HGF-induced MET activity promotes TNBC breast cancer invasion through invadopodia-mediated ECM degradation.

(A) Bright-field images of MDA-MB-231 spheroids embedded in the collagen matrix at 0h and 48h in the presence or absence of HGF or complete media. The ratio of the invaded area to the core area was quantified and plotted. The core has been outlined in green, and the invaded area has been outlined in yellow. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, One-way ANOVA with multiple comparisons. **** P < 0.0001, * P < 0.05, Scale bar: 100µm. N=3, n=30. (B) MDA-MB-231 cells were seeded on Alexa568-labeled gelatin-coated coverslip with or without HGF for 3h. The degradation index was quantified and plotted. The arrowhead represents degradation spots. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Unpaired t-test, **** P < 0.0001. scale bar: 10µm. N=3, n=200. (C) MDA-MB-231 cells were seeded on a gelatin-coated glass-bottom dish and allowed to attach for 3 hours, followed by incubation with or without HGF for an additional 3 hours. Cells were fixed and immunostained for TKS5 and Cortactin. Images were captured using a TIRF microscope. TKS5 puncta positive for Cortactin were considered as invadopodia. Arrowheads indicate TKS5-Cortactin positive puncta. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Paired t-test, ** P < 0.01. scale bar: 10µm. N = 4, n = 150. (D, E) MDA-MB-231 cells were seeded on gelatin-coated coverslips for 3h, followed by incubation with vehicle or PHA665752 (5 µM) with or without HGF for an additional 3h. Cells were fixed, immunostained with anti-TKS5 antibody, Phalloidin to stain F-actin, and DAPI. Images were captured using a confocal microscope. TKS5 puncta positive for Actin were considered as invadopodia. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm. Inset: 2µm. The error bar represents Mean ±SEM, One-way ANOVA with multiple comparisons. **** P < 0.0001, ** P < 0.01, ns P > 0.05, N=3, n=200. (F) MDA-MB-231 cells were transfected with control SHC002 or MET shRNA clone #3, and after 36h of transfection, cells were seeded on Alexa568-labeled gelatin-coated coverslip for 6h. The cells were fixed and immunostained with anti-MET antibody, Phalloidin, and DAPI. Degradation spots immunostained for Actin were quantified and plotted. The inset shows a magnified view of the region indicated by the box. The Arrowheads indicate the degradation spots that were immunostained for Actin. scale bar: 10µm. Inset: 2µm. The error bar represents Mean ±SEM, Unpaired t-test. *** P < 0.001, N=3, n=200.

Enhanced MET recruitment to Invadopodia upon HGF stimulation.

(A) MDA-MB-231 cells were seeded on gelatin-coated imaging dishes for 6h. Cells were fixed and immunostained for Cortactin and MET. Images were captured using a TIRF microscope. Colocalized Cortactin-MET puncta were quantified and plotted. The arrowheads indicate the MET-positive Cortactin puncta. The error bar represents Mean ±SD, scale bar: 10µm, n= 60. (B) GFP-TKS5-expressing MDA-MB-231 cells were seeded on gelatin-coated imaging dishes for 6h. Cells were fixed, immunostained for MET and imaged with TIRFM. The arrowhead indicates the MET-positive TKS5 puncta. scale bar: 10µm. (C) Cherry-TKS5-expressing MDA-MB-231 cells were seeded on gelatin-coated glass-bottom imaging dishes for 6h. Cells were fixed, immunostained with anti-pY-MET antibody and imaged with TIRFM. The arrowhead indicates the pY-MET-positive TKS5 puncta. scale bar: 10µm. (D) Colocalized TKS5-MET or pY-MET puncta were quantified and plotted. The error bar represents Mean ±SD, N=3, n ≤ 50. (E) The fluorescence intensity of TKS5 and MET at individual invadopodia was quantified using Motiontracking and normalized with their respective mean intensities. The normalized fluorescence intensities of TKS5 (X-axis) and MET (Y-axis) were plotted. The points were fitted with a smoothing spline. n=800. (F) MDA-MB-231 cells were seeded on Alexa568-labeled gelatin-coated coverslips for 12h. Cells were fixed and immunostained for F-actin and MET. The inset shows a magnified view of the region indicated by the box. The white Arrowhead represents Actin at the degradation spots, and the yellow arrowhead represents Actin-MET puncta without degradation. scale bar: 10µm. Inset: 2µm. (G) BT-549 cells were seeded on gelatin-coated coverslips for 3h followed by incubation with or without HGF for 3h. Cells were fixed, immunostained for TKS5, MET and Nucleus. Images were taken using the confocal microscope. Colocalized TKS5-MET puncta were quantified and plotted. The error bar represents Mean ±SEM/SD, Unpaired t-test scale bar: 10µm, *** P < 0.001, N=3, n = 200. (H) BT-549 cells expressing GFP-MET were seeded on glass-bottom dishes. The cells were imaged using a TIRF microscope in the presence or absence of HGF. The GFP-MET vesicles moving in the TIRF field for a minimum of 4 consecutive frames were quantified and plotted as MET tracks. The error bar represents Mean ±SEM, Unpaired t-test. scale bar: 10µm. ** P < 0.01, N=3, n=30.

Intracellular trafficking of MET promotes invadopodia-associated ECM degradation and breast cancer cell invasion.

(A) Graphical representation of WT MET and mutants’ endocytosis and degradation. (B) BT-549 cells expressing WT or the mutants of V5-MET were treated with or without HGF for 2 hours, followed by cell lysis. The lysates were subjected to immunoblotting with anti-V5 and anti-vinculin antibodies. The V5 signal intensity was quantified and normalized with Vinculin. Error bar represents Mean ±SEM. Unpaired t-test. * P < 0.05. (C) BT-549 cells expressing WT or M1250T V5-MET were lysed and incubated with Ni-NTA beads. The pulled-down samples were processed for immunoblotting for MET and pY-MET. (D, D’) WT or mutant V5-MET expressing BT-549 cells were seeded on glass coverslips with or without HGF. Cells were fixed, immunostained for V5 and EEA1. Images were captured with a confocal microscope. V5-MET puncta localizing with EEA1 were quantified and plotted. scale bar: 10µm. The error bar represents Mean ±SEM. ** P < 0.01. ns P > 0.05 N=3, n=60. (E, E’) BT-549 cells expressing V5-MET WT or mutants were seeded on Alexa568-labeled gelatin-coated coverslips with or without HGF for 6h. Cells were fixed, then immunostained for V5 and the Nucleus and imaged with the confocal microscope. Gelatin degradation by the V5-MET overexpressing cells was calculated and plotted. scale bar: 10µm. The error bar represents Mean ±SEM, Unpaired t-test. ** P < 0.01, ns P > 0.05, N=3, n=120. (F) V5-MET WT or M1250T mutant expressing BT-549 cells were seeded on a gelatin-coated coverslip for 6h and fixed. Cells were immunostained for V5, TKS5, and images were captured with the confocal microscope. The error bar represents Mean ±SEM, Unpaired t-test. scale bar: 10µm. ** P < 0.01, N=3, n=120 (G) Bright-field images of GFP-MET WT or M1250T expressing BT-549 spheroids embedded in the collagen matrix at 0h and 24h. The ratio of the invaded area to the core area was quantified and plotted. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 100µm. **** P < 0.0001, N=3, n=40.

HGF promotes RAB4 & RAB14 mediated MET delivery to the plasma membrane MDA-MB-231 cells, seeded on glass coverslips, were transfected with.

(A) GFP-RAB4 or (B) Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with or without HGF, then fixed and immunostained for MET. Images were captured using a confocal microscope. scale bar: 10µm. (C) Graph showing the percent colocalization of MET with GFP-RAB4 and Cherry-RAB14 in the presence of absence of HGF treatment. The error bar represents Mean ±SD, One-way ANOVA. ** P< 0.01. N=3, n=200 (D) BT-549 cells were transfected with siRNA against RAB4, RAB14, RAB22, or control siRNA. After 60 hours, cells were transfected with GFP-MET and seeded on the gelatin-coated glass-bottom dish. The GFP-MET-transfected cells were imaged in the presence of HGF using a TIRF microscope for 30 frames without an interval. scale bar: 10µm (E) The GFP-MET vesicles forming the track were identified as mentioned in the Method section. The numbers of GFP-MET tracks moving for at least 4 consecutive frames were quantified for each condition and plotted. In addition, the total number of GFP-MET vesicles was quantified. The error bars represent Mean±SD. Unpaired t-test. *** P< 0.001, ** P< 0.01, ns P > 0.05, N=3, n = 30 (F) BT-549 cells were depleted with RAB4 or RAB14, and after 60h, cells were seeded on gelatin-coated coverslips for 6h with HGF stimulation. Cells were fixed and immunostained for MET, TKS5, and F-actin. Images were captured in a confocal microscope. MET colocalizing with invadopodia was quantified and plotted. The number of cells forming lamellipodia was quantified. scale bar: 10µm. The error bar represents Mean ±SEM, One-way ANOVA, *** P< 0.001, ** P< 0.01. N=3, n=200.

RCP-RAB14-KIF16B trafficking axis regulates MET recycling.

(A) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with or without HGF, then fixed and immunostained for MET. Images were captured using a confocal microscope. scale bar: 10µm. The percent colocalization of GFP-RCP with RAB14 or RAB14-MET in the presence or absence of HGF treatment was calculated and plotted. scale bar: 10µm. The error bar represents Mean ±SD. Paired t-test. * P< 0.05, N=3, n=200. (B) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with HGF stimulation, then fixed and immunostained for MET. Images were captured using a confocal microscope. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm. Inset: 2µm. (C) BT-549 cells were transfected with GFP-RAB14 and seeded on a glass-bottom imaging dish. Time-lapse images were captured using a confocal microscope. The inset shows the events over time, where the tubule is formed and, upon fission, dissociates from the vesicular body. Scalebar: 10µm, Inset: 2µm (D) BT-549 cells co-transfected with GFP-RCP and Cherry-RAB14 were seeded on a glass-bottom imaging. Time-lapse movies were captured using a confocal microscope. Arrowheads indicate the endosomal tubules containing RCP and RAB14. (E) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with HGF stimulation, then fixed and immunostained for MET. Images were captured using a confocal microscope. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm, Inset: 2µm. (F) BT-549 cells were treated with control or SMARTpool siRNA against RCP. 60h after transfection, cells were transfected with Cherry-RAB14. After providing 2 hours of HGF stimulation, cells were fixed and immunostained for MET and EEA1. Images were captured using a confocal microscope. The percentage colocalization of RAB14 endosomes containing MET with EEA1 was calculated. scale bar: 10µm. The error bar represents Mean ±SD. Paired t-test. ** P< 0.001, ns P > 0.05, N=3, n=200. (G) BT-549 cells seeded on glass coverslips were transfected with YFP-KIF16B and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with HGF stimulation, then fixed and immunostained for MET. Images were captured using a confocal microscope. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm, inset: 2µm (H) BT-549 cells seeded on glass coverslips were transfected with YFP-KIF16B and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with or without HGF, then fixed and immunostained for MET. Images were captured using a confocal microscope. scale bar: 10µm. The percent colocalization of YFP-KIF16B with RAB14 or RAB14-MET in the presence or absence of HGF treatment was calculated and plotted. scale bar: 10µm. The error bar represents Mean ±SD. Unpaired t-test. ** P< 0.01, * P< 0.05, ns P > 0.05, N=3, n=200. (I) BT-549 cells co-transfected with YFP-KIF16B WT or S109A mutant and Cherry-RAB14 were seeded on a glass-bottom imaging dish. Time-lapse images were captured using a confocal microscope. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm, Inset: 2µm. N=3, n=20.

HGF/MET facilitates ECM degradation by recycling MT1-MMP to the surface.

(A) MDA-MB-231 cells seeded on coverslips, surface labeled with MT1-MMP antibody at 4°C for 1 h Complete media was added, and cells were shifted to 37°C for 30 minutes to allow endocytosis. To remove surface-bound antibodies, an acid wash was given. After washing with PBS, serum-free media was added with or without HGF, and the cells were shifted to 37°C for 10 minutes to allow recycling from the endocytosed pool. Cells were fixed and immunostained for EEA1. The integral intensity of the MT1-MMP and the percentage of MT1-MMP antibody recycling were calculated. N = 3, n= 300; scale bars = 10 µm. The error bar represents means ± SD. Paired t-test, *** P< 0.001, Unpaired t-test, * P < 0.05. (B) MDA-MB-231 cells transfected with pHluorin MT1-MMP were seeded on gelatin-coated glass-bottom dishes. Time-lapse imaging was performed in the presence or absence of HGF stimulation using a Nikon TIRF microscope without any intervals for 300 frames. The flashes of pHluorin MT1-MMP, which represent exocytic events, were calculated using Motiontracking. scale bars = 10 µm. The error bar represents Means ± SEM. Unpaired t-test, ** P < 0.001. N = 4, n= 35. (C) MDA-MB-231 cells were seeded on gelatin-coated glass-bottom dishes and allowed to attach for 3h, followed by incubation with or without HGF for another 3h. Cells were fixed and immunostained for TKS5, MT1-MMP, and F-actin. Images were captured using a Nikon TIRF microscope. TKS5, MT1-MMP, and Actin-positive structures were identified using Motiontracking. The error bar represents Mean ±SEM, Unpaired t-test, * P < 0.01. scale bar: 10µm. N=3, n=120. (D) GFP-MT1-MMP expressing MDA-MB-231 cells were fixed and immunostained for MET. Images were captured using an LSM900 Airyscan super-resolution microscope. Images were processed using the Airyscan Joint deconvolution (jDCV) method. The image was converted to Maximum Intensity projection for representation. The inset shows a magnified view of the region indicated by the box. Scale bar: 10 µm, inset: 2 µm. (E) GFP or GFP-MT1-MMP expressing BT-549 cells were lysed and incubated with GST agarose beads bound to 25µg of GFP binding protein (GBP). Next washes were given to remove unbound proteins and proceeded for immunoblotting with GFP and MET. WCL: whole-cell lysates (F) MDA-MB-231 cells co-expressing GFP-MET and Cherry MT1-MMP were subjected to live-cell TIRF imaging. The inset represents an event at different time points. Scale bar: 10 µm, Inset: 2 µm. (G) BT-549 cells expressing MT1-MMP WT or a catalytic mutant alone or with V5-MET WT or M1250T mutant were seeded on gelatin-coated coverlips for 6h and fixed. Cells were immunostained for V5 and TKS5. Images were captured with the confocal microscope. The number of TKS5 puncta in the MT1-MMP and/or MET expressing cells was calculated and plotted. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 10µm. ** P<0.01, * P < 0.05, N=3, n=150.

Proposed model showing RAB14-mediated MET trafficking to invadopodia along with MT1-MMP in the presence of HGF stimulation.

HGF stimulation triggers invadopodia formation and ECM degradation by promoting invadopodia formation and MT1-MMP recycling. HGF also increases the recruitment of MET to invadopodia by stimulating the trafficking of the RTK in the RAB4 and RAB14-dependent trafficking pathway. HGF stimulation increases the phosphorylation (34) and colocalization of RCP with MET-containing RAB14 vesicles. RCP and MET are present on the endosomal budding site of RAB14, which further elongate and form tubules. KIF16B gets recruited to these endosomes through RAB14 and drives the endosomal tubulation for efficient cargo trafficking. MT1-MMP interacts with MET, presumably promoting their co-trafficking to invadopodia.

HGF-induced MET activity promotes TNBC breast cancer invasion through invadopodia-mediated ECM degradation.

(A) From the publicly available database cBioportal, various breast tumor datasets were analyzed for the frequency of alterations for MET. Graph showing frequency for different alteration types in the various breast tumor cohorts (B) From the publicly available database cBioportal, various breast tumor datasets were analyzed for the survival of patients with altered EGFR or MET. (C) Immunoblotting of indicated cell lines with MET and Tubulin. (D) Bright-field images of BT-549 spheroids embedded in the collagen matrix at 0h and 48h in the presence or absence of HGF or complete media. The ratio of the invaded area to the core area was quantified and plotted. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, One-way ANOVA with multiple comparisons. scale bar: 100µm. **** P < 0.0001, *** P < 0.001 N=3, n=30. (E) BT-549 cells were seeded on Alexa568-labeled gelatin-coated coverslip for 3h, followed by incubation with or without HGF for an additional 3h. Cells were fixed, immunostained for TKS5, Cortactin, and imaged with a confocal microscope. The degradation index and number of colocalized TKS5-Cortactin puncta were quantified and plotted. The inset shows a magnified view of the region indicated by the box. The error bar represents Mean ±SEM, Unpaired t-test. scale bar: 10µm. Inset: 2µm, *** P < 0.001, ** P < 0.01, N=3, n=200. (F) MCF10A DCIS cells were seeded on Alexa568-labeled gelatin-coated coverslip with or without HGF for 6h. Degradation Index was quantified and plotted. The Arrowhead represents degradation spots. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 10µm. **** P < 0.0001, N=3, n=200. (G) MCF10A DCIS cells were seeded on a gelatin-coated glass-bottom dish for 3h, followed by incubation with or without HGF for 3h. Cells were fixed and immunostained for Cortactin and F-actin. Images were captured using a TIRF microscope. Actin-positive Cortactin puncta were quantified and plotted. The error bar represents Mean ±SEM, Unpaired t-test. scale bar: 10µm. ** P < 0.01, N=3, n=100 (H) MDA-MB-231 cells were seeded on a gelatin-coated glass bottom dish for 3h, followed by incubation with or without EGF stimulation for an additional 3h. Cells were fixed and immunostained for TKS5 and Cortactin. Images were captured using a TIRF microscope. TKS5 puncta positive for Cortactin were considered as invadopodia. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 10µm. **** P < 0.0001, N=3, n=90 (I) MDA-MB-231 cells were transfected with SHC002 control shRNA or 5 shRNA clones against MET. After 48 hours, cells were lysed and subjected to immunoblotting with anti-MET antibody. The numbers indicate the normalized signal intensity of MET. (J) Immunoblot of MDA-MB-231 cell lysate treated with or without HGF in the presence of vehicle or PHA665752, probed for pY1234-35 MET or MET and vinculin.

HGF promotes MET recycling that drives breast cancer invasion.

(A) BT-549 cells were seeded on gelatin-coated coverslips and allowed to adhere for 3h, followed by 3 hours with HGF stimulation. Cells were fixed, immunostained for TKS5, MET, and the Nucleus. Images were captured using the confocal microscope. The inset shows a magnified view of the region indicated by the box. Scale bar: 10µm. Inset: 2µm. (B) The ratio of the integral intensity of MET to TKS5 was calculated, and the frequency distribution was plotted. n= 800 invadopodia. (C) MDA-MB-231 cells were seeded on gelatin-coated glass-bottom dishes in the presence or absence of HGF, after 6h, fixed, and immunostained for TKS5 and MET. The cells were imaged using a TIRF microscope. The percentage of TKS5 puncta positive for MET was quantified and plotted. scale bar: 10µm. Unpaired t-test. *** P < 0.001, N=3, n=120. (D) MDA-MB-231 cells were seeded on gelatin-coated glass coverslips for 3h, followed by incubation with or without HGF for an additional 3h. The cells were fixed and immunostained with anti-MET, anti-Cortactin antibody, and Phalloidin. The cells were imaged using a confocal microscope. Numbers of Actin-Cortactin puncta positive for MET were quantified and plotted. Scale bar: 10µm. Unpaired t-test. *** P < 0.001, N=3, n=200. (E) MDA-MB-231 cells seeded on glass coverslips were stimulated with or without HGF for 2h. Cells were fixed and immunostained for MET and RAB5. The percent colocalization of MET with RAB5 was calculated. scale bar: 10µm. The error bar represents Mean ±SEM. N=3, n=200. (F) MDA-MB-231 cells were treated with HGF for a period of 0-3h in the presence of cycloheximide, lysed at the indicated time points, and subjected to immunoblotting. The numbers represent the relative MET expression at the mentioned time point. (G) BT-549 cells were seeded on gelatin-coated coverslips for 6h and fixed. Cells were immunostained for EGFR, F-actin, and Cortactin. Images were captured with the confocal microscope. scale bar: 10µm. (H) BT-549 cells were seeded on gelatin-coated coverslips for 6h with or without EGF stimulation and fixed. Cells were immunostained for EGFR, F-actin, and Cortactin. Images were captured with the confocal microscope. The percentage of Actin-Cortactin puncta that immunostained for EGFR was quantified and plotted. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm. Inset: 2µm. Unpaired t-test. ** P < 0.01, N=3, n=100. (I) WT or M1250T V5-MET expressing BT-549 cells were seeded on glass coverslips cells fixed, and immunostained for V5 and pY-MET. Images were acquired with a confocal microscope. Corrected total cell fluorescence (CTCF) for V5 and pY-MET was calculated using ImageJ. The CTCF of pY-MET was normalized with that of V5 and plotted. scale bar: 10µm. The error bar represents Mean ±SEM, Unpaired t-test. ** P < 0.01. N=3, n=10. (J) MDA-MB-231 cells expressing V5-MET WT or mutants were seeded on Alexa568-labeled gelatin-coated coverslips for 12h. Cells were fixed, then immunostained for V5, TKS5, and the Nucleus. Cells were imaged with the confocal microscope. Gelatin degradation by the V5-MET overexpressing cells was calculated. The error bar represents Mean ±SEM, Unpaired t-test. * P < 0.05 ns P > 0.05 scale bar: 10µm. N=3, n=120 (K, K’) V5-MET WT or M1250T or LL/AA mutant expressing MDA-MB-231 cells were seeded on a gelatin-coated glass-bottom dish for 6h and fixed. Cells were immunostained for TKS5, and images were captured with the TIRF microscope. Data points from individual biological replicates are distinctly color-coded. The error bar represents Mean ±SD, Unpaired t-test. Scale. ** P < 0.01, ns P > 0.05 bar: 10µm. N=3, n=60.

HGF promotes RAB4 and RAB14-mediated MET delivery to the plasma membrane.

MDA-MB-231 cells seeded on glass coverslips were transfected with (A) GFP-RAB22 or (B) GFP-RAB11. After 14h of transfection, cells were incubated for 2h with or without HGF, then fixed and immunostained for MET. Images were captured using a confocal microscope. The percent colocalization of MET with GFP-RAB22 or GFP-RAB11 in the presence or absence of HGF treatment was calculated and plotted. Scale bar: 10µm. The error bar represents Mean ±SD. Unpaired t-test. ns P > 0.05 N=3, n=200. (C) MDA-MB-231 cells seeded on glass coverslips were incubated for 2h with or without HGF. Cells were fixed and immunostained for MET and VPS35. The percent colocalization of MET with VPS35 was calculated. scale bar: 10µm. The error bar represents Mean ±SEM. Unpaired t-test. ns P> 0.05. N = 3, n = 200 (D). The KD efficiency of RAB4, RAB14, and RAB22 was confirmed by quantitative RT-PCR. N = 3. Values in the graph represent means ± SEM. Paired t-test, ** P < 0.01, * P < 0.05. (E) MDA-MB-231 cells were transfected with control or RAB4, RAB14, or RAB22 siRNA. After 60 hours, cells were harvested and processed for immunoblotting to analyse total MET levels. The relative MET levels were quantified. The numbers indicate the normalized MET signal intensity, N=3. (F) MDA-MB-231 cells were transfected with control or VPS26A siRNA. After 60 hours of transfection, cells were given a pulse of HGF for 5 min, and excess HGF was washed off. The cells were incubated at 37°C for 30 min and fixed. The cells were immunostained for MET and F-actin. Images were captured in a confocal microscope, and MET localized to Actin at the cell periphery was quantified. scale bar: 10µm. The error bar represents Mean ±SD. Unpaired t-test. ns P>0.05. N=3, n=200 (G) MDA-MB-231 cells were transfected with control or VPS26A siRNA. After 60 hours of transfection, cells were harvested and processed for immunoblotting to analyse total MET levels. (H) MDA-MB-231 cells transfected with GFP-RAB14 and incubated with or without HGF. Cells were fixed, immunostained for MET, and then imaged using a confocal microscope. The mean Integral intensity and area of GFP-RAB14 vesicles were calculated and plotted. The error bar represents Mean ±SEM. Unpaired t-test. **** P< 0.0001, ns P>0.05, N=3, n=200. (I) BT549 cells grown on coverslips were transfected with Cherry-RAB14. Cells were treated with HGF for 2h and fixed, followed by immunostaining with antibodies against MET and EEA1. Images were captured using a confocal microscope. The white arrowhead indicate the MET-RAB14-EEA1 positive puncta, yellow arrowhead indicates the MET-RAB14 endosomes devoid of EEA1. scale bar: 10µm. (J) BT-549 cells co-expressing GFP-RAB4 and Cherry-RAB14 were fixed, immunostained for MET and imaged with the super-resolution microscope. The inset shows a magnified view of the region indicated by the box. The white arrowhead represents RAB4-MET vesicles, and the yellow arrowhead represents RAB14-MET vesicles. scale bar: 10µm, Inset: 2µm. (K) BT-549 cells expressing Cherry-RAB14 were fixed, then immunostained with anti-MET and anti-GOLGIN 97 antibodies. Images were captured using the Olympus SpinSR super-resolution microscope. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm, Inset: 2µm.

RCP-RAB14-KIF16B trafficking axis regulates MET recycling.

(A) The frequency of alteration of MET in 27 breast cancer datasets was analyzed using the publicly available database cBioportal. The graph shows the frequency of alterations for different breast tumor cohorts. (B) Graph showing the probability of survival of the patients with RCP alteration compared to the unaltered groups. (C) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP. After 14h of transfection, cells were incubated for 2h with HGF stimulation, then fixed and immunostained for MET and EEA1. Images were captured using a confocal microscope. scale bar: 10μm. (D) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP S435A and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with or without HGF, then fixed and immunostained for MET. Images were captured using a confocal microscope. The percent colocalization of GFP-RCP S435A with RAB14 or RAB14-MET in the presence or absence of HGF treatment was calculated and plotted. scale bar: 10µm. The error bar represents Mean ±SD. Unpaired t-test. ** P< 0.01, ns P > 0.05 N=3, n=200. (E) BT-549 cells depleted with RAB14 or control siRNA-treated cells were transfected with GFP-RCP for 14 hours, followed by 2 hours of HGF stimulation. Cells were fixed and immunostained for MET and EEA1. Images were captured using a confocal microscope, and the percentage colocalization of RCP with MET was quantified and plotted. The graph represents Mean ±SD. Unpaired t-test. ns P > 0.05, N=3, n=200. (F) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP and Cherry-RAB14. After 14h of transfection, cells were incubated for 2h with HGF stimulation, then fixed and immunostained for MET. Images were captured using a confocal microscope. The inset shows a magnified view of the region indicated by the box. scale bar: 10µm. Inset: 2µm. (G) BT-549 cells seeded on glass coverslips were transfected with GFP-RCP. After 14h of transfection, cells were incubated for 2h with HGF stimulation, then fixed and immunostained for MET and WASH. Images were captured using a confocal microscope. scale bar: 10μm. (H, H’) BT-549 cells were treated with control or RCP siRNA. The efficiency of gene silencing was estimated by qRT-PCR and immunoblotting. N=2. (I) BT-549 cells were treated with control or SMARTpool siRNA against RCP. 60h after transfection, cells were transfected with Cherry-RAB14. After providing 2 hours of HGF stimulation, cells were fixed and immunostained for MET and EEA1. Images were captured using a confocal microscope. The percentage colocalization of RAB14 or MET endosomes that do not EEA1 was calculated. scale bar: 10µm. The error bar represents Mean ±SD. Paired t-test, ns P > 0.05, N=3, n=200. (J) BT-549 cells depleted of RAB14 or control siRNA-treated cells were transfected with YFP-KIF16B. After 14 hours of transfection, cells were treated with HGF, followed by fixing and immunostaining for MET and EEA1. Images were captured using a confocal microscope, and the percentage colocalization of KIF16B with MET was quantified and plotted. The graph represents Mean ±SD. Unpaired t-test. * P < 0.05 N=3, n=200.

HGF/MET facilitates ECM degradation by recycling MT1-MMP to the surface.

(A) MDA-MB-231 cells treated with or without 100ng/ml of HGF were lysed and subjected to immunoblotting with anti-MT1-MMP antibody. Vinculin was taken as a loading control. (A’) MDA-MB-231 cells treated with 10 µg/ml of cycloheximide, either in the presence or absence of HGF, were lysed and subjected to immunoblotting with an anti-MT1-MMP antibody. Tubulin was taken as a loading control. (B, B’) Untreated or HGF-treated MDA-MB-231 cells were subjected to surface biotinylation. HGF stimulation was given for 10 minutes, followed by biotin labeling at 4°C for 45 minutes. Washes were given to remove excess biotin; subsequently, biotin was quenched, and cells were lysed. The lysate was allowed to bind with Neutravidin beads, followed by immunoblotting. The numbers indicate the Normalized signal intensity of MT1-MMP. In (B’), the MT1-MMP signal intensity has been normalized with CIMPR. Scale bar: The error bar represents Mean ±SD, Paired t-test, * P < 0.05. N=4. (C) MDA-MB-231 cells were treated with control or MET siRNA. The efficiency of gene silencing was estimated by immunoblotting. To analyze total MT1-MMP levels in MET-depleted cells, the membrane was probed with an MT1-MMP antibody. Actin was used as a loading control. (D) MDA-MB-231 cells seeded on coverslips were incubated with MT1-MMP antibody at 4° C. After PBS washes, cells were shifted to 37° C to allow endocytosis in the presence or absence of HGF. The cells were fixed and immunostained with anti-EEA1 antibody. Images were captured using Zeiss LSM780. The integral intensity of the MT1-MMP antibody was calculated and plotted. Scale bar: 10µm. The error bar represents Mean ±SEM, ns P > 0.05, N=3, n = 200. (E) MDA-MB-231 cells were transfected with control or MET siRNA. After 60 hours of transfection, cells were seeded on a gelatin-coated glass-bottom dish and allowed to attach for 3h, followed by incubation with or without HGF for another 3h. Cells were fixed and immunostained for TKS5, MT1-MMP, and F-actin. Images were captured using a Nikon TIRF microscope. TKS5, MT1-MMP, and Actin-positive structures were identified and plotted. The inset shows a magnified view of the region indicated by the box. The error bar represents Mean ±SEM, Unpaired t-test, *** P < 0.001. scale bar: 10µm. Inset: 2µm. N=3, n=120. (F) MDA-MB-231 cells seeded on glass coverslips were incubated with or without HGF. Cells were fixed and immunostained for MT1-MMP and MET. The percentage colocalization was quantified using Motiontracking. Scale bar: 10µm. The error bar represents Mean ±SD, Unpaired t-test. **** P<0.0001. N=3, n=200. (G) Quantification of GBP pulldown assay of GFP-MT1-MMP in 3 different cell lines. The value represents the signal intensity of trapped MET normalized by pulled-down GFP or GFP-MT1-MMP per assay. GFP and GFP-MT1-MMP bars are represented by distinct colors. (H) GFP or GFP-MT1-MMP and V5/H6-MET co-expressing HeLa cell lysates were incubated with Ni-NTA beads. Washes were given, and the pull-down samples were processed for immunoblotting with anti-GFP and anti-V5 antibodies. WCL: whole-cell lysates. (I) BT-549 cells co-expressing pHluorin MT1-MMP with V5-MET WT or M1250T mutant were seeded on gelatin-coated coverslips for 6h and fixed. Cells were immunostained for V5 and TKS5. Images were captured with the confocal microscope. The number of MT1-MMP puncta localizing with TKS5 was calculated and plotted. The inset shows a magnified view of the region indicated by the box. The error bar represents Mean ±SD, Unpaired t-test. scale bar: 10µm. Inset: 2µm. ** P<0.01, * P < 0.05, N=3, n=100 (J, J’) SCR and MT1-MMP knock-out MDA-MB-231 cells were seeded on gelatin-coated glass-bottom dishes for 6 hours. Cells were fixed and immunostained for TKS5 and F-actin. Images were acquired using a TIRF microscope. The number of Actin-TKS5 puncta was quantified and plotted. Scale bar: 10µm. The error bar represents Mean±SD, Unpaired t-test. **** P<0.0001. N=3, n=60. (K, K’) BT-549 cells expressing WT or catalytically inactive (E240A) MT1-MMP were seeded on the gelatin-coated glass-bottom dish for 6h, then fixed and immunostained for TKS5. Images were captured using a confocal microscope. The number of TKS5 puncta in MT1-MMP-expressing cells was quantified and plotted. The error bar represents Mean ±SEM, One-way ANOVA, ** P < 0.01, * P < 0.05. scale bar: 10µm. N=3, n=150. (L) BT-549 cells coexpressing V5-MET WT or M1250T with GFP or GFP-MT1-MMP were lysed and incubated with Ni-NTA beads. Washes were given to remove unbound proteins. The pulled-down samples were processed for immunoblotting with antibodies against V5 and GFP. (M) BT-549 cells expressing V5-MET WT or M1250T were lysed and subjected to immunoblotting with V5, MT1-MMP, and Actin. (N) MDA-MB-231 cells were treated with control or MT1-MMP siRNA and subjected to immunoblotting. The total level of the mature form of MET with a molecular weight of 140 kDa was analyzed in control and MT1-MMP-depleted conditions. Vinculin was taken as a loading control.