Small cell lung cancer (SCLC) is a highly aggressive and metastatic neuroendocrine carcinoma which progresses rapidly. There is growing evidence that the chemokine receptor CXCR4 and its ligand CXCL12 are involved in migration and metastasis of SCLC. As STAT3 and Rho GTPases have been shown to be involved in the CXCR4 signaling pathway, we are investigating these pathways in SCLC cell lines and the potential therapeutic use of inhibitors of CXCR4 and/or downstream signaling pathway components in SCLC metastasis processes in vitro and in vivo.
Analysing the CXCR4/CXCL12 axis we could demonstrate different STAT3 activation types in SCLC cell lines. STAT3 was constitutively phosphorylated on both tyrosine and serine residues, which could be further increased upon CXCL12 stimulation and was completely inhibited by TN14003, a CXCR4 antagonist and/or by CDDO-Me, a synthetic triterpenoid which inhibits STAT3 directly. STAT3 tyrosine phosphorylation could also be inhibited by AG490, a Jak2 inhibitor. In addition to tyrosine phosphorylation, STAT3 acetylation on a lysine residue has also been associated in the initiation of STAT3 dimerization. CXCL12 stimulation of SCLC cells led to an increased STAT3 acetylation specific for the nuclear fraction.
STAT3 may also be involved in Rho GTPases mediated signalling cascades regulating cellular responses like actin organization, proliferation and cell migration. We could show a strong CXCR4 mediated RhoA and Rac1 activation as potential upstream signaling of STAT3 activation in SCLC cell lines. Further functional investigations (e.g. chemotaxis, pseudoemperipolesis, and cell viability assessment) will reveal the involvement of STAT3 and Rho GTPases in CXCR4 mediated metastatic processes in SCLC.
To investigate the effects of CXCR4 inhibitors and inhibitors of downstream pathways on metastasis formation in vivo, an orthotopic tumor mouse model is being established. Human SCLC cells are injected intrathoracically into the pleural space of Rag2γc mice. Tumor engraftment can be observed using BL-Imaging, MRI and PET. Six weeks after inoculation of tumor cells the animals are sacrificed in order to investigate metastasis formation in specific CXCL12 expressing organs.
To summarise, we have shown constitutive serine and tyrosine phosphorylation of STAT3 and CXCL12 induced regulation. CXCL12 also induced acetylation of STAT3 and activation of RhoA and Rac1. These results are clarifying the CXCR4 downstream cascade in small cell lung cancer and underline the interest in inhibitors for this receptor and its targets in this pathway for potential therapeutic use.