Novel Ultrastructural Insights into the Clear-Cell Carcinoma of the Pancreas: A Case Report.

Pancreatic cancer, most frequently as ductal adenocarcinoma (PDAC), is the third leading cause of cancer death. Clear-cell primary adenocarcinoma of the pancreas (CCCP) is a rare, aggressive, still poorly characterized subtype of PDAC. We report here a case of a 65-year-old male presenting with pancreatic neoplasia. A histochemical examination of the tumor showed large cells with clear and abundant intracytoplasmic vacuoles. The clear-cell foamy appearance was not related to the hyperproduction of mucins. Ultrastructural characterization with transmission electron microscopy revealed the massive presence of mitochondria in the clear-cell cytoplasm. The mitochondria showed disordered cristae and various degrees of loss of structural integrity. Immunohistochemistry staining for NADH dehydrogenase [ubiquinone] 1 alpha subcomplex, 4-like 2 (NDUFA4L2) proved specifically negative for the clear-cell tumor. Our ultrastructural and molecular data indicate that the clear-cell nature in CCCP is linked to the accumulation of disrupted mitochondria. We propose that this may impact on the origin and progression of this PDAC subtype.

The Dual Role of Necroptosis in Pancreatic Ductal Adenocarcinoma.

Pancreatic cancer (PC) is the seventh leading cause of cancer-related death. PC incidence has continued to increase by about 1% each year in both men and women. Although the 5-year relative survival rate of PC has increased from 3% to 12%, it is still the lowest among cancers. Hence, novel therapeutic strategies are urgently needed. Challenges in PC-targeted therapeutic strategies stem from the high PC heterogeneity and from the poorly understood interplay between cancer cells and the surrounding microenvironment. Signaling pathways that drive PC cell growth have been the subject of intense scrutiny and interest has been attracted by necroptosis, a distinct type of programmed cell death. In this review, we provide a historical background on necroptosis and a detailed analysis of the ongoing debate on the role of necroptosis in PC malignant progression.

Cancer-Homing CAR-T Cells and Endogenous Immune Population Dynamics.

Chimeric antigen receptor (CAR) therapy is based on patient blood-derived T cells and natural killer cells, which are engineered in vitro to recognize a target antigen in cancer cells. Most CAR-T recognize target antigens through immunoglobulin antigen-binding regions. Hence, CAR-T cells do not require the major histocompatibility complex presentation of a target peptide. CAR-T therapy has been tremendously successful in the treatment of leukemias. On the other hand, the clinical efficacy of CAR-T cells is rarely detected against solid tumors. CAR-T-cell therapy of cancer faces many hurdles, starting from the administration of engineered cells, wherein CAR-T cells must encounter the correct chemotactic signals to traffic to the tumor in sufficient numbers. Additional obstacles arise from the hostile environment that cancers provide to CAR-T cells. Intense efforts have gone into tackling these pitfalls. However, we argue that some CAR-engineering strategies may risk missing the bigger picture, i.e., that a successful CAR-T-cell therapy must efficiently intertwine with the complex and heterogeneous responses that the body has already mounted against the tumor. Recent findings lend support to this model.

p53, cathepsin D, Bcl-2 are joint prognostic indicators of breast cancer metastatic spreading.

BACKGROUND: Traditional prognostic indicators of breast cancer, i.e. lymph node diffusion, tumor size, grading and estrogen receptor expression, are inadequate predictors of metastatic relapse. Thus, additional prognostic parameters appear urgently needed. Individual oncogenic determinants have largely failed in this endeavour. Only a few individual tumor growth drivers, e.g. mutated p53, Her-2, E-cadherin, Trops, did reach some prognostic/predictive power in clinical settings. As multiple factors are required to drive solid tumor progression, clusters of such determinants were expected to become stronger indicators of tumor aggressiveness and malignant progression than individual parameters. To identify such prognostic clusters, we went on to coordinately analyse molecular and histopathological determinants of tumor progression of post-menopausal breast cancers in the framework of a multi-institutional case series/case-control study. METHODS: A multi-institutional series of 217 breast cancer cases was analyzed. Twenty six cases (12 %) showed disease relapse during follow-up. Relapsed cases were matched with a set of control patients by tumor diameter, pathological stage, tumor histotype, age, hormone receptors and grading. Histopathological and molecular determinants of tumor development and aggressiveness were then analyzed in relapsed versus non-relapsed cases. Stepwise analyses and model structure fitness assessments were carried out to identify clusters of molecular alterations with differential impact on metastatic relapse. RESULTS: p53, Bcl-2 and cathepsin D were shown to be coordinately associated with unique levels of relative risk for disease relapse. As many Ras downstream targets, among them matrix metalloproteases, are synergistically upregulated by mutated p53, whole-exon sequence analyses were performed for TP53, Ki-RAS and Ha-RAS, and findings were correlated with clinical phenotypes. Notably, TP53 insertion/deletion mutations were only detected in relapsed cases. Correspondingly, Ha-RAS missense oncogenic mutations were only found in a subgroup of relapsing tumors. CONCLUSIONS: We have identified clusters of specific molecular alterations that greatly improve prognostic assessment with respect to singularly-analysed indicators. The combined analysis of these multiple tumor-relapse risk factors promises to become a powerful approach to identify patients subgroups with unfavourable disease outcome.

A seven-gene CpG-island methylation panel predicts breast cancer progression.

BACKGROUND: DNA methylation regulates gene expression, through the inhibition/activation of gene transcription of methylated/unmethylated genes. Hence, DNA methylation profiling can capture pivotal features of gene expression in cancer tissues from patients at the time of diagnosis. In this work, we analyzed a breast cancer case series, to identify DNA methylation determinants of metastatic versus non-metastatic tumors. METHODS: CpG-island methylation was evaluated on a 56-gene cancer-specific biomarker microarray in metastatic versus non-metastatic breast cancers in a multi-institutional case series of 123 breast cancer patients. Global statistical modeling and unsupervised hierarchical clustering were applied to identify a multi-gene binary classifier with high sensitivity and specificity. Network analysis was utilized to quantify the connectivity of the identified genes. RESULTS: Seven genes (BRCA1, DAPK1, MSH2, CDKN2A, PGR, PRKCDBP, RANKL) were found informative for prognosis of metastatic diffusion and were used to calculate classifier accuracy versus the entire data-set. Individual-gene performances showed sensitivities of 63-79 %, 53-84 % specificities, positive predictive values of 59-83 % and negative predictive values of 63-80 %. When modelled together, these seven genes reached a sensitivity of 93 %, 100 % specificity, a positive predictive value of 100 % and a negative predictive value of 93 %, with high statistical power. Unsupervised hierarchical clustering independently confirmed these findings, in close agreement with the accuracy measurements. Network analyses indicated tight interrelationship between the identified genes, suggesting this to be a functionally-coordinated module, linked to breast cancer progression. CONCLUSIONS: Our findings identify CpG-island methylation profiles with deep impact on clinical outcome, paving the way for use as novel prognostic assays in clinical settings.

A non-mutated TROP2 fingerprint in cancer genetics.

The advent of high throughput DNA sequencing is providing massive amounts of tumor-associated mutation data. Implicit in these analyses is the assumption that, by acquiring a series of hallmark changes, normal cells evolve along a neoplastic path. However, the lack of correlation between cancer risk and global exposure to mutagenic factors provides arguments against this model. This suggested that additional, non-mutagenic factors are at work in cancer development. A candidate determinant is TROP2, that stands out for its expression in the majority of solid tumors in human, for its impact on the prognosis of most solid cancers and for its role as driver of cancer growth and metastatic diffusion, through overexpression as a wild-type form. The Trop-2 signaling network encompasses CREB1, Jun, NF-κB, Rb, STAT1 and STAT3, through induction of cyclin D1 and MAPK/ERK. Notably, Trop-2-driven pathways vastly overlap with those activated by most functionally relevant/most frequently mutated RAS and TP53, and are co-expressed in a large fraction of individual tumor cases, suggesting functional overlap. Mutated Ras was shown to synergize with the TROP2-CYCLIND1 mRNA chimera in transforming primary cells into tumorigenic ones. Genomic loss of TROP2 was found to promote carcinogenesis in squamous cell carcinomas through modulation of Src and mutated Ras pathways. DNA methylation and TP53 status were shown to cause genome instability and TROP gene amplification, together with Trop-2 protein overexpression. These findings suggest that mutagenic and the TROP2 non-mutagenic pathways deeply intertwine in driving transformed cell growth and malignant progression of solid cancers.

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.

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.

A deterministic code for transcription factor-DNA recognition through computation of binding interfaces.

The recognition code between transcription factor (TF) amino acids and DNA bases remains poorly understood. Here, the determinants of TF amino acid-DNA base binding selectivity were identified through the analysis of crystals of TF-DNA complexes. Selective, high-frequency interactions were identified for the vast majority of amino acid side chains ('structural code'). DNA binding specificities were then independently assessed by meta-analysis of random-mutagenesis studies of Zn finger-target DNA sequences. Selective, high-frequency interactions were identified for the majority of mutagenized residues ('mutagenesis code'). The structural code and the mutagenesis code were shown to match to a striking level of accuracy (P = 3.1 × 10-33), suggesting the identification of fundamental rules of TF binding to DNA bases. Additional insight was gained by showing a geometry-dictated choice among DNA-binding TF residues with overlapping specificity. These findings indicate the existence of a DNA recognition mode whereby the physical-chemical characteristics of the interacting residues play a deterministic role. The discovery of this DNA recognition code advances our knowledge on fundamental features of regulation of gene expression and is expected to pave the way for integration with higher-order complexity approaches.

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.

Letter to the editor: efficacy and safety of anti-Trop antibodies, R. Cubas, M. Li, C. Chen and Q. Yao, Biochim Biophys Acta 1796 (2009) 309-1.

Still little information is available on the efficacy and toxicity of anti-Trop-2 antibodies in man. Findings on antibodies anti-Trop-1/Ep-CAM, a paralog molecule of Trop-2, may provide preliminary indications, and a low-affinity anti-Trop-1/Ep-CAM monoclonal antibody, failed to show any benefit in colon cancer patients. Other anti-Trop-1 antibodies, e.g. MT201, may bear more promise, as may more advanced molecular forms, e.g. a BiTE design (MT110) or trifunctional antibodies (catumaxomab). However, the efficacy of these reagents in cancer patients still needs to be proven in randomized clinical trials. As for toxicity, the administration of ING-1, a high-affinity, human-engineered anti-Trop-1/Ep-CAM monoclonal antibody, caused cases of acute pancreatitis. The exocrine pancreas also expresses Trop-2. Hence, similar concerns should apply to the administration of anti-Trop-2 monoclonal antibodies to patients. More in general, contrary to the statements by Cubas et al., Trop-2/T-16/Mov-16 is expressed by several normal tissues in man, e.g. epidermis, exocervix, esophagus, tongue, urothelium, kidney, pancreas, trophoblast and breast. Hence, additional effort is required to develop Trop-2 into a useful immunotherapeutic target, by increasing anti-Trop-2 antibody efficacy, while keeping under control toxicity on expressing normal tissues.

Trop-2 cleavage by ADAM10 is an activator switch for cancer growth and metastasis.

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 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.

Cell growth stimulation by CRASH, an asparaginase-like protein overexpressed in human tumors and metastatic breast cancers.

The gene encoding CRASH, a human asparaginase-like protein, has been cloned and its transcriptional activation has been detected in gynecologic cancers. To define the expression of CRASH in human tumors and its possible functional role, monoclonal antibodies against the CRASH protein have been generated. In non-transformed tissues CRASH was only detected in testis, brain, esophagus, prostate and proliferating endometrium. On the other hand, 36/50 ovarian carcinomas, 16/78 mammary carcinomas, 6/6 uroepithelial bladder carcinomas and 5/33 colon carcinomas scored positive for CRASH, with the absence of reactivity in the corresponding normal tissues. Strikingly, 11 out of the 16 breast cancers that expressed CRASH were metastatic, nominating CRASH to be functionally relevant in tumor progression. Twenty-eight out of 42 endometrium tumors expressed CRASH at high levels as did 5/41 prostate carcinomas, as well as ovary and breast cancers, indicating a regulation of CRASH expression by sex hormones. A bona fide estrogen responsive element was detected at bases -201/-183. This proved to be highly preserved across species, supporting an actual functional role. Asparaginase-like proteins play a role in growth regulation and signaling by p70 S6 kinase. The somatic knock-out of CRASH resulted in significant inhibition of growth of KM12L4A colon carcinoma cells, which abundantly express CRASH, whereas the proliferation of the syngeneic, weakly-expressing, slowly-growing KL12SM was not affected. These results are consistent with a selective growth advantage for aggressive cancers expressing CRASH, and nominate CRASH as a novel diagnostic and therapeutic tumor target.

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.