Trop-2 induces ADAM10-mediated cleavage of E-cadherin and drives EMT-less metastasis in colon cancer

Abstract

We recently reported that activation of Trop-2 through its cleavage at R87-T88 by ADAM10 underlies Trop-2-driven progression of colon cancer. However, the mechanism of action and pathological impact of Trop-2 in metastatic diffusion remain unexplored. Through searches for molecular determinants of cancer metastasis, we identified TROP2 as unique in its up-regulation across independent colon cancer metastasis models. Overexpression of wild-type Trop-2 in KM12SM human colon cancer cells increased liver metastasis rates in vivo in immunosuppressed mice. Metastatic growth was further enhanced by a tail-less, activated ΔcytoTrop-2 mutant, indicating the Trop-2 tail as a pivotal inhibitory signaling element. In primary tumors and metastases, transcriptome analysis showed no down-regulation of CDH1 by transcription factors for epithelial-to-mesenchymal transition, thus suggesting that the pro-metastatic activity of Trop-2 is through alternative mechanisms. Trop-2 can tightly interact with ADAM10. Here, Trop-2 bound E-cadherin and stimulated ADAM10-mediated proteolytic cleavage of E-cadherin intracellular domain. This induced detachment of E-cadherin from β-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of β-catenin signaling, which were further enhanced by the ΔcytoTrop-2 mutant. This Trop-2/E-cadherin/β-catenin program led to anti-apoptotic signaling, increased cell migration, and enhanced cancer-cell survival. In patients with colon cancer, activation of this Trop-2-centered program led to significantly reduced relapse-free and overall survival, indicating a major impact on progression to metastatic disease. Recently, the anti-Trop-2 mAb Sacituzumab govitecan-hziy was shown to be active against metastatic breast cancer. Our findings define the key relevance of Trop-2 as a target in metastatic colon cancer.

Keywords: E-cadherin; Epithelial-mesenchymal transition; Metastasis; Signaling networks; Trop-2; proteolytic cleavage; β-catenin.

Fig. 1

Transcriptome profiling of metastatic colon cancer cells. (A) Transcriptome profiling of metastatic colon cancer cells: HCT116U5.5 vs HCT116 (HCT column) and KM12L4A (A) or KM12SM (B) vs KM12C (KM12 columns). The profiles of the two independent colon cancer metastatic models showed largely mutually exclusive expression changes, indicating widely divergent transcriptomic scenarios. Pseudo-color-coded change factors (CF), according to the scale bar (green, increased expression in metastatic cells; red, decreased expression; white, no change). Genes are sorted by descending CF values in HCT116 cells. Yellow highlight: only two genes, TROP2/TACSTD2 and VIMENTIN, were upregulated in both systems. (B) Expression levels of TROP2 mRNA in KM12C and HCT116 versus KM12SM, KM12L4A or HCT116-U5.5 colon cancer cells. DNA microarray and Northern blot (left), and RT-PCR (upper right) analyses, with ACTB and 28S as loading controls. Western blot analysis (lower right) for corresponding Trop-2 protein expression were also performed, with β-actin as loading controls. (C) IHC and immunofluorescence assessment of Trop-2 expression in KM12SM and HCT116 U5.5 versus HCT116 colon cancer cells (arrows). Size bars: 50 μm.

Fig. 2

Trop-2 drives metastasis in vivo. (A) Primary spleen tumors and metastatic livers from nude mice injected intra-spleen with KM12SM cells transfected with vector alone, wtTrop-2 or ΔcytoTrop-2. Representative images are shown, full data analysis is in Table S2A. (B, top) Histopathology analysis of spleen and liver tumors from KM12SM transfectants. Control KM12SM/vector cells (left), with central necrotic areas (red arrowheads), defined fibrous capsules and glandular lumen rudiments with apoptotic bodies (inset, black arrowheads); KM12SM/wtTrop-2 (middle), showing a pleomorphic appearance, numerous mitotic figures and lesser necrosis compared with vector-alone (red arrowheads) and instances of endoluminal vascular growth (bottom panel, inset); ΔcytoTrop-2 induced further loss of differentiation: KM12SM/∆cytoTrop-2 (right), showing an invasive growth pattern (bottom panel, inset), frequent mitoses and peripheral haemorrhagic necrosis. (B, bottom) Decreased cell-cell adhesion in liver metastases from KM12SM/∆cytoTrop-2 (right) compared to liver metastases from KM12SM/vector cells (left) (arrows). Nodular growth borders are indicated by arrowheads. A representative image of the extent of pseudoglandular differentiation in parental (vector), wtTrop-2 (wtT2), ∆cytoTrop-2 (∆cT2) cells is shown in the central panel, with data analysis as distribution boxplots of small and large gland-like structures shown below. (C) Spleen tumors and liver metastases from control parental KM12SM, wtTrop-2 and ∆cytoTrop-2 were scored for occurrence of apoptotic bodies (top) and mitotic figures (bottom) (arrowheads). Representative images are shown on the left, with data analysis as distribution boxplots shown on the right. Tissue sections were stained with haematoxylin and eosin. Size bars = 50 μm. Individual counts for psuedoglandular structures, apoptotic bodies and mitotic counts were obtained per independent 20x optical field and are listed in Table S2B. Statistical analysis was conducted as in Material and Methods. Vector: n = 6; wtTrop-2: n = 25; ΔcytoTrop-2: n = 35.

Fig. 3

Trop-2 drives functional inactivation of E-cadherin (A) E-cadherin immunoprecipitates from KM12SM transfectants were analysed by Western blotting for β-catenin (β-cat), Trop-2 and β-actin (β-act). E-cadherin (E-cad) was also probed as control (left). Corresponding cell lysates were also analysed, with Akt as protein loading control (right). (B) Matrigel invasion by KM12SM vector-alone transfectants compared to KM12SM cells expressing either the wtTrop-2 or the ΔcytoTrop-2 at 5 days from seeding (left). Representative images of three independent experiments are shown. The arrow indicates the direction of invasion through the Matrigel layer. Clusters of invading cells are detected only upon overexpression of Trop-2. The total number of invading clusters was not significantly different between wtTrop-2 and ΔcytoTrop-2. The distance of each invading cluster from the matrigel surface along the Z axis was measured; average values for each group are showm in the histogram on the right. (C) Cell-cell aggregation in KM12SM transfectants, assessed 1 h and 24 h after seeding on low-attachment plates. Representative phase-contrast images are shown (top). Distribution of cell aggregation states after 1 h from seeding was profiled as % of cells in each aggregate class.

Fig. 4

Trop-2 induces E-cadherin cleavage by ADAM10 (A) Western blot analysis of KM12SM/Trop-2 and vector-only transfectants with antibodies against the E-cadherin N-terminal extracellular domain (top) or C-terminal cytoplasmic tail (bottom). Molecular weight markers are indicated on the right. Red arrowhead: cleaved E-cadherin 80 kD extracellular domain and 30 kD cytoplasmic domain. Ponceau red staining is shown as protein loading control. (B) HT29 colon camcer cell subpopulations were selected from the parental cell line by flow cytometry for low/nihl or high expression of Trop-2 (top) and analysed for E-cadherin cleavage by Western blotting as above on full length gels (bottom). Uncleaved E-cadherin provide loading control. (C) KM12SM/Trop-2 cells were infected with a lentiviral shRNA targeting the ADAM10 transcript (ADAM10-sh1) or with a scramble shRNA (scr-sh) as control. ADAM10 levels (top) and E-cadherin cleavage (middle, bottom) were revealed by Western blotting as above. Ponceau red staining is shown as protein loading control. (D) Trop-2 immunoprecipitates from KM12SM transfectants were analysed by Western blotting for ezrin (top). Ezrin levels in corresponding whole lysates are shown (bottom).

Fig. 5

β-catenin release and activation by Trop-2. Activation of β-catenin transcriptional activity requires transit at the cell membrane and transport to the nucleus. Levels and localization of β-catenin were analyzed in KM12C, KM12SM cells transfected with wtTrop-2, ΔcytoTrop-2 or control vector. (A) Confocal microscopy analysis of active unphosphorylated β-catenin in KM12SM transfectants. Active β-catenin colocalization with E-cadherin at the cell membrane (left). Nuclear localization (arrowheads): nuclei were identified by counterstaining with DRAQ5 (blue pseudo-color) (middle). Co-staining with Trop-2 (left) (B) Histogram plots of active β-catenin in the nuclei of KM12SM transfectants (see Table S5 for the full dataset) (top). Histogram plots of GFP-positive/β-catenin-responsive cells in KM12C non-metastatic and KM12SM metastatic transfectants; subgroup percent values are indicated. (C) Flow cytometry analysis of GFP expression in KM12SM cells co-transfected with the pTOPFLASH-EGFP β-catenin-reporter and the empty vector (top) or the expression vector for wtTrop-2 (tmiddle) or ΔcytoTrop-2 (bottom). GFP average fluorescence intensity (FI) is indicated as normalized for Trop-2 transfection efficiency.

Fig. 6

Impact of the Trop-2/E-cadherin/β-catenin module on colon cancer patient survival. (A) Colon cancer IHC staining over a series of cases from high (i) to low (iv) expression of Trop-2 (magnification 40x) (left). (B) ROC analysis was applied to obtain the Trop-2 positivity threshold according to the 0-1 criterion. The optimal cut-off parameter for Trop-2 positive expression was 88% (Trop-2^high: score above the cut-off threshold; Trop-2^low score below the threshold). Kaplan-Meier analysis of overall survival (OS) according to Trop-2 expression levels: 57.1% of patients harboring Trop-2^hi. (C) DNA microarray data from colon cancer patients were pre-processed and meta-analyzed through the KMPlot database (www.kmplot.com). Kaplan-Meier (KM) curves showing the individual impact of TROP2, CTNNB1 and CDH1 transcript levels on overall survival (OS) are depicted on the left, while the impact of TROP2, CTNNB1 and CDH1 coexpression is shown on the right. Vertical bars are shown for assessment at 3 and 5 years from diagnosis.