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. 2012 Aug 1;72(15):3764-74.
doi: 10.1158/0008-5472.CAN-11-3990. Epub 2012 Jun 1.

The oncogenic lung cancer fusion kinase CD74-ROS activates a novel invasiveness pathway through E-Syt1 phosphorylation

Hyun Jung Jun  1 , Hannah Johnson, Roderick T Bronson, Sebastien de Feraudy, Forest White, Alain Charest
Affiliations

The oncogenic lung cancer fusion kinase CD74-ROS activates a novel invasiveness pathway through E-Syt1 phosphorylation

Hyun Jung Jun et al. Cancer Res. .

Abstract

Patients with lung cancer often present with metastatic disease and therefore have a very poor prognosis. The recent discovery of several novel ROS receptor tyrosine kinase molecular alterations in non-small cell lung cancer (NSCLC) presents a therapeutic opportunity for the development of new targeted treatment strategies. Here, we report that the NSCLC-derived fusion CD74-ROS, which accounts for 30% of all ROS fusion kinases in NSCLC, is an active and oncogenic tyrosine kinase. We found that CD74-ROS-expressing cells were highly invasive in vitro and metastatic in vivo. Pharmacologic inhibition of CD74-ROS kinase activity reversed its transforming capacity by attenuating downstream signaling networks. Using quantitative phosphoproteomics, we uncovered a mechanism by which CD74-ROS activates a novel pathway driving cell invasion. Expression of CD74-ROS resulted in the phosphorylation of the extended synaptotagmin-like protein E-Syt1. Elimination of E-Syt1 expression drastically reduced invasiveness both in vitro and in vivo without modifying the oncogenic activity of CD74-ROS. Furthermore, expression of CD74-ROS in noninvasive NSCLC cell lines readily conferred invasive properties that paralleled the acquisition of E-Syt1 phosphorylation. Taken together, our findings indicate that E-Syt1 is a mediator of cancer cell invasion and molecularly define ROS fusion kinases as therapeutic targets in the treatment of NSCLC.

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Conflict of interest statement

No potential conflicts of interest were disclosed.

Figures

Figure 1
Figure 1
ROS fusion kinases are oncogenic. A, schematic representation of the ROS fusion kinases used in this study. The numbering refers to the amino acid position for the indicated domains of the fusion proteins. All constructs are Myc epitope tagged. B, growth curves of ROS fusion kinases expressing Rat1 clones. For each indicated ROS fusion construct, results from three independent clones are averaged and plotted as the mean ± S.D. Representative photographs of Rat1 cells expressing the indicated ROS fusion kinase variants in (C) focus formation and (D) anchorage-independent growth in soft agar assays. Quantitation of the focus formation assay is depicted in Supplementary Fig. 1B. E, growth of the indicated ROS fusion expressing Rat1 clones as subcutaneous xenografts in immunocompromised mice. 106 Rat1 cells were injected into the flanks of NIHnu/nu mice and tumors developed within 20–25 days. Mice that did not developed tumors were observed for a period of 50 days. Kinase-inactive (KM) versions of these ROS fusions proteins were included as controls. The construct SLC34A2-ROS is abbreviated to SLC-ROS. Scale bar= 250 μm.
Figure 2
Figure 2
Cells expressing ROS fusion kinases are sensitive to ALK inhibitors. A, ROS fusion kinases-expressing Rat1 cells were grown in the presence of NVP-TAE684 to determine IC50 values. B, western blot analysis of total cell lysates from pooled Rat1 clones of ROS fusion kinases treated with NVP-TAE684 for 24 hours and probed for the indicated signaling proteins using phospho-specific antibodies. Blots were stripped and rehybridized with the indicated antibodies to control for expression levels and loading. (C–E) NVP-TAE684 reverses ROS fusion kinase-expressing Rat1 cells transformed phenotype. Cells were treated with NVP-TAE684 (10 nM) or vehicle and assayed for (C) saturation density growth inhibition, (D) focus formation and (E) growth in soft agar. Quantitation of the focus formation assay is depicted in Supplementary Fig. 4B.
Figure 3
Figure 3
CD74-ROS expressing cells are highly invasive and metastatic. (A, B) Representative photomicrographs of H&E stained paraffin embedded cross sections of subcutaneous xenograft tumors from clonal FIG-ROS and CD74-ROS expressing Rat1 cells. Arrows point to invasive areas. Stars mark the locations of ribs. T; tumor, LN; lymph node. Scale bars: (A) 500 μm (B) 250 μm. C, graphical representation of CD74-ROS invasiveness as measured by in vitro Boyden chamber matrigel invasion assays.
Figure 4
Figure 4
Quantitative phosphotyrosine profiling reveals significant differences between FIG-ROS and CD74-ROS expressing Rat1 cells. A, thirty five tyrosine phosphorylated peptides that were found to have significantly different (p=≤0.05) levels between FIG-ROS and CD74-ROS expressing cells are represented in a heat map. Peptides were sorted based on the relative iTRAQ ratios found in CD74-ROS relative to the mean. Protein names are abbreviated and phosphorylation site numbering is based on that present in phosphosite. SLC34A2-(S)ROS expressing cells are abbreviated as SLC-ROS. Ratios shown on the axis refer to iTRAQ ratios relative to the mean. B, bar chart depicting the relative iTRAQ ratios across 3 biological replicates. Error bars are representative of the standard deviation. FIG-ROS-WT and CD74-ROS-WT ratios are significantly different based on students t-test. * p=2.07 e−5. C, a representative manually validated tandem MS spectrum of E-SYT1 peptide DLPDPYVSVLLLPDK indicating the presence of phosphorylation site pY993 (mascot score; 37). D, lysates from parental Rat1 cells (pLXSN vector control), FIG-ROS and CD74-ROS expressing cells were immunoprecipitated (IP) with either E-Syt1 or anti phosphotyrosine antibodies. The immunoprecipitates were analyzed for phosphotyrosine content (pTyr) or for the presence of E-Syt1 protein respectively by immunoblotting (IB).
Figure 5
Figure 5
E-SYT1 is a driver of invasion. A, western blot analysis of E-Syt1 expression knock down from control pLXSN, FIG-ROS and CD74-ROS expressing Rat1 cells using a scrambled control and two short hairpin RNAs. B, graphical representation of the in vitro invasiveness of parental CD74-ROS expressing Rat1 cells and E-Syt1 shRNA knock down clones and controls. C, elimination of E-Syt1 expression in CD74-ROS Rat1 cells abolishes invasion in vivo. Arrows point to invasive areas. Stars mark the locations of ribs. T; tumor. Scale bar: 500 μm.
Figure 6
Figure 6
Expression of CD74-ROS in NSCLC cells induces invasion and E-Syt1 phosphorylation. A, cell lysates from the indicated NSCLC cell lines expressing CD74-ROS-WT or CD74-ROS-KM fusion proteins were immunoblotted for expression levels (9E10) and immunoprecipitated (IP) with an anti phosphotyrosine antibody and analyzed for the presence of E-Syt1 protein by immunoblotting (IB). B, graphical representation of in vitro invasion of NSCLC cell lines expressing CD74-ROS-WT or CD74-ROS-KM fusion proteins. The data is shown as fold increase in invasion when compared to empty pLXSN vector controls for the respective lines and is calculated using three independent clones for each construct.
Figure 7
Figure 7
Expression of CD74-ROS in NSCLC cells induces SHP-2 signaling. Immunoblot analysis of total cell lysates from pools of three independent NSCLC clones from each of the NSCLC cell lines probed for the indicated proteins using phospho-specific antibodies. A, activation of the tyrosine phosphatase SHP-2 and the mTORC1 pathways. B, the STAT-3 and MAPK pathways. All blots were stripped and rehybridized with the indicated antibodies to control for expression levels and loading.
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