Trop-2 is a calcium signal transducer that is associated with transformed cell growth in experimental systems. However, its role in human cancer remains essentially unknown. In this study, we profiled Trop-2 expression in normal human tissues at the mRNA and protein levels. We then systematically compared Trop-2 mRNA and protein levels in tumours with their tissues of origin. We find that Trop-2 expression is invariably upregulated in tumours, regardless of baseline expression in normal tissues, which suggests a corresponding selective advantage. Thus, we investigated the outcome of Trop-2 upregulation on tumour growth. Overexpression of wild-type Trop-2 was shown to be necessary and sufficient to drive cancer growth in a widely invariant manner across cell type and species. Upregulation of Trop-2 was shown to quantitatively stimulate tumour growth, as proportional to expression levels in vivo, and tumour cell growth was abrogated by somatic knockdown of Trop-2 expression. On the other hand, we found no evidence of tumour-associated TROP2 mutations, nor of TROP2 induction of oncogenic transformation per se. Our data support a model where above-baseline expression of wild-type Trop-2 is a key driver of human cancer growth.

Our findings show that upregulation of a wild-type Trop-2 has a key controlling role in human cancer growth, and that tumour development is quantitatively driven by Trop-2 expression levels. However, little is known about the regulation of expression of the TROP2 gene. Hence, we investigated the TROP2 transcription control network. TROP2 expression was shown to depend on a highly interconnected web of transcription factors: TP63/TP53L, ERG, GRHL1/Get-1 (grainyhead-like epithelial transactivator), HNF1A/TCF-1 (T-cell factor), SPI1/PU.1, WT (Wilms' tumour)1, GLIS2, AIRE (autoimmune regulator), FOXM1 (forkhead box M1) and FOXP3, with HNF4A as the major network hub. TROP2 upregulation was shown to subsequently drive the expression and activation of CREB1 (cyclic AMP-responsive-element binding protein), Jun, NF-kB, Rb, STAT1 and STAT3 through induction of the cyclin D1 and ERK (extracellular signal regulated kinase)/MEK (MAPK/ERK kinase) pathways. Growth-stimulatory signalling through NF-kB, cyclin D1 and ERK was shown to require an intact Trop-2 cytoplasmic tail. Network hubs and interacting partners are co-expressed with Trop-2 in primary human tumours, supporting a role of this signalling network in cancer growth.

Trop-2 is a transmembrane signal transducer that can induce cancer growth. Using antibody targeting and N-terminal Edman degradation, we show here that Trop-2 undergoes cleavage in the first thyroglobulin domain loop of its extracellular region, between residues R87 and T88. Molecular modeling indicated that this cleavage induces a profound rearrangement of the Trop-2 structure, which suggested a deep impact on its biological function. No Trop-2 cleavage was detected in normal human tissues, whereas most tumors showed Trop-2 cleavage, including skin, ovary, colon, and breast cancers. Coimmunoprecipitation and mass spectrometry analysis revealed that ADAM10 physically interacts with Trop-2. Immunofluorescence/confocal time-lapse microscopy revealed that the two molecules broadly colocalize at the cell membrane. We show that ADAM10 inhibitors, siRNAs and shRNAs abolish the processing of Trop-2, which indicates that ADAM10 is an effector protease. Proteolysis of Trop-2 at R87-T88 triggered cancer cell growth both in vitro and in vivo. A corresponding role was shown for metastatic spreading of colon cancer, as the R87A-T88A Trop-2 mutant abolished xenotransplant metastatic dissemination. Activatory proteolysis of Trop-2 was recapitulated in primary human breast cancers. Together with the prognostic impact of Trop-2 and ADAM10 on cancers of the skin, ovary, colon, lung, and pancreas, these data indicate a driving role of this activatory cleavage of Trop-2 on malignant progression of tumors.

Trop-2 is a transmembrane signal transducer that is overexpressed in most human cancers, and drives malignant progression. To gain knowledge on the higher-order molecular mechanisms that drive Trop-2 signaling, we applied next-generation sequencing, proteomics, and high-resolution microscopy to models and primary cases of human colorectal cancer (CRC). We had previously shown that Trop-2 induces a Ca 2+ signal. We reveal here that Trop-2 binds the cell membrane Na + /K + -ATPase, and that clustering of Trop-2 induces an intracellular Ca 2+ rise followed by membrane translocation of PKCα, which in turn phosphorylates the Trop-2 cytoplasmic tail. This feed-forward signaling is promoted by the binding of Trop-2 to the PKCα membrane-anchor CD9. CRISPRbased inactivation of CD9 in CRC cells shows that CD9 is required by Trop-2 for recruiting PKCα and cofilin-1 to the cell membrane. This induces malignant progression through proteolytic cleavage of E-cadherin, remodeling of the β-actin cytoskeleton, and activation of Akt and ERK. The interaction between Trop-2 and CD9 was validated in vivo in murine models of CRC growth and invasion. Overexpression of the components of this Trop-2-driven super-complex significantly worsened disease-free and overall survival of CRC patients, supporting a pivotal relevance in CRC malignant progression. Our findings demonstrate a previously unsuspected layer of cancer growth regulation, which is dormant in normal tissues, and is activated by Trop-2 in cancer cells.

Purpose: Inhibition of AKT is a key target area for personalized cancer medicine. However, predictive markers of response to AKT inhibitors are lacking. Correspondingly, the AKT-dependent chain of command for tumor growth, which will mediate AKTdependent therapeutic responses, remains unclear.Experimental Design: Proteomic profiling was utilized to identify nodal hubs of the Trop-2 cancer growth-driving network. Kinase-specific inhibitors were used to dissect Trop-2-dependent from Trop-2-independent pathways. In vitro assays, in vivo preclinical models, and case series of primary human breast cancers were utilized to define the mechanisms of Trop-2-driven growth and the mode of action of Trop-2-predicted AKT inhibitors.Results: Trop-2 and AKT expression was shown to be tightly coordinated in human breast cancers, with virtual overlap with AKT activation profiles at T308 and S473, consistent with functional interaction in vivo. AKT allosteric inhibitors were shown to only block the growth of Trop-2-expressing tumor cells, both in vitro and in preclinical models, being ineffective on Trop-2-null cells. Consistently, AKTtargeted siRNA only impacted on Trop-2-expressing cells. Lentiviral downregulation of endogenous Trop-2 abolished tumor response to AKT blockade, indicating Trop-2 as a mandatory activator of AKT.Conclusions: Our findings indicate that the expression of Trop-2 is a stringent predictor of tumor response to AKT inhibitors. They also support the identification of target-activatory pathways, as efficient predictors of response in precision cancer therapy. Clin Cancer Res; 22(16); 4197-205. Ó2016 AACR.