The CXCR4/CXCL12 axis plays a role in cancer metastases, stem cell mobilization and chemosensitization. Proof of concept for efficient CXCR4 inhibition has been demonstrated in stem cell mobilization prior to autologous transplantation in hematological malignancies. Nevertheless CXCR4 inhibitors suitable for prolonged use as required for anticancer therapy are not available. To develop new CXCR4 antagonists a rational, ligand-based approach was taken, distinct from the more commonly used development strategy. A three amino acid motif (Ar-Ar-X) in CXCL12, also found in the reverse orientation (X-Ar-Ar) in the vMIP-II inhibitory chemokine formed the core of nineteen cyclic peptides evaluated for inhibition of CXCR4-dependent migration, binding, P-ERK1/2-induction and calcium efflux. Peptides R, S and I were chosen for evaluation in in vivo models of lung metastases (B16-CXCR4 and KTM2 murine osteosarcoma cells) and growth of a renal cells xenograft. Peptides R, S, and T significantly reduced the association of the 12G5-CXCR4 antibody to the receptor and inhibited CXCL12-induced calcium efflux. The four peptides efficiently inhibited CXCL12-dependent migration at concentrations as low as 10 nM and delayed CXCL12-mediated wound healing in PES43 human melanoma cells. Intraperitoneal treatment with peptides R, I or S drastically reduced the number of B16-CXCR4-derived lung metastases in C57/BL mice. KTM2 osteosarcoma lung metastases were also reduced in Balb/C mice following CXCR4 inhibition. All three peptides significantly inhibited subcutaneous growth of SN12C-EGFP renal cancer cells. A novel class of CXCR4 inhibitory peptides was discovered. Three peptides, R, I and S inhibited lung metastases and primary tumor growth and will be evaluated as anticancer agents.

CXCR4 is a chemokine receptor implicated in the metastatic process. The CXCR4 ligand, CXCL12, was shown to bind also the CXCR7 receptor, a recently deorphanized chemokine receptor whose signalling pathway and function are still controversial. This study was conducted to determine patients clinic-pathological factors and outcome according to the expressions of CXCR4 and CXCR7 in renal cell carcinoma (RCC). CXCR4 and CXCR7 expression was evaluated in 223 RCC patients through immunohistochemistry; moreover CXCR4 and CXCR7 was detected in 49 others consecutive RCC patients trough RT- PCR. CXCR4 expression was low in 42/223 RCC (18.8%), intermediate in 71/223 (31.9%) and high in 110/223 (49.3%). CXCR7 expression was low in 44/223 RCC patients (19.8%), intermediate in 65/223 (29.1%) and high in 114/223 (51.1%). High CXCR4 and high CXCR7 expression predicted shorter disease free survival. In multivariate analysis, high CXCR4 expression (p= 0.0061), high CXCR7 (p= 0.0194) expression and the concomitant high expression of CXCR4 and CXCR7 (p= 0.0235) are independent prognosis factors. Through RT-PCR, CXCR4 was overexpressed in 36/49 and CXCR7 in 33/49 samples correlating with symptoms at diagnosis and lymph nodes status. So we can hypothesize that CXCR4 and CXCR7, singularly evaluated and in combination, are valuable prognostic factors in RCC patients.

Hepatocellular carcinoma (HCC) is a heterogeneous disease with a poor prognosis and limited markers for predicting patient survival. Because chemokines and chemokine receptors play numerous and integral roles in HCC disease progression, the CXCR4–CXCL12–CXCR7 axis was studied in HCC patients. CXCR4 and CXCR7 expression was analyzed by immunohistochemistry in 86 HCC patients (training cohort) and validated in 42 unrelated HCC patients (validation cohort). CXCR4 levels were low in 22.1% of patients, intermediate in 30.2%, and high in 47.7%, whereas CXCR7 levels were low in 9.3% of patients, intermediate in 44.2% and high in 46.5% of the patients in the training cohort. When correlated to patient outcome, only CXCR4 affected overall survival (P=0.03). CXCR4–CXCL12–CXCR7 mRNA levels were examined in 33/86 patients. Interestingly, the common CXCR4–CXCR7 ligand CXCL12 was expressed at significantly lower levels in tumor tissues compared to adjacent normal liver (P=0.032). The expression and function of CXCR4 and CXCR7 was also analyzed in several human HCC cell lines. CXCR4 was expressed in Huh7, Hep3B, SNU398, SNU449 and SNU475 cells, whereas CXCR7 was expressed in HepG2, Huh7, SNU449 and SNU475 cells. Huh7, SNU449 and SNU475 cells migrated toward CXCL12, and this migration was inhibited by AMD3100/anti-CXCR4 and by CCX771/anti-CXCR7. Moreover, SNU449 and Huh7 cells exhibited matrix invasion in the presence of CXCL12 and CXCL11, a ligand exclusive to CXCR7. In conclusion, CXCR4 affects the prognosis of HCC patients but CXCR7 does not. Therefore, the CXCR4–CXCL12–CXCR7 axis plays a role in the interaction of HCC with the surrounding normal tissue and represents a suitable therapeutic target.

Inhibition of the axis CXCR4/CXCL12 can affect tumor growth and metastases. Moreover, CXCR4 inhibition increases hematopoietic stem cell mobility in patients undergoing autologous transplantation for myeloma and non-Hodgkin lymphoma. Although several inhibitors were described neither one reached satisfactory efficacy, bioavailability and acceptable toxicity. To develop new CXCR4 inhibitors through a rationale design approach, comparative studies were undertaken evaluating the N-terminal structure of the ligand CXCL12 and v-MIP II, an inhibitory chemokine like produced by the herpes virus 8-Kaposi associated. A common amino acidic motif (RFF) was identified in both structures although in inverted orientation. Having as a core this small domain, a new library (20 units) of cycle-peptide molecules was generated. 20 units of cycle-peptide molecules were synthesized that consists of 5 and 7 amino-acid residues cycled by a S-S bridge designed to interact with the receptor CXCR4. The peptides were characterized for functionally CXCR4 interaction through: 1. inhibition of CXCR4 binding; 2. inhibition of migration CXCL12-induced; 3. inhibition of calcium CXCL12-induced; 4. Inhibition of P-Erk CXCL12-induced. Four peptides were identified as possible CXCR4 inhibitors. These four CXCR4 inhibitory peptides were further evaluated for the in vivo efficacy through inhibition of metastasis formation. B16 mouse melanoma cells transfected with CXCR4 were injected in the vein tail in C57 Bl mice and peptide treatment followed for 10 days. Dramatic reduction in number and size of lung metastases were registered in mice peptides-treated. Moreover, xenograft of human renal cancer cells SN12C-EGFP were subcutaneously injected and systemic peptides treatment followed for 10 days. Statistically significant decrease in tumor growth was showed in peptides treated mice. Taken together the data define three new CXCR4 inhibitory peptides, designed through innovative strategy, effective in “in vivo” deserving further development. Citation Information: Clin Cancer Res 2010;16(7 Suppl):B50

Modulation of the axis CXCR4/CXCL12 affected primary tumor growth and secondary lesions development. Moreover, CXCR4 inhibition increases hematopoietic stem cell mobility in patients undergoing autologous transplantation for myeloma and non-Hodgkin lymphoma. Although several CXCR4 inhibitors were described neither one reached satisfactory efficacy, bioavailability and acceptable toxicity. To develop new CXCR4 inhibitors through a rationale design approach, comparative studies were undertaken evaluating the N-terminal structure of the ligand CXCL12 and v-MIP II, an inhibitory chemokine like produced by the herpes virus 8-Kaposi associated. A common amino acidic motif (RFF) was identified in both structures although in inverted orientation. Having as a core this small domain, a new library (20 units) of cycle-peptide molecules was generated. 20 units of cycle-peptide molecules were synthesized that consists of 5 and 7 amino-acid residues cycled by a S-S bridge designed to interact with the receptor CXCR4. The peptides were characterized for functionally CXCR4 interaction through: 1. inhibition of CXCR4 binding; 2. inhibition of migration CXCL12-induced; 3. inhibition of calcium release CXCL12-induced; 4. Inhibition of P-Erk CXCL12-induced. Four peptides were identified as CXCR4 inhibitors. These four CXCR4 inhibitory peptides were further evaluated for the in vivo efficacy through inhibition of metastasis formation. B16 mouse melanoma cells transfected with CXCR4 were injected in the vein tail in C57 Bl mice and peptide treatment followed for 10 days. Dramatic reduction in number and size of lung metastases were registered in mice peptides-treated. Moreover, xenograft of human renal cancer cells SN12C-EGFP were subcutaneously injected and systemic peptides treatment followed for 10 days. Statistically significant decrease in tumor growth was showed in peptides treated mice. Taken together the data define four new CXCR4 inhibitory peptides, designed through innovative strategy, effective in “in vivo” systems deserving further development. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2270.