Wilson disease, ABCC2 c.3972C > T polymorphism and primary liver cancers: suggestions from a familial cluster.

BACKGROUND: Polymorphisms in genes modulating xenobiotics metabolism, in particular the ABCC2 c.3972C > T single nucleotide polymorphism (SNP) at exon 28, have been suggested to increase primary liver cancer (PLC) risk. Conversely, the occurrence of PLCs in Wilson disease patients is a rare event, in contrast with the occurrence observed in other chronic liver diseases. Here we report the clinical case of five siblings carrying the ABCC2 c.3972C > T SNP; three of them were affected by Wilson disease and two brothers with Wilson disease also developed PLCs. METHODS: The presence of the ABCC2 c.3972C > T SNP was assessed by Sanger sequencing and the exposure of PLC risk factors by standardized questionnaires. RESULTS: Notably, PLCs occurred only in the two brothers with the ABCC2 c.3972C > T SNP and Wilson disease who resulted exposed to asbestos and cigarette smoking, but not in the other siblings with the ABCC2 c.3972C > T SNP, alone or in association with Wilson disease, not exposed to these carcinogens and/or to other known risk factors for PLCs. CONCLUSIONS: These findings suggest that ABCC2 c.3972C > T SNP and WD, also in association, may not represent a sufficient condition for PLC development, but that co-occurrence of further host/exogenous risk factors are needed to drive this process, reinforcing the notion that liver carcinogenesis is the result of a complex interplay between environmental and host genetic determinants. Due to the sporadic cases of this study and the paucity of data currently available in literature on this issue, future investigations in a larger population are needed to confirm our findings.

Epigenetic inheritance and the missing heritability.

Genome-wide association studies of complex physiological traits and diseases consistently found that associated genetic factors, such as allelic polymorphisms or DNA mutations, only explained a minority of the expected heritable fraction. This discrepancy is known as "missing heritability", and its underlying factors and molecular mechanisms are not established. Epigenetic programs may account for a significant fraction of the "missing heritability." Epigenetic modifications, such as DNA methylation and chromatin assembly states, reflect the high plasticity of the genome and contribute to stably alter gene expression without modifying genomic DNA sequences. Consistent components of complex traits, such as those linked to human stature/height, fertility, and food metabolism or to hereditary defects, have been shown to respond to environmental or nutritional condition and to be epigenetically inherited. The knowledge acquired from epigenetic genome reprogramming during development, stem cell differentiation/de-differentiation, and model organisms is today shedding light on the mechanisms of (a) mitotic inheritance of epigenetic traits from cell to cell, (b) meiotic epigenetic inheritance from generation to generation, and (c) true transgenerational inheritance. Such mechanisms have been shown to include incomplete erasure of DNA methylation, parental effects, transmission of distinct RNA types (mRNA, non-coding RNA, miRNA, siRNA, piRNA), and persistence of subsets of histone marks.

Activated FGFR2 signalling as a biomarker for selection of intrahepatic cholangiocarcinoma patients candidate to FGFR targeted therapies.

FGFR inhibitors have been developed to inhibit FGFR activation and signal transduction; notwithstanding, currently the selection of intrahepatic cholangiocarcinoma (iCCA) patients for these drugs only relies on the detection of FGFR2 genetic alterations (GAs) in tumor tissues or circulating tumor DNAs, without concomitant assessment of FGFR2 signalling status. Accordingly, we performed multi-omic analyses of FGFR2 genes and FGFR2 signalling molecules in the tissue samples from 36 iCCA naïve patients. Gain-of-function FGFR2 GAs were detected in 7 patients, including missense mutations (n = 3; p.F276C, p.C382R and p.Y375C), translocations (n = 1) and copy number gain (n = 4; CNV ≥ 4). In contrast, among 29 patients with wild-type FGFR2, 4 cases showed activation of FGFR2 signalling, as they expressed the FGFR2 ligand FGF10 and phosphorylated FGFR2/FRS2α proteins; the remaining 25 cases resulted negative for activated FGFR2 signalling, as they lacked FGFR2 (n = 8) or phosphorylated FRS2α (n = 17) expression. Overall, we found that activation of FGFR2 signalling occurs not only in iCCA naïve patients with FGFR2 GAs, but also in a subgroup carrying wild-type FGFR2. This last finding entails that also this setting of patients could benefit from FGFR targeted therapies, widening indication of these drugs for iCCA patients beyond current approval. Future clinical studies are therefore encouraged to confirm this hypothesis.

The 2EF Antibody Targets a Unique N-Terminal Epitope of Trop-2 and Enhances the In Vivo Activity of the Cancer-Selective 2G10 Antibody.

Trop-2 proteolytic processing in cancer cells exposes epitopes that were specifically targeted by the 2G10 antibody. We sought additional recognition of Trop-2 within difficult-to-reach, densely packed tumor sites. Trop-2 deletion mutants were employed in immunization and screening procedures, and these led to the recognition of a novel epitope in the N-terminal region of Trop-2, by the 2EF antibody. The 2EF mAb was shown to bind Trop-2 at cell-cell junctions in MCF-7 breast cancer cells, and in deeply seated sites in prostate cancer, that were inaccessible to benchmark anti-Trop-2 antibodies. The 2EF antibody was shown to inhibit the growth of HT29 colon tumor cells in vitro, with the highest activity at high cell density. In vivo, 2EF showed anticancer activity against SKOv3 ovarian, Colo205, HT29, HCT116 colon and DU-145 prostate tumors, with the highest impact on densely packed tumor sites, whereby 2EF outcompeted benchmark anti-Trop-2 antibodies. Given the different recognition modes of Trop-2 by 2EF and 2G10, we hypothesized the effective interaction of the two mAb in vivo. The 2EF mAb was indeed demonstrated to enhance the activity of 2G10 against tumor xenotransplants, opening novel avenues for Trop-2-targeted therapy. We humanized 2EF by state-of-the-art CDR grafting/re-modeling, yielding the Hu2EF for therapy of Trop-2-expressing tumors in patients.

Asbestos exposure as an additional risk factor for small duct intrahepatic cholangiocarcinoma: a pilot study.

Intrahepatic cholangiocarcinoma (iCCA) is a rare malignancy, recently classified in small duct and large duct morphological subtypes. Growing evidence suggests asbestos as a putative risk factor for iCCA, albeit no correlation between asbestos and iCCA morphology has been investigated so far. The aim of the present study was to assess the relationship between asbestos exposure and iCCA morphological subtype. Forty patients with surgically removed iCCA were prospectively enrolled: asbestos exposure was assessed according to the Italian National Mesothelioma Register questionnaire. From the surgical iCCA specimens the main histopathological variables were collected, including the small duct (sd-iCCA, 32 patients) and large duct subtypes (ld-iCCA, 8 patients). Five sd-iCCA cases had a definite/probable occupational exposure to asbestos, while no cases of ld-iCCA were classified as being occupationally exposed (definite/probable). Other kind of asbestos exposure (i.e. possible occupational, familial, environmental) were recorded in 16 sd-iCCA and 3 ld-iCCA. Cases with unlikely exposure to asbestos were 11 sd-iCCA (35.5%) and 5 ld-iCCA (62.5%). In conclusion, these findings seem to indicate that sd-iCCA might be more frequently associated to asbestos exposure rather than ld-iCCA, suggesting that asbestos fibres might represent a parenchymal, rather than a ductal risk factor for iCCA. This pilot study must be confirmed by further case-control studies or large independent cohorts.

Phylogenetic conservation of Trop-2 across species-rodent and primate genomics model anti-Trop-2 therapy for pre-clinical benchmarks.

A phylogenetic conservation analysis of Trop-2 across vertebrate species showed a high degree of sequence conservation, permitting to explore multiple models as pre-clinical benchmarks. Sequence divergence and incomplete conservation of expression patterns were observed in mouse and rat. Primate Trop-2 sequences were found to be 95%-100% identical to the human sequence. Comparative three-dimension primate Trop-2 structures were obtained with AlphaFold and homology modeling. This revealed high structure conservation of Trop-2 (0.66 ProMod3 GMQE, 0.80-0.86 ± 0.05 QMEANDisCo scores), with conservative amino acid changes at variant sites. Primate TACSTD2/TROP2 cDNAs were cloned and transfectants for individual ORF were shown to be efficiently recognized by humanized anti-Trop-2 monoclonal antibodies (Hu2G10, Hu2EF). Immunohistochemistry analysis of Macaca mulatta (rhesus monkey) tissues showed Trop-2 expression patterns that closely followed those in human tissues. This led us to test Trop-2 targeting in vivo in Macaca fascicularis (cynomolgus monkey). Intravenously injected Hu2G10 and Hu2EF were well tolerated from 5 to 10 mg/kg. Neither neurological, respiratory, digestive, urinary symptoms, nor biochemical or hematological toxicities were detected during 28-day observation. Blood serum pharmacokinetic (PK) studies were conducted utilizing anti-idiotypic antibodies in capture-ELISA assays. Hu2G10 (t1/2 = 6.5 days) and Hu2EF (t1/2 = 5.5 days) were stable in plasma, and were detectable in the circulation up to 3 weeks after the infusion. These findings validate primates as reliable models for Hu2G10 and Hu2EF toxicity and PK, and support the use of these antibodies as next-generation anti-Trop-2 immunotherapy tools.

3D-Informed Targeting of the Trop-2 Signal-Activation Site Drives Selective Cancer Vulnerability.

Next-generation Trop-2-targeted therapy against advanced cancers is hampered by expression of Trop-2 in normal tissues. We discovered that Trop-2 undergoes proteolytic activation by ADAM10 in cancer cells, leading to the exposure of a previously inaccessible protein groove flanked by two N-glycosylation sites. We designed a recognition strategy for this region, to drive selective cancer vulnerability in patients. Most undiscriminating anti-Trop-2 mAbs recognize a single immunodominant epitope. Hence, we removed it by deletion mutagenesis. Cancer-specific, glycosylation-prone mAbs were selected by ELISA, bio-layer interferometry, flow cytometry, confocal microscopy for differential binding to cleaved/activated, wild-type and glycosylation site-mutagenized Trop-2. The resulting 2G10 mAb family binds Trop-2-expressing cancer cells, but not Trop-2 on normal cells. We humanized 2G10 by state-of-the-art complementarity determining region grafting/re-modeling, yielding Hu2G10. This antibody binds cancer-specific, cleaved/activated Trop-2 with Kd < 10-12 mol/L, and uncleaved/wtTrop-2 in normal cells with Kd 3.16×10-8 mol/L, thus promising an unprecedented therapeutic index in patients. In vivo, Hu2G10 ablates growth of Trop-2-expressing breast, colon, prostate cancers, but shows no evidence of systemic toxicity, paving the way for a paradigm shift in Trop-2-targeted therapy.

Trop-2, Na+/K+ ATPase, CD9, PKCα, cofilin assemble a membrane signaling super-complex that drives colorectal cancer growth and invasion.

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 Ca2+ signal. We reveal here that Trop-2 binds the cell membrane Na+/K+-ATPase, and that clustering of Trop-2 induces an intracellular Ca2+ 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. CRISPR-based 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 ß-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.

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

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 ß-actin, and loss of cell-cell adhesion, acquisition of invasive capability, and membrane-driven activation of ß-catenin signaling, which were further enhanced by the ΔcytoTrop-2 mutant. This Trop-2/E-cadherin/ß-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.

Molecular Features and Targeted Therapies in Extrahepatic Cholangiocarcinoma: Promises and Failures.

Biliary tract cancers (BTCs) include a heterogenous group of aggressive malignancies with limited therapeutic options. According to their anatomical location, these hepatobiliary tumors are usually classified into intrahepatic cholangiocarcinoma (iCCA), extrahepatic cholangiocarcinoma (eCCA), and gallbladder cancer (GBC). Unfortunately, BTCs are often diagnosed when already metastatic, and although the advent of genomic sequencing has led to a deeper understanding of iCCA pathogenesis, very little data are currently available about the molecular landscape of eCCA. Moreover, despite novel systemic treatments emerging in BTC, the grim prognosis of eCCA patients has not changed in the past decade, and no targeted therapies have been approved so far. The aim of the current review is to provide an overview regarding molecular features and potential targeted therapies in eCCA, together with novel therapeutic approaches and future directions of translational and clinical research on this highly aggressive disease that poses many unanswered questions.

Abandoning the Notion of Non-Small Cell Lung Cancer.

Non-small cell lung cancers (NSCLCs) represent 85% of lung tumors. NSCLCs encompass multiple cancer types, such as adenocarcinomas (LUADs), squamous cell cancers (LUSCs), and large cell cancers. Among them, LUADs and LUSCs are the largest NSCLC subgroups. LUADs and LUSCs appear sharply distinct at the transcriptomic level, as well as for cellular control networks. LUADs show distinct genetic drivers and divergent prognostic profiles versus LUSCs. Therapeutic clinical trials in NSCLC indicate differential LUAD versus LUSC response to treatments. Hence, LUAD and LUSC appear to be vastly distinct diseases at the molecular, pathological, and clinical level. Abandoning the notion of NSCLC may critically help in developing novel, more effective subtype-specific molecular alteration-targeted therapeutic procedures.

Distinct lung cancer subtypes associate to distinct drivers of tumor progression.

The main non-small-cell lung cancer (NSCLC) histopathological subtypes are lung adenocarcinomas (LUAD) and lung squamous cell carcinomas (LUSC). To identify candidate progression determinants of NSCLC subtypes, we explored the transcriptomic signatures of LUAD versus LUSC. We then investigated the prognostic impact of the identified tumor-associated determinants. This was done utilizing DNA microarray data from 2,437 NSCLC patients. An independent analysis of a case series of 994 NSCLC was conducted by next-generation sequencing, together with gene expression profiling from GEO (https://www.ncbi.nlm.nih.gov/geo/). This work led us to identify 69 distinct tumor prognostic determinants, which impact on LUAD or LUSC clinical outcome. These included key drivers of tumor growth and cell cycle, transcription factors and metabolic determinants. Such disease determinants appeared vastly different in LUAD versus LUSC, and often had opposite impact on clinical outcome. These findings indicate that distinct tumor progression pathways are at work in the two NSCLC subtypes. Notably, most prognostic determinants would go inappropriately assessed or even undetected when globally investigating unselected NSCLC. Hence, differential consideration for NSCLC subtypes should be taken into account in current clinical evaluation procedures for lung cancer.

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.

Trop-2 is a determinant of breast cancer survival.

Trop-2 is a calcium signal transducer that drives tumor growth. Anti-Trop-2 antibodies with selective reactivity versus Trop-2 maturation stages allowed to identify two different pools of Trop-2, one localized in the cell membrane and one in the cytoplasm. Of note, membrane-localized/functional Trop-2 was found to be differentially associated with determinants of tumor aggressiveness and distinct breast cancer subgroups. These findings candidated Trop-2 states to having an impact on cancer progression. We tested this model in breast cancer. A large, consecutive human breast cancer case series (702 cases; 8 years median follow-up) was analyzed by immunohistochemistry with anti-Trop-2 antibodies with selective reactivity for cytoplasmic-retained versus functional, membrane-associated Trop-2. We show that membrane localization of Trop-2 is an unfavorable prognostic factor for overall survival (1+ versus 0 for all deaths: hazard ratio, 1.63; P = 0.04), whereas intracellular Trop-2 has a favorable impact on prognosis, with an adjusted hazard ratio for all deaths of 0.48 (high versus low; P = 0.003). A corresponding impact of intracellular Trop-2 was found on disease relapse (high versus low: hazard ratio, 0.51; P = 0.004). Altogether, we demonstrate that the Trop-2 activation states are critical determinants of tumor progression and are powerful indicators of breast cancer patients survival.