Role of c-Met/β1 integrin complex in the metastatic cascade in breast cancer

Metastases cause 90% of human cancer deaths. The metastatic cascade involves local invasion, intravasation, extravasation, metastatic site colonization, and proliferation. Although individual mediators of these processes have been investigated, interactions between these mediators remain less well defined. We previously identified a complex between receptor tyrosine kinase c-Met and β1 integrin in metastases. Using cell culture and in vivo assays, we found that c-Met/β1 complex induction promoted intravasation and vessel wall adhesion in triple-negative breast cancer cells, but did not increase extravasation. These effects may have been driven by the ability of the c-Met/β1 complex to increase mesenchymal and stem cell characteristics. Multiplex transcriptomic analysis revealed upregulated Wnt and hedgehog pathways after c-Met/β1 complex induction. A β1 integrin point mutation that prevented binding to c-Met reduced intravasation. OS2966, a therapeutic antibody disrupting c-Met/β1 binding, decreased breast cancer cell invasion and mesenchymal gene expression. Bone-seeking breast cancer cells exhibited higher levels of c-Met/β1 complex than parental controls and preferentially adhered to tissue-specific matrix. Patient bone metastases demonstrated higher c-Met/β1 complex than brain metastases. Thus, the c-Met/β1 complex drove intravasation of triple-negative breast cancer cells and preferential affinity for bone-specific matrix. Pharmacological targeting of the complex may have prevented metastases, particularly osseous metastases.


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that mammospheres express breast cancer stem cell genes.Related to Figure 1E.Use of qPCR to verify that mammospheres derived from MDA-MB-231 breast cancer cells expressed breast cancer stem cell genes at higher levels than adherent MDA-MB-231 cells.Shown are individual results from the five stem cell genes.n=3/group.*P<0.05;**P<0.01;***P<0.001.Supplemental Figure 3. Assessment of c-Met/b1 complex formation in MCF7-iDimerize-c-Met-b1 cells.Related to Figure 1G.(A) Co-Immunoprecipitation showing gradual increase in complex formation upon addition of Ac ligand.Exogenous ITGB1 was pulled down using HA.(B) Endogenous Immunoprecipitation of ITGB1 shows gradual increase in complex formation.of c-Met/b1 complex formation in MCF-7 cells does not promote intravasation of breast cancer cells.Related to Figure 2B.Shown are immunostainings of CMRA-labeled luminal A MCF-7 breast cancer cells which were incubated in a cell culture intravasation assay for 48 hours in the absence or presence of A/C ligand.n=3/group.Scale bar, 50 µm.c-Met/b1 complexes detected in brain metastases derived form triplenegative versus luminal breast cancer.Related to Figure 2B.Shown are example PLA results for patient breast cancer brain metastases, revealing more c-Met/b1 complex in brain metastases from triple-negative versus luminal breast cancer (P=0.02;n=7).conditioned media promotes intravasation of breast cancer cells.Related to Figure 2D.Shown are immunostainings of CMRA-labeled MDA-MB-231 breast cancer cells which were incubated in a cell culture intravasation assay for 48 hours in the absence or presence of mammosphere-conditioned media (MCM).Results are quantified in Figure 2D.n=3/group.Scale bar, 200 µm.Supplemental Figure 7. Bevacizumab increases c-Met/b1 complex formation in breast cancer cells.Related to Figs. 2E-F.Immunoprecipitation to pull down b1 integrin revealed more c-Met/b1 complex formation after treating MDA-MB-231 breast cancer cells with 2.5 mg/mL bevacizumab for 24 hours.cancer cells.Related to Figure 2F.Shown are immunostainings of CMRAlabeled MDA-MB-231 breast cancer cells and CMFDAlabeled HUVEC cells with DAPI nuclear staining in blue at the 48 hour time point after intravasation assays.n=3/group.Results are quantified in Figure 2F.Scale bar, 20 µm.iDim iDim + A/C Cancer Cells Supplemental Figure 9. c-Met/b1 complex does not alter extravasation of breast cancer cells.Related to Fig. 2Induction of Shown are immunostainings of CMRA-labeled MDA-MB-231 cells at the 48 hour time point after a cell culture extravasation assay.-ligation assays of patient samples reveal c-Met/b1 complexes in different types of metastases.Related to Figure 3G.Shown are PLA immunostainings of breast, renal cell cancer (RCC), and prostate cancer metastases to brain versus bony structures.Results were quantified in Figure 3G.p h a g u s b r a i n B r e a s t b r a i n B r e a s t c l i v u s L u n g b r a i n P r o s t a t e b r a i n Supplemental Figure 11.Immunoprecipitation of patient samples reveals c-Met/b1 complexes in different types of metastases.Related to Fig. 3G.Shown are immunoprecipitations of human tumors in which c-Met is precipitated and blotted for b1 integrin.The ratio of b1 integrin to c-Met quantified band intensity was higher in patient bony metastases (n=11) relative to brain metastases (n=12) from different patients (left two bars; P<0.01) and in paired bone metastases relative to brain metastases from the same patients (n=3; right panel; P<0.001).*P<0.05;**P<0.01;***P<0.001.b1 integrin knockdown via CRISPRi in breast cancer cells.Related to Fig. 4. MDA-MB-231 breast cancer cells were engineered to express KRAB CAS, followed by guide RNAs targeting b1 integrin.Western blot revealed loss of b1 integrin expression in the resulting cells.reveals point mutations that lower binding of b1 integrin to c-Met.Related to Fig. 4. Shown are results when immunoprecipitating for b1 integrin and blotting for c-Met as well as confirmatory blot for b1 integrin in MDA-MB-231 cells engineered for b1 integrin loss via CRISPR followed by restoration of wild-type b1 integrin (ctrl) or restoring b1 integrin with point mutations D246A and D287A which we previously demonstrated to reduce binding to c-Met in glioblastoma cells.Results confirmed reduced binding to c-Met in MDA-MB-231 breast cancer cells.Supplemental Figure 14.Individual mesenchymal transcription factors do not change in expression after OS2966 treatment of MDA-MB-231 iDim cells.Related to Figure 5B.Shown are qPCR results for six mesenchymal transcription factors in MDA-MB-231-iDimerize-c-Met-β1 cells after 24 hours of OS2966 treatment (n=3/group).*P<0.05;**P<0.01;***P<0.001.Supplemental Figure 15.Bone and lung-seeking breast cancer cells have elevated expression of mesenchymal genes compared to brain-seeking and parental breast cancer cells.Related to Figure 5D.Shown are relative expression of seven mesenchymal genes in MDA-MB-231 parental breast cancer cells and MDA-MB-231-BR brain-seeking, MDA-MB-231-BO bone-seeking, and MDA-MB-231-LM2 lungseeking cells derived from the parental MDA-MB-231 cells.*P<0.05;**P<0.01;***P<0.001. .Individual mesenchymal transcription factors do not change in expression after OS2966 treatment of MDA-MB-231 breast cancer cells.Related to Figure 5D.Shown are qPCR results for six mesenchymal transcription factors in MDA-MB-231 lines after 48 hours of OS2966 treatment (n=3/group).*P<0.05;**P<0.01;***P<0.001.Supplemental Figure 17.Individual mesenchymal transcription factors do not change in expression after OS2966 treatment of MDA-MB-231-BR brain-seeking breast cancer cells.Related to Figure 5D.Shown are qPCR results for six mesenchymal transcription factors in MDA-MB-231 BR lines after 48 hours of OS2966 treatment (n=3/group).*P<0.05;**P<0.01;***P<0.001.mesenchymal transcription factors do not change in expression after OS2966 treatment of lung-seeking MDA-MB-231 breast cancer cells.Related to Figure 5D.Shown are qPCR results for six mesenchymal transcription factors in MDA-MB-231 lung lines after 48 hours of OS2966 treatment (n=3/group).*P<0.05;**P<0.01;***P<0.001.

Table S1 .
Genes related to canonical cancer pathways that are upregulated by c-Met/b1 compelx formation.

Table S2 . Pathways activated by c-Met/b1 complex induction in breast cancer cells.
Shown are the pathways activated when MDA-MB-231-iDimerize-c-Met-β1 cells were treated with AP21967 based on assessed in the NanoString nCounter platform using a 770 gene multiplex related to 13 cancer-associated canonical pathways

Genes whose expression is altered when b1 integrin cannot bind c-Met in breast cancer cells.
Shown are the genes whose expression is altered when MDA-MB-231 breast cancer cells undergo CRISPRi knockdown of b1 integrin followed by lentiviral transduction of the b1D246A mutant vs. wild-type b1 integrin, as assessed in the NanoString nCounter platform using a 770 gene multiplex related to each step in the cancer progression process.

Table S4 .
Primers used for qPCR.Shown are primers used for qPCR to assess expression of genes in breast cancer cells.