Sarah A. Lewin

Scavenging of CXCL12 by CXCR7 promotes tumor growth and metastasis of CXCR4-positive breast cancer cells

Chemokine CXCL12 and receptor CXCR4 control multiple steps in primary tumor growth and metastasis in breast cancer and more than 20 other human malignancies. Mechanisms that regulate availability of CXCL12 in tumor microenvironments will substantially impact cancer progression and ongoing efforts to target the CXCL12-CXCR4 pathway for cancer chemotherapy. We used dual luciferase imaging to investigate CXCR7-dependent scavenging of CXCL12 in breast tumors in vivo and quantify effects of CXCR7 on tumor growth and metastasis of a separate population of CXCR4+ breast cancer cells. In a mouse xenograft model of human breast cancer, in vivo imaging showed that malignant cells expressing CXCR7 reduced bioluminescent CXCL12 secreted in the primary tumor microenvironment. Capitalizing on sensitive detection of bioluminescent CXCL12, we also demonstrated that CXCR7+ cells reduced amounts of chemokine released from orthotopic tumors into the circulation. Immunofluorescence staining of human primary breast cancers showed expression of CXCR4 and CXCR7 on malignant cells in ≈30% of cases. In most cases, CXCR4 and CXCR7 predominantly were expressed on separate populations of malignant cells in a tumor. We modeled these cases of human breast cancer by co-implanting tumor xenografts with CXCR4+ breast cancer cells, human mammary fibroblasts secreting CXCL12, and CXCR7+ or control breast cancer cells. Bioluminescence imaging showed that CXCR7+ breast cancer cells enhanced proliferation of CXCR4+ breast cancer cells in orthotopic tumors and spontaneous metastases. Treatment with a small-molecule inhibitor of CXCR7 chemokine limited the growth of CXCR4+ breast cancer cells in tumors that also contained malignant CXCR7+ cells. These studies establish a new in vivo imaging method to quantify chemokine scavenging by CXCR7 in the tumor microenvironment and identify that CXCR7+ cells promote growth and metastasis of CXCR4+ breast cancer cells.

In vivo imaging of ligand receptor binding with Gaussia luciferase complementation

Studies of ligand-receptor binding and the development of receptor antagonists would benefit greatly from imaging techniques that translate directly from cell-based assays to living animals. We used Gaussia luciferase protein fragment complementation to quantify the binding of chemokine (C-X-C motif) ligand 12 (CXCL12) to chemokine (C-X-C motif) receptor 4 (CXCR4) and CXCR7. Studies established that small-molecule inhibitors of CXCR4 or CXCR7 specifically blocked CXCL12 binding in cell-based assays and revealed differences in kinetics of inhibiting chemokine binding to each receptor. Bioluminescence imaging showed CXCL12-CXCR7 binding in primary and metastatic tumors in a mouse model of breast cancer. We used this imaging technique to quantify drug-mediated inhibition of CXCL12-CXCR4 binding in living mice. We expect this imaging technology to advance research in areas such as ligand-receptor interactions and the development of new therapeutic agents in cell-based assays and small animals.

Secreted CXCL12 (SDF-1) forms dimers under physiological conditions.

Chemokine CXCL12 (CXC chemokine ligand 12) signalling through CXCR (CXC chemokine receptor) 4 and CXCR7 has essential functions in development and underlies diseases including cancer, atherosclerosis and autoimmunity. Chemokines may form homodimers that regulate receptor binding and signalling, but previous studies with synthetic CXCL12 have produced conflicting evidence for homodimerization. We used bioluminescence imaging with GL (Gaussia luciferase) fusions to investigate dimerization of CXCL12 secreted from mammalian cells. Using column chromatography and GL complementation, we established that CXCL12 was secreted from mammalian cells as both monomers and dimers. Secreted CXCL12 also formed homodimers in the extracellular space. Monomeric CXCL12 preferentially activated CXCR4 signalling through Gαi and Akt, whereas dimeric CXCL12 more effectively promoted recruitment of β-arrestin 2 to CXCR4 and chemotaxis of CXCR4-expressing breast cancer cells. We also showed that CXCR7 preferentially sequestered monomeric CXCL12 from the extracellular space and had minimal effects on dimeric CXCL12 in cell-based assays and an orthotopic tumour xenograft model of human breast cancer. These studies establish that CXCL12 secreted from mammalian cells forms homodimers under physiological conditions. Since monomeric and dimeric CXCL12 have distinct effects on cell signalling and function, our results have important implications for ongoing efforts to target CXCL12 pathways for therapy.

Marijuana Use in Potential Liver Transplant Candidates

Concern exists that liver transplant center substance abuse policies may have an inappropriate and disproportionate impact on marijuana users. Our hypothesis is that patients with chronic liver disease who were marijuana users will have inferior survival. This is a retrospective (1999–2007) cohort study. The primary outcome measure is time‐dependent, adjusted patient survival from the time of liver transplant evaluation. The primary exposure variable is a positive cannabinoid toxicology screen during the liver transplant evaluation period. Overall, 155 patients qualified as marijuana users while 1334 patients were marijuana non‐users. Marijuana users were significantly (p < 0.05) younger (48.3 vs. 52.1), more likely to be male (78.1% vs. 63.0%), have hepatitis C (63.9% vs. 40.6%) and were less likely to receive a transplant (21.8% vs. 14.8%). Marijuana users were more likely to use tobacco, narcotics, benzodiazepines, amphetamines, cocaine or barbiturates (p < 0.05). Unadjusted survival rates were similar between cohorts. Upon multivariate analysis, MELD score, hepatitis C and transplantation were significantly associated with survival, while marijuana use was not (HR 1.09, 95% CI 0.78–1.54). We conclude that patients who did and did not use marijuana had similar survival rates. Current substance abuse policies do not seen to systematically expose marijuana users to additional risk of mortality.

Effects of Smoking on Survival for Patients with End-Stage Liver Disease

BACKGROUND Smokers with chronic liver disease can become eligible for transplantation, but some insurers refuse reimbursement pending smoking cessation. STUDY DESIGN Our hypothesis is that liver transplantation candidates and recipients who smoke have inferior survival compared with nonsmokers. Using a retrospective cohort study design, three Cox proportional hazards models were constructed to determine covariate-adjusted mortality from transplantation evaluation and transplantation based on smoking status at evaluation, transplantation, and posttransplantation followup. RESULTS From 1999 to 2007, 2,260 patients were evaluated. Seven hundred sixty were active smokers, and 1,500 were nonsmokers. Smokers at evaluation were younger (49.3 versus 51.7 years), were more likely to be men (65.9% versus 58.7%), have hepatitis C (54.2% versus 30.1%), have a lower Model for End-Stage Liver Disease score (10.5 versus 12.3), and less likely to receive transplant (12.2% versus 18.6%) (all p < 0.05). The postevaluation multivariate model indicated that substance use, higher Model for End-Stage Liver Disease score, hepatitis C, and older age increased mortality risk (all p < 0.05), and liver transplantation (hazards ratio = 0.986; 95% CI, 0.977 to 0.994) was associated with lower mortality. Smoking was not associated with increased mortality risk at any time point in those evaluated or receiving transplants. CONCLUSIONS Providers should continue encouraging potential liver transplantation candidates to stop smoking, but insurer-driven mandated smoking cessation might not improve survival.

Abstract 4056: Gaussia luciferase complementation demonstrates that secreted CXCL12 (SDF-1) forms dimer under physiologic conditions

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Chemokine CXCL12 signaling through receptors CXCR4 and/or CXCR7 promotes tumor growth and metastasis in more than 20 different human malignancies, establishing CXCL12 as a promising therapeutic target in cancer. Under physiologic conditions, many chemokines form homodimers, which may activate signaling pathways distinct from those regulated by monomers of the same chemokine. Prior studies with recombinant, mutant forms of CXCL12 engineered to form either monomers or dimers show that monomeric and dimeric CXCL12 preferentially activate different downstream signaling molecules. However, the extent to which CXCL12 forms homodimers under physiologic conditions and resultant effects on CXCR4 and CXCR7 signaling remain to be determined. To analyze dimerization of CXCL12 secreted by mammalian cells, we used bioluminescence imaging of CXCL12 fused to either full length or complementation fragments of Gaussia luciferase (GL). Using column chromatography and GL protein fragment complementation, we demonstrated that CXCL12 is secreted from mammalian cells as both monomers and homodimers, and the homodimer remains stable in the extracellular environment. In 3D spheroid cultures, we also established that CXCL12 secreted from two independent cell populations can form homodimers in the extracellular space. Relative to dimeric CXCL12, monomeric CXCL12 preferentially signaled via CXCR4 to Gαi proteins to activate AKT and suppress intracellular cAMP. By comparison, dimeric CXCL12 was more potent at recruiting α-arrestin 2 to CXCR4 and regulating chemotaxis CXCR4+ breast cancer cells. In cell-based assays and an orthotopic tumor xenograft model of human breast cancer, we also found that CXCR7 preferentially sequestered monomeric CXCL12 and had minimal accumulation of the dimer. From these studies, we conclude that CXCL12 secreted from mammalian cells forms homodimers under physiologic conditions, and monomeric versus homodimeric CXCL12 regulate distinct signaling pathways. These results inform ongoing efforts to target CXCL12 and its receptors for cancer therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4056. doi:1538-7445.AM2012-4056