin-coated plates.

Figure 6

Integrin α2 binding to collagen type IV mediates FAK activation. A, Western blotting for total FAK and tyrosine-phosphorylated (Y397–) FAK in three cell lines. Plating B16-KY8 and B16-SKY but not B16-F0 on collagen type IV–coated plates increases activation of FAK (phospho-FAK). Total FAK remains unchanged. B, time course of total FAK expression and Y397 phosphorylation. Tyrosine-phosphorylation of FAK (Y397) with time following plating on collagen type IV until a peak of 90 min. Expression of integrin α2 and patterns of metastases of human colon cancer.