Team finds CXCL12 monomers and dimers lead to increased survival despite different mechanisms of action.
Treatment using the chemokine CXCL12 could represent a new approach to halting metastasis in colon cancer and melanoma, researchers claim. A team headed by investigators at the Medical College of Wisconsin found that injecting tumor-bearing mice with either CXCL12 monomers or dimers improved survival by inhibiting CXCR4-mediated metastasis, even though the monomeric and dimeric CXCL12 had very distinct mechanisms of action.
Micheal B. Dwinell, Ph.D., Luke J. Drury, Ph.D., and colleagues report their findings in PNAS in a paper titled “Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways.”
Chemokine receptor signaling is linked with cancer metastasis and the infiltration of tumor-associated immune cells, neoangiogenesis, and proliferation, the researchers explain. The involvement of chemokines as primary mediators of cancer metastasis—and in particular the role of CXCR4 in tumor cell trafficking—was first reported back in 2001. Indeed, some 23 different cancers have been shown to express elevated levels of CXCR4, which sensitizes the tumors to CXCL12 gradients in distant tissues. However, while small molecule antagonists of CXCR4 have been developed as a means to inhibit metastasis, the approach hasn’t been easy to progress to the clinic.
Work by Dr. Dwinell et al. has previously demonstrated that epigenetic silencing of the CXCL12 promoter increases metastasis of colon and breast cancers, while restoring CXCL12 expression reduced metastasis in vivo. These results led to the notion that CXCL12 secretion might act as a tumor suppressor that prevents metastasis by shielding the primary tumor from remote chemotactic gradients.
To investigate this role of CXCL12 further, the team administered exogenous CXCL12 variants to immunodeficient mice that had been orthotopically engrafted with human colonic carcinoma cells stably expressing firefly luciferase. Mice were given intraperitoneal injections of either wild-type (CXCL12WT), preferentially monomeric (CXCL12H25R), or constitutively dimeric (CXCL122) chemokine. Colon cancer cells engineered to produce endogenous CXCL12 (CSCL12-luc) were used as a positive control, as the previous work had shown that these cell demonstrate a significantly reduced metastatic potential.
The results confirmed that treatment with each of the three test CXCL12 proteins boosted survival, and while imaging studies showed little change in overall primary tumor formation, treated mice developed far fewer liver tumors, suggesting the survival benefit reflected decreased metastasis. “Indeed, administration of exogenous CXCL12 proteins was comparable to that of CXCL12-luc cells in decreasing metastasis and improving survival,” the authors state.
Treating the colon cancer-grafted mice with the CXCL12WT and CXCL122 variants at different concentrations showed that while both were effective at medium to high concentrations, only the dimer decreases metastasis when administered at the lowest concentration. Monomeric CXCL12 was also ineffective at low concentrations at inhibiting liver metastasis.
Encouragingly, preincubation of CXCR4-expressing B16 murine melanoma cells with the optimal 5-μM dose of CXCL122 similarly blocked the metastasis of melanoma tumors to the lungs. “Together, these data indicate that CXCL12 limits the ability of either orthotopic colonic or intravenously implanted melanoma cells to metastasize,” the researchers state. However, the increased potency of CXCL122 suggested that CXCL12 in different oligomeric states blocked metastasis through distinct mechanisms.
The team therefore looked more closely at the effects of the CXCL12 variants, using cell culture systems to study colorectal cancer cell migration. They found that CXCL12WT and CXCL12H25R induced chemotaxis in HCT116 cells, and HT29 epithelial sheet migration, but neither 10-nM CXCL122 nor a 1,000-nM concentration of CXCL12WT was able to stimulate migration: native PAGE showed there was accumulation of homodimers at increasing concentrations of CXCL12WT, “consistent with the notion that the lack of migration reflects formation of nonmotogenic CXCL12 dimers,” the researchers remark. Indeed, CXCL122 competitively blocked CXCL12WT-mediated, but not CXCL8-induced, carcinoma migration.
Migration involves cytoskeletal rearrangement and the formation of newly branched filamentous-actin (F-actin), and imaging studies confirmed that lamellipodia formation and F-actin were elevated in HCT116 cells treated with motogenic concentrations of CXCL12WT or CXCL12H25R but not when treated using 10-nM CXCL122 or 1,000-nM CXCL12WT ligands.
In summary, Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited β-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12, on the other hand, activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2. However it only weakly (if at all) recruited arrestin, stimulated actin polymerization, or chemotaxis.
The researchers then evaluated the ability of the different CXCL12 variants to interact with the chemokine receptors CXCR4 and CXCR7. Radioligand binding assays indicated that CXCL12WT and CXCL12H25R each bound to CXCR4 and CXCR7 with high affinity, whereas CXCL122 preferentially interacted with CXCR4. In fact, relative to its binding to CXCR4, CXCL122 bound to CXCR7 with 1,000-fold lower affinity than CXCL12WT, which meant that CXCR7 was an unlikely target for CXCL122.
Under the two-step, two-site model for chemokine receptor activation, CXCL12 binds first to the N terminus of CXCR4, and the team’s previous work had shown that the CXCR4 N terminus bridges the CXCL12 dimer interface and forms contacts that are absent in the monomer. This supports the notion that the different CXCL12 oligomers do interact differently with the receptor. Moreover, NMR measurements of CXCR41-38 dynamics indicated that residues 5–10 of the receptor are flexible when complexed with CXCL122, but are more ordered when bound to CXCL12H25R. “Based on these structural data, we speculated that oligomer-specific binding modes lead to qualitatively different signaling,” the investigators remark. Indeed, the results of NMR titration studies, combined with the data generated on the differences in backbone mobility, suggested that CXCL12 monomor makes specific contacts with CXCR4 that are lost when CXCL12 dimerizes.
“In contrast to early studies focused largely on CXCR4 receptor antagonism, our data establish that exogenous CXCL12 is a potent inhibitor of colorectal and melanoma metastasis,” the authors conclude. “Our data suggest CXCL12 interruption in metastasis results from formation of dimers at elevated concentrations activating distinct ‘cellular idling’ signaling pathways, desensitizing tumor cell-expressed CXCR4, and/or ligand-induced disruption in chemoattractant gradients…Our work supports a cancer progression model in which locally produced CXCL12 limits the migration potential by either desensitizing cells to endocrine ligand or by exceeding the optimal level for chemotaxis, resulting in the absence of actin polymerization and cell migration characteristic of dimeric CXCL12. Alternatively, tumor-cell–produced CXCL12 limits spread of cells by nullifying chemokine gradients produced in distant tissues.
The overall data suggest CXCL12 could represent a biological drug that specifically target metastasis, they stress. “Differential activation of CXCR4 provides an exciting avenue for therapeutic intervention using biologic agonists tailored to the monomer or dimer.”