Alternative implication of CXCR4 in JAK2/STAT3 activation in small cell lung cancer

Abstract

Small cell lung cancer (SCLC) is an aggressive, rapidly metastasising tumour. Previously, we demonstrated the influence of CXCL12-CXCR4 interaction on processes involved in metastasis and chemoresistance in SCLC. We show here that STAT3 is expressed in both primary SCLC tumour tissues and SCLC cell lines. We investigated the function of STAT3 upon CXCL12 stimulation in SCLC cell lines. Small cell lung cancer cell lines present constitutive phosphorylation of STAT3, and in the reference cell lines NCI-H69 and NCI-H82 constitutive phosphorylation was further increased by CXCL12 stimulation. Further investigating this signalling cascade, we showed that it involves interactions between CXCR4 and JAK2 in both cell lines. However CXCL12-induced adhesion to VCAM-1 could be completely inhibited by the JAK2 inhibitor AG490 only in NCI-H82. Furthermore, CXCR4 antagonist but not AG490 inhibited cell adhesion whereas both antagonisms were shown to inhibit growth of the cells in soft agar, indicating the central involvement of this signalling in anchorage-independent growth of SCLC cells. Most interestingly, while using primary tumour material, we observed that in contrast to non-small-cell lung cancer samples from primary tumour tissues, all analysed samples from SCLC were strongly positive for tyrosine-phosphorylated STAT3. Taken together, these data indicate that STAT3 is constitutively phosphorylated in SCLC and is important in SCLC growth and spreading thus presenting an interesting target for therapy.

Figure 1

Characterisation of JAK2/STAT3 pathway in SCLC cells. Nuclear extracts of untreated SCLC cells were analysed by western blot analysis and showed STAT3 phosphorylation at different degrees. (A) CXCL12-induced phosphorylation of STAT3 occurred in a time-dependent manner, as observed in western blot analysis with nuclear extracts of NCI-H69 treated with 100 ng ml⁻¹ CXCL12 (B). All tested cell lines but NCI-N592 showed induction of STAT3 phosphorylation by CXCL12 stimulation (C). JAK2 was stained in a western blot analysis following co-immunoprecipitation with a CXCR4 antibody after stimulating NCI-H69 (left panel) and NCI-H82 (right panel) with 100 ng ml⁻¹ CXCL12 for the indicated period of time (D). Each blot shown is representative for at least three independent experiments.

Figure 2

Expression of integrin subunits in SCLC cell lines. Fluorescence histograms depicting the expression of β1-integrin (CD29) and α1-, α2-, α3-, α4-, α5-integrin (CD49a, CD49b, CD49c, CD49d, CD49e) subunits by SCLC cell lines NCI-H69 and NCI-H82 are shown by black lines. The respective isotype controls are represented by grey shaded histograms.

Figure 3

Adhesion to extracellular matrix proteins. Relative adhesion rates of NCI-H82 (A) and NCI-H69 (B) to VCAM-1, fibronectin and collagen I measured after 30 min are shown for untreated controls, cells stimulated with co-immobilised CXCL12 (8 μg ml⁻¹), cells preincubated with AG490 (60 min, 100 μM), and cells that were both preincubated with the inhibitor and stimulated by CXCL12. Error bars represent s.e.m. Differences were accepted as significant when P<0.05 (^*), n=5, each experiment in quadruplicates.

Figure 4

Adhesion to stromal cell line M2-10B4. NCI-H82 (A) and NCI-H69 (B) were allowed to adhere to M2-10B4 cells for 3 h after pretreatment with AG490 (100 μM, 60 min) or TN14003 (100 μg ml⁻¹, 30 min), respectively, then submitted to vigorous washes to remove non-adherent cells from the medium, the whole-cell complex was then submitted to trypsinisation to put it in suspension before assessment of the proportion of each cell type by FACS (n=3).

Figure 5

Anchorage-independent growth in soft-agar. SCLC cells NCI-H82 (A, B and E, F) and NCI-H69 (C, D and G, H) were singularised, put in a 0.3% agarose gel and observed in the absence (A, C) or presence of TN14003 (100 μgml⁻¹) (E, G) and AG490 (100 μM) (F, H). Appropriate controls were done with DMSO (B, D) to rule out any involvement of this solvant in the observed results. Characteristic cell aggregates were photographed after 14 days (n=3).

Figure 6

Representative immunohistochemical staining of primary tumour samples from 10 patients with SCLC (A and C) and 13 patients with NSCLC (B and D). Slides were stained with phospho-Tyr705-STAT3 antibody (upper row: A and B) and STAT3 antibody (inferior row: C and D), respectively.