Emanuela Guerra

A Bicistronic CYCLIN D1-TROP2 mRNA Chimera Demonstrates a Novel Oncogenic Mechanism in Human Cancer

A chimeric CYCLIN D1-TROP2 mRNA was isolated from human ovarian and mammary cancer cells. The CYCLIN D1-TROP2 mRNA was shown to be a potent oncogene as it transforms naïve, primary cells in vitro and induces aggressive tumor growth in vivo in cooperation with activated RAS. Silencing of the chimeric mRNA inhibits the growth of breast cancer cells. The CYCLIN D1-TROP2 mRNA was expressed by a large fraction of the human gastrointestinal, ovarian, and endometrial tumors analyzed. It is most frequently detected in intestinal cell aneuploid cancers and it is coexpressed with activated RAS oncogenes, consistent with a cooperative transforming activity in human cancers. The chimeric mRNA is a bicistronic transcript of post transcriptional origin that independently translates the Cyclin D1 and Trop-2 proteins. This is a novel mechanism of CYCLIN D1 activation that achieves the truncation of the CYCLIN D1 mRNA in the absence of chromosomal rearrangements. This leads to a higher CYCLIN D1 mRNA stability, with inappropriate expression during the cell cycle. The stabilized CYCLIN D1 mRNA cooperates with TROP2 in stimulating the growth of the expressing cells. These findings show a novel epigenetic, oncogenic mechanism, which seems to be widespread in human cancers.

The Trop-2 signalling network in 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-κB, 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-κB, 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.

mTrop1/Epcam Knockout Mice Develop Congenital Tufting Enteropathy through Dysregulation of Intestinal E-cadherin/β-catenin

Congenital tufting enteropathy (CTE) is a life-threatening hereditary disease that is characterized by enteric mucosa tufting degeneration and early onset, severe diarrhea. Loss-of-function mutations of the human EPCAM gene (TROP1, TACSTD1) have been indicated as the cause of CTE. However, loss of mTrop1/Epcam in mice appeared to lead to death in utero, due to placental malformation. This and indications of residual Trop-1/EpCAM expression in cases of CTE cast doubt on the role of mTrop1/Epcam in this disease. The aim of this study was to determine the role of TROP1/EPCAM in CTE and to generate an animal model of this disease for molecular investigation and therapy development. Using a rigorous gene-trapping approach, we obtained mTrop1/Epcam -null (knockout) mice. These were born alive, but failed to thrive, and died soon after birth because of hemorrhagic diarrhea. The intestine from the mTrop1/Epcam knockout mice showed intestinal tufts, villous atrophy and colon crypt hyperplasia, as in human CTE. No structural defects were detected in other organs. These results are consistent with TROP1/EPCAM loss being the cause of CTE, thus providing a viable animal model for this disease, and a benchmark for its pathogenetic course. In the affected enteric mucosa, E-cadherin and β-catenin were shown to be dysregulated, leading to disorganized transition from crypts to villi, with progressive loss of membrane localization and increasing intracellular accumulation, thus unraveling an essential role for Trop-1/EpCAM in the maintenance of intestinal architecture and functionality. Supporting information is available for this article.

Trop-1 are conserved growth stimulatory molecules that mark early stages of tumor progression

Trop‐1 is a cell‐cell adhesion regulatory molecule that is overexpressed by a large fraction of tumors in man.

Mutations of TP53 induce loss of DNA methylation and amplification of the TROP1 gene.

p53 and DNA methylation play key roles in the maintenance of genome stability. In this work, we demonstrate that the two mechanisms are linked and that p53 plays a role in the maintenance of the DNA methylation levels. The loss of p53 was shown to induce loss of DNA methylation in the TROP1 gene, a human cancer-expressed locus that undergoes amplification when hypomethylated. This demethylation was reverted by the reintroduction of a wild-type TP53 (wtTP53) in the TP53-null cells. Using a gene-amplification assay in vivo, we demonstrate that the loss of p53 leads to a demethylation-dependent TROP1 gene amplification. The induction of gene amplification was reverted by the expression of a wtTP53 gene or by in vitro methylation of the transfected DNA with the Sss I DNA methylase. Taken together, these findings demonstrate that the inactivation of TP53 induces loss of DNA methylation and DNA methylation-dependent gene amplification.

Comment on “Cancer chemoprevention: Evidence of a nonlinear dose response for the protective effects of resveratrol in humans and mice”

A report that low doses of resveratrol can suppress intestinal adenoma in a mouse model appears confounded because in a slightly different protocol, resveratrol has the opposite effect. In ApcMin mice (an animal model of colorectal carcinogenesis) fed a high-fat diet, low doses of resveratrol suppress intestinal adenoma development more potently than high doses do; however, these findings appear affected by multiple confounding factors, as resveratrol alone added to a standard diet has opposite outcomes.

Trop-2 Induces Tumor Growth Through AKT and Determines Sensitivity to AKT Inhibitors.

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 AKT-dependent 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, AKT-targeted 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. ©2016 AACR.

The trophoblast cell surface antigen 2 and miR-125b axis in urothelial bladder cancer

Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro.The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue.In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches.Human trophoblast cell surface antigen 2 (Trop-2) is a 40-kDa transmembrane glycoprotein that was first identified as a marker of human trophoblast cells. Trop-2 acts on cell proliferation, adhesion, and migration by activating a number of intracellular signalling pathways. Elevated Trop-2 expression has been demonstrated in several types of cancer and correlated with aggressiveness and poor prognosis. Since no data are available on Trop-2 in bladder cancer (BC), the purpose of the study was to determine its levels in tissue specimens from normal individuals and patients with BC at different stages. Moreover, since according to recent evidence Trop-2 is a miR-125b target, miR-125b expression was also assessed in tissue specimens. Finally, the effect of the Trop-2/miR-125b axis on the proliferation and migration of BC cells was evaluated in vitro. The Trop-2/miR-125b axis was seen to be differentially expressed in normal urothelium, non-invasive BC and invasive BC tissue. Significant miR-125b down-regulation was associated with a significant increase in Trop-2 protein levels in BC tissue and correlated with disease severity. In vitro analysis confirmed the role of miR-125b in down-modulation of Trop-2 protein levels and showed that Trop-2/miR-125b axis affects cellular proliferation in bladder tissue. In conclusion, our findings highlight a role for the Trop-2/miR-125b axis in BC progression and suggest Trop-2 and miR-125b as diagnostic/prognostic marker candidates as well as druggable targets for innovative therapeutic approaches.

Abstract 4588: Novel anti-Trop-2 monoclonal antibodies with unique binding specificities show therapeutic synergy against most human cancers

Trop-2 is an epithelial transmembrane glycoprotein that transduces a calcium signal and activates a growth-signaling network that converges on AKT. Trop-2 is overexpressed in the majority of carcinomas, where it drives tumor cell proliferation, and in its mature, glycosylated/functionally-competent form associates with worse prognosis. Trop-2 extracellular domain contains an N-terminal cysteine-rich globular region followed by a cysteine-less region as a connecting “stem” to the transmembrane domain. Trop-2 molecules engage in homophylic interactions between adjacent cells and establish multimeric complexes with tight junction proteins, which may hinder accessibility by therapeutic antibodies. Up to now, Trop-2-targeted approaches have employed anti-Trop-2 monoclonal antibodies (mAb) which essentially recognize a single immunodominant epitope poised between the globular and stem regions. Such mAb have limited or no therapeutic efficacy. In order to untap the potential of anti-Trop-2 immunotherapy we generated novel anti-Trop-2 mAb with tailored specificity towards the globular versus stem regions. Hybridoma diversity was maximized by immunization with soluble human Trop-2 extracellular region produced in different transformed mammalian cell lines (human 293 and murine L) and in insect cells/baculovirus expressing system. These were expected to provide native folding of Trop-2 together with a broad spectrum of differential glycosylation. Trop-2-binding hybridomas were further selected by multiple rounds of flow cytometry analysis using live 293 cells expressing different Trop-2 extracellular portions. Two classes of mAb were identified, that bound the stem versus the globular region. These mAb efficiently bound Trop-2 expressing cancer cells and were able to inhibit cell growth in vitro. In vivo the naked anti-globular OX-G64 and anti-stem OX-S55 mAb were most effective in inhibiting the growth of distinct tumors, including colon, ovary and prostate cancers. Notably, they showed differential efficacy for established tumors versus isolated-cell models of metastatic dissemination, consistent with our strategy of maximizing differential accessibility of Trop-2 according to growth mode. Most remarkably, we demonstrated in vivo synergy of these anti-Trop-2 mAb, paving the way for game-changing anti-cancer mAb therapy. The differential efficacy of the OX-G64 and OX-S55 anti-Trop2 mAb against different tumor histotypes and growth stages further allows to exploit their cancer-killing potential in pathological stage-tailored therapeutic approaches. Citation Format: Emanuela Guerra, Marco Trerotola, Valeria Relli, Chiara Pedicone, Antonella D9 Amore, Francesca Dini, Silvia Fratarcangeli, Saverio Alberti. Novel anti-Trop-2 monoclonal antibodies with unique binding specificities show therapeutic synergy against most human cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4588. doi:10.1158/1538-7445.AM2017-4588

Abstract 5042: Trop-2 is a universal cancer growth stimulator through a ubiquitous signaling platform

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Trop-2 is a transmembrane calcium signal transducer. Our findings show that Trop-2 is overexpressed by the majority of human cancers, suggesting strong selective pressure for a conserved function. Trop-2 was then demonstrated to be necessary and sufficient to stimulate cancer growth, with a linear relationship between growth rates and Trop-2 expression levels. Cell growth stimulation was shown to be conserved across cell-types and species. These findings indicated impingement on a ubiquitous downstream signal-transduction module. The Trop-2 signaling pathway was investigated by yeast 2 hybrid screening, coimmunoprecipitation / mass-spectrometry and proteomic chip analysis. Trop-2 was demonstrated to bind CD44 and tetraspanins, triggering their growth-promoting ability via a feed-forward activation loop of CD9-recruited PKCα and phosphorylation of the Trop-2 cytoplasmic tail. We demonstrate that PKCα stimulates growth in a Trop-2-restricted manner and that PKCα and Trop-2 are coordinately transported in recurrent waves to membrane ruffles and podosomes. Trop-2 induction was shown to activate the ERK pathway, to up-regulate NF-κB, and to modulate apoptotic factors, including p53 and Rb. Members of the Trop-2 pathway (CD9, CD44 and p53) were found coordinately upregulated in retrospective studies of human cancer case series. These findings reveal the existence of a novel, strikingly widespread mechanism of stimulation of cancer growth. This is quantitatively driven by a wild-type Trop-2 and acts upon ready-to-signal, ubiquitous signal-transduction platforms. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5042.