Collagen fibers in stroma and vasculature as well as fibers produced by cancer-associated (recruited) fibroblasts form an invasion barrier. At the same time, collagen fibers are essential for maintaining the invasive phenotype and supporting migration and proliferation of cancer cells. Massive production of both collagen and MMPs that degrade collagen, mostly by cancer-associated fibroblasts, may be one solution to this dilemma. However, the present study suggests that invasive cells may benefit even more from producing fibers of MMP-resistant homotrimeric type I collagen. Not only are the homotrimers resistant to all collagenolytic MMPs in solution (MMP-1, 2, 8, 13; ), but the homotrimer matrix is resistant to cleavage by fibroblasts () and cancer cells (Supp. Fig. S2
), even when the cells are stimulated with proinflammatory cytokines.
Because the homotrimer fibers appear to be produced only by cancer cells and not by cancer-associated fibroblasts ( and Fig. , ), they comprise a small fraction of tumor collagen. As a result, cancer cells may utilize these fibers as MMP-resistant roadways for invasion rather than as building materials for the tumor stroma. Our data suggest that these collagenase-resistant fiber roadways may provide the necessary support for proliferation and migration of the cancer cells without forming invasion barriers (Suppl. Fig. S6
). They may also promote better organization and migration of all tumor cells and more directed and efficient degradation of surrounding stroma.
In addition to MMP resistance, type I collagen homotrimer fibers have distinct mechanical properties (33
). Matrix mechanics plays an important role in malignancy, significantly affecting the behavior of cancer cells (35
). For instance, higher rigidity of the homotrimer fibers (34
) may contribute to faster proliferation and migration of cancer cells. Potential differences in binding of proteoglycans, cytokines, and other matrix molecules to homotrimer fibers may also affect cancer microenvironment.
Note that collagenase-resistant fibers can still be degraded by other enzymes that cleave non-helical terminal peptides. Such cleavage is likely involved in normalizing collagen turnover in Col1a1r/r
and oim mice; which make collagenase-resistant type I collagen but exhibit only mild localized fibrosis (39
). (Type I collagen has an altered MMP cleavage site in Col1a1r/r
and is homotrimeric in oim mice). Alternative collagen cleavage does not prevent severe, generalized collagen turnover deficiency in MMP-14-knockout mice (6
); although this may be related to other functions of the multifunctional (43
) MMP-14. Alternative cleavage may also be less important in cancer progression.
Consistent with our hypothesis for the role of type I collagen homotrimers in cancer invasion, the same cancer cells seem to produce a higher fraction of the homotrimers in vivo
(~50%, ) than in vitro
(25-35%, ). The higher homotrimer content of cancer cell derived collagen in xenograft tumors may result from selective degradation of the heterotrimers by collagenases. However, it may also be caused by selective proliferation of cancer cell subpopulations producing more homotrimers. Indeed, a several fold reduction in the relative amount of the α2(I) chain mRNA compared to the α1(I) chain mRNA was observed after several cycles of selection for more aggressive TC-1 cells in C57BL/6 mice, as reported in the Gene Expression Omnibus database, NCBI, accession # GSE2774 (44
What are the factors that enable cancer cells to produce homotrimeric type I collagen? These cells are exposed to the same environment as cancer-associated fibroblasts, which do not make the homotrimers. Thus, the answer likely lies within the cells themselves. One possibility is insufficient expression of the α2(I) chain, e.g., due to methylation of the α2(I) gene (45
). However, similar homotrimer synthesis by different cancer cells () would then mean similar α2(I) chain expression deficiency. Such similarity between different cancers seems unlikely.
Another clue to answering this question may be contained in comparing different types of cells producing the homotrimers. From literature analysis, we found no convincing evidence for the homotrimer production by cells normally responsible for collagen synthesis, except for cases with deficient α2(I) chain synthesis caused by rare mutations. In our own measurements, we also observed no detectable homotrimers in (i) fibroblast and osteoblast cultures (human and murine); (ii) murine skin, tendons, and bone; and (iii) normal and fibrotic human skin. In contrast, the homotrimer synthesis by embryonic cells (46
), dedifferentiated cells (47
), non-osteogenic bone marrow cells (48
), chemically transformed cells (15
), cancer cells (17
), and stressed mesangial cells (49
) has been well documented. These observations suggest that mature collagen-producing cells may have a mechanism for preventing the α1(I) homotrimer formation when a sufficient number of the α2(I) chains is synthesized, e.g., a specialized chaperone that promotes association and folding of two α1(I) with one α2(I) C-propeptide chains. This mechanism may be absent in cells that normally produce little or no type I collagen, in which the corresponding chaperone may not be expressed. This mechanism may also be absent or not fully functional in fetal cells.
Regardless of the underlying mechanism, homotrimeric type I collagen appears to be produced only in fetal or pathological tissues. This property may be utilized for diagnostic and therapeutic targeting of cancer and other pathologies involving the homotrimer synthesis, e.g., for visualizing peripheral areas invaded by cancer cells during surgery. Indeed, insoluble, collagenase-resistant homotrimer fibers may present an ideal target, provided that a molecule that selectively binds to the homotrimers but not heterotrimers can be designed. This task may be challenging since the homotrimers do not contain unique peptide sequences. However, some triple helix regions are much less stable within the homotrimers than within the heterotrimers (26
). Targeting these regions by molecules that recognize unfolded but not folded chains may present one possible solution.