Genetic Ablation of the MET Oncogene Defines a Crucial Role of the HGF/MET Axis in Cell-Autonomous Functions Driving Tumor Dissemination.

Cancer cell dissemination is sustained by cell-autonomous and non-cell-autonomous functions. To disentangle the role of HGF (Hepatocyte Growth Factor) and MET ligand/receptor axis in this complex process, we genetically knocked out the MET gene in cancer cells in which MET is not the oncogenic driver. In this way, we evaluated the contribution of the HGF/MET axis to cancer cell dissemination independently of its direct activities in cells of the tumor microenvironment. The lack of MET expression in MET-/- cells has been proved by molecular characterization. From a functional point of view, HGF stimulation of MET-/- cancer cells was ineffective in eliciting intracellular signaling and in sustaining biological functions predictive of malignancy in vitro (i.e., anchorage-independent growth, invasion, and survival in the absence of matrix adhesion). Cancer cell dissemination was assessed in vivo, evaluating: (i) the ability of MET-/- lung carcinoma cells to colonize the lungs following intravenous injection and (ii) the spontaneous dissemination to distant organs of MET-/- pancreatic carcinoma cells upon orthotopic injection. In both experimental models, MET ablation affects the time of onset, the number, and the size of metastatic lesions. These results define a crucial contribution of the HGF/MET axis to cell-autonomous functions driving the metastatic process.

Exploring circular MET RNA as a potential biomarker in tumors exhibiting high MET activity.

BACKGROUND: MET-driven acquired resistance is emerging with unanticipated frequency in patients relapsing upon molecular therapy treatments. However, the determination of MET amplification remains challenging using both standard and next-generation sequencing-based methodologies. Liquid biopsy is an effective, non-invasive approach to define cancer genomic profiles, track tumor evolution over time, monitor treatment response and detect molecular resistance in advance. Circular RNAs (circRNAs), a family of RNA molecules that originate from a process of back-splicing, are attracting growing interest as potential novel biomarkers for their stability in body fluids. METHODS: We identified a circRNA encoded by the MET gene (circMET) and exploited blood-derived cell-free RNA (cfRNA) and matched tumor tissues to identify, stratify and monitor advanced cancer patients molecularly characterized by high MET activity, generally associated with genomic amplification. RESULTS: Using publicly available bioinformatic tools, we discovered that the MET locus transcribes several circRNA molecules, but only one candidate, circMET, was particularly abundant. Deeper molecular analysis revealed that circMET levels positively correlated with MET expression and activity, especially in MET-amplified cells. We developed a circMET-detection strategy and, in parallel, we performed standard FISH and IHC analyses in the same specimens to assess whether circMET quantification could identify patients displaying high MET activity. Longitudinal monitoring of circMET levels in the plasma of selected patients revealed the early emergence of MET amplification as a mechanism of acquired resistance to molecular therapies. CONCLUSIONS: We found that measurement of circMET levels allows identification and tracking of patients characterized by high MET activity. Circulating circMET (ccMET) detection and analysis could be a simple, cost-effective, non-invasive approach to better implement patient stratification based on MET expression, as well as to dynamically monitor over time both therapy response and clonal evolution during treatment.

Tumor plasticity and therapeutic resistance in oncogene-addicted non-small cell lung cancer: from preclinical observations to clinical implications.

The identification of actionable targets in oncogene-addicted non-small cell lung cancer (NSCLC) has fueled biomarker-directed strategies, especially in advanced stage disease. Despite the undeniable success of molecular targeted therapies, duration of clinical response is relatively short-lived. While extraordinary efforts have defined the complexity of tumor architecture and clonal evolution at the genetic level, not equal interest has been given to the dynamic mechanisms of phenotypic adaptation engaged by cancer during treatment. At the clinical level, molecular targeted therapy of EGFR-mutant and ALK-rearranged tumors often results in epithelial-to-mesenchymal transition (EMT) and histological transformation of the original adenocarcinoma without the acquisition of additional genetic lesions, thus limiting subsequent therapeutic options and patient outcome. Here we provide an overview of the current understanding of the genetic and non-genetic molecular circuits governing this phenomenon, presenting current strategies and potentially innovative therapeutic approaches to interfere with lung cancer cell plasticity.

Water and microbial monitoring technologies towards the near future space exploration.

Space exploration is demanding longer lasting human missions and water resupply from Earth will become increasingly unrealistic. In a near future, the spacecraft water monitoring systems will require technological advances to promptly identify and counteract contingent events of waterborne microbial contamination, posing health risks to astronauts with lowered immune responsiveness. The search for bio-analytical approaches, alternative to those applied on Earth by cultivation-dependent methods, is pushed by the compelling need to limit waste disposal and avoid microbial regrowth from analytical carryovers. Prospective technologies will be selected only if first validated in a flight-like environment, by following basic principles, advantages, and limitations beyond their current applications on Earth. Starting from the water monitoring activities applied on the International Space Station, we provide a critical overview of the nucleic acid amplification-based approaches (i.e., loop-mediated isothermal amplification, quantitative PCR, and high-throughput sequencing) and early-warning methods for total microbial load assessments (i.e., ATP-metry, flow cytometry), already used at a high readiness level aboard crewed space vehicles. Our findings suggest that the forthcoming space applications of mature technologies will be necessarily bounded by a compromise between analytical performances (e.g., speed to results, identification depth, reproducibility, multiparametricity) and detrimental technical requirements (e.g., reagent usage, waste production, operator skills, crew time). As space exploration progresses toward extended missions to Moon and Mars, miniaturized systems that also minimize crew involvement in their end-to-end operation are likely applicable on the long-term and suitable for the in-flight water and microbiological research.

Future perspectives from lung cancer pre-clinical models: new treatments are coming?

Lung cancer currently stands out as both the most common and the most lethal type of cancer, the latter feature being partly explained by the fact that the majority of lung cancer patients already display advanced disease at the time of diagnosis. In recent years, the development of specific tyrosine kinase inhibitors (TKI) for the therapeutic benefit of patients harboring certain molecular aberrations and the introduction of prospective molecular profiling in the clinical practice have revolutionized the treatment of advanced non-small cell lung cancer (NSCLC). However, the identification of the best strategies to enhance treatment effectiveness and to avoid the critical phenomenon of drug tolerance and acquired resistance in patients with lung cancer still remains an unmet medical need. Circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) are two complementary approaches to define tumor heterogeneity and clonal evolution in a non-invasive manner and to perform functional studies on metastatic cells. Finally, the recent discovery that the tumor microenvironment architecture can be faithfully recapitulated in vitro represents a novel pre-clinical frontier with the potential to optimize more effective immunology-based precision therapies that could rapidly move forward to the clinic.

Stromal Microenvironment Shapes the Intratumoral Architecture of Pancreatic Cancer.

Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.

Water Quality and Total Microbial Load: A Double-Threshold Identification Procedure Intended for Space Applications.

During longer-lasting future space missions, water renewal by ground-loaded supplies will become increasingly expensive and unmanageable for months. Space exploration by self-sufficient spacecrafts is thus demanding the development of culture-independent microbiological methods for in-flight water monitoring to counteract possible contamination risks. In this study, we aimed at evaluating total microbial load data assessed by selected early-warning techniques with current or promising perspectives for space applications (i.e., HPC, ATP-metry, qPCR, flow cytometry), through the analysis of water sources with constitutively different contamination levels (i.e., chlorinated and unchlorinated tap waters, groundwaters, river waters, wastewaters). Using a data-driven double-threshold identification procedure, we presented new reference values of water quality based on the assessment of the total microbial load. Our approach is suitable to provide an immediate alert of microbial load peaks, thus enhancing the crew responsiveness in case of unexpected events due to water contamination and treatment failure. Finally, the backbone dataset could help in managing water quality and monitoring issues for both space and Earth-based applications.

AR Expression in Breast Cancer CTCs Associates with Bone Metastases.

Molecular drivers underlying bone metastases in human cancer are not well understood, in part due to constraints in bone tissue sampling. Here, RNA sequencing was performed of circulating tumor cells (CTC) isolated from blood samples of women with metastatic estrogen receptor (ER)+ breast cancer, comparing cases with progression in bone versus visceral organs. Among the activated cellular pathways in CTCs from bone-predominant breast cancer is androgen receptor (AR) signaling. AR gene expression is evident, as is its constitutively active splice variant AR-v7. AR expression within CTCs is correlated with the duration of treatment with aromatase inhibitors, suggesting that it contributes to acquired resistance to endocrine therapy. In an established breast cancer xenograft model, a bone-tropic derivative displays increased AR expression, whose genetic or pharmacologic suppression reduces metastases to bone but not to lungs. Together, these observations identify AR signaling in CTCs from women with bone-predominant ER+ breast cancer, and provide a rationale for testing androgen inhibitors in this subset of patients.Implications: This study highlights a role for the AR in breast cancer bone metastasis, and suggests that therapeutic targeting of the AR may benefit patients with metastatic breast cancer. Mol Cancer Res; 16(4); 720-7. ©2018 AACR.

Deep Sequencing Reveals a Novel miR-22 Regulatory Network with Therapeutic Potential in Rhabdomyosarcoma.

Current therapeutic options for the pediatric cancer rhabdomyosarcoma have not improved significantly, especially for metastatic rhabdomyosarcoma. In the current work, we performed a deep miRNA profiling of the three major human rhabdomyosarcoma subtypes, along with cell lines and normal muscle, to identify novel molecular circuits with therapeutic potential. The signature we determined could discriminate rhabdomyosarcoma from muscle, revealing a subset of muscle-enriched miRNA (myomiR), including miR-22, which was strongly underexpressed in tumors. miR-22 was physiologically induced during normal myogenic differentiation and was transcriptionally regulated by MyoD, confirming its identity as a myomiR. Once introduced into rhabdomyosarcoma cells, miR-22 decreased cell proliferation, anchorage-independent growth, invasiveness, and promoted apoptosis. Moreover, restoring miR-22 expression blocked tumor growth and prevented tumor dissemination in vivo Gene expression profiling analysis of miR-22-expressing cells suggested TACC1 and RAB5B as possible direct miR-22 targets. Accordingly, loss- and gain-of-function experiments defined the biological relevance of these genes in rhabdomyosarcoma pathogenesis. Finally, we demonstrated the ability of miR-22 to intercept and overcome the intrinsic resistance to MEK inhibition based on ERBB3 upregulation. Overall, our results identified a novel miR-22 regulatory network with critical therapeutic implications in rhabdomyosarcoma. Cancer Res; 76(20); 6095-106. ©2016 AACR.

A tunable delivery platform to provide local chemotherapy for pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating and painful cancers. It is often highly resistant to therapy owing to inherent chemoresistance and the desmoplastic response that creates a barrier of fibrous tissue preventing transport of chemotherapeutics into the tumor. The growth of the tumor in pancreatic cancer often leads to invasion of other organs and partial or complete biliary obstruction, inducing intense pain for patients and necessitating tumor resection or repeated stenting. Here, we have developed a delivery device to provide enhanced palliative therapy for pancreatic cancer patients by providing high concentrations of chemotherapeutic compounds locally at the tumor site. This treatment could reduce the need for repeated procedures in advanced PDAC patients to debulk the tumor mass or stent the obstructed bile duct. To facilitate clinical translation, we created the device out of currently approved materials and drugs. We engineered an implantable poly(lactic-co-glycolic)-based biodegradable device that is able to linearly release high doses of chemotherapeutic drugs for up to 60 days. We created five patient-derived PDAC cell lines and tested their sensitivity to approved chemotherapeutic compounds. These in vitro experiments showed that paclitaxel was the most effective single agent across all cell lines. We compared the efficacy of systemic and local paclitaxel therapy on the patient-derived cell lines in an orthotopic xenograft model in mice (PDX). In this model, we found up to a 12-fold increase in suppression of tumor growth by local therapy in comparison to systemic administration and reduce retention into off-target organs. Herein, we highlight the efficacy of a local therapeutic approach to overcome PDAC chemoresistance and reduce the need for repeated interventions and biliary obstruction by preventing local tumor growth. Our results underscore the urgent need for an implantable drug-eluting platform to deliver cytotoxic agents directly within the tumor mass as a novel therapeutic strategy for patients with pancreatic cancer.

Hepatocyte Growth Factor-mediated satellite cells niche perturbation promotes development of distinct sarcoma subtypes.

Embryonal Rhabdomyosarcoma (ERMS) and Undifferentiated Pleomorphic Sarcoma (UPS) are distinct sarcoma subtypes. Here we investigate the relevance of the satellite cell (SC) niche in sarcoma development by using Hepatocyte Growth Factor (HGF) to perturb the niche microenvironment. In a Pax7 wild type background, HGF stimulation mainly causes ERMS that originate from satellite cells following a process of multistep progression. Conversely, in a Pax7 null genotype ERMS incidence drops, while UPS becomes the most frequent subtype. Murine EfRMS display genetic heterogeneity similar to their human counterpart. Altogether, our data demonstrate that selective perturbation of the SC niche results in distinct sarcoma subtypes in a Pax7 lineage-dependent manner, and define a critical role for the Met axis in sarcoma initiation. Finally, our results provide a rationale for the use of combination therapy, tailored on specific amplifications and activated signaling pathways, to minimize resistance emerging from sarcomas heterogeneity.

Pericentromeric satellite repeat expansions through RNA-derived DNA intermediates in cancer.

Aberrant transcription of the pericentromeric human satellite II (HSATII) repeat is present in a wide variety of epithelial cancers. In deriving experimental systems to study its deregulation, we observed that HSATII expression is induced in colon cancer cells cultured as xenografts or under nonadherent conditions in vitro, but it is rapidly lost in standard 2D cultures. Unexpectedly, physiological induction of endogenous HSATII RNA, as well as introduction of synthetic HSATII transcripts, generated cDNA intermediates in the form of DNA/RNA hybrids. Single molecule sequencing of tumor xenografts showed that HSATII RNA-derived DNA (rdDNA) molecules are stably incorporated within pericentromeric loci. Suppression of RT activity using small molecule inhibitors reduced HSATII copy gain. Analysis of whole-genome sequencing data revealed that HSATII copy number gain is a common feature in primary human colon tumors and is associated with a lower overall survival. Together, our observations suggest that cancer-associated derepression of specific repetitive sequences can promote their RNA-driven genomic expansion, with potential implications on pericentromeric architecture.

SMYD1 and G6PD modulation are critical events for miR-206-mediated differentiation of rhabdomyosarcoma.

Rhadomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood. RMS cells resemble fetal myoblasts but are unable to complete myogenic differentiation. In previous work we showed that miR-206, which is low in RMS, when induced in RMS cells promotes the resumption of differentiation by modulating more than 700 genes. To better define the pathways involved in the conversion of RMS cells into their differentiated counterpart, we focused on 2 miR-206 effectors emerged from the microarray analysis, SMYD1 and G6PD. SMYD1, one of the most highly upregulated genes, is a H3K4 histone methyltransferase. Here we show that SMYD1 silencing does not interfere with the proliferative block or with the loss anchorage independence imposed by miR-206, but severely impairs differentiation of ERMS, ARMS, and myogenic cells. Thus SMYD1 is essential for the activation of muscle genes. Conversely, among the downregulated genes, we found G6PD, the enzyme catalyzing the rate-limiting step of the pentose phosphate shunt. In this work, we confirmed that G6PD is a direct target of miR-206. Moreover, we showed that G6PD silencing in ERMS cells impairs proliferation and soft agar growth. However, G6PD overexpression does not interfere with the pro-differentiating effect of miR-206, suggesting that G6PD downmodulation contributes to - but is not an absolute requirement for - the tumor suppressive potential of miR-206. Targeting cancer metabolism may enhance differentiation. However, therapeutic inhibition of G6PD is encumbered by side effects. As an alternative, we used DCA in combination with miR-206 to increase the flux of pyruvate into the mitochondrion by reactivating PDH. DCA enhanced the inhibition of RMS cell growth induced by miR-206, and sustained it upon miR-206 de-induction. Altogether these results link miR-206 to epigenetic and metabolic reprogramming, and suggest that it may be worth combining differentiation-inducing with metabolism-directed approaches.

Alternative lengthening of telomeres renders cancer cells hypersensitive to ATR inhibitors.

Cancer cells rely on telomerase or the alternative lengthening of telomeres (ALT) pathway to overcome replicative mortality. ALT is mediated by recombination and is prevalent in a subset of human cancers, yet whether it can be exploited therapeutically remains unknown. Loss of the chromatin-remodeling protein ATRX associates with ALT in cancers. Here, we show that ATRX loss compromises cell-cycle regulation of the telomeric noncoding RNA TERRA and leads to persistent association of replication protein A (RPA) with telomeres after DNA replication, creating a recombinogenic nucleoprotein structure. Inhibition of the protein kinase ATR, a critical regulator of recombination recruited by RPA, disrupts ALT and triggers chromosome fragmentation and apoptosis in ALT cells. The cell death induced by ATR inhibitors is highly selective for cancer cells that rely on ALT, suggesting that such inhibitors may be useful for treatment of ALT-positive cancers.

Bioengineered implantable scaffolds as a tool to study stromal-derived factors in metastatic cancer models.

Modeling the hematogenous spread of cancer cells to distant organs poses one of the greatest challenges in the study of human metastasis. Both tumor cell-intrinsic properties as well as interactions with reactive stromal cells contribute to this process, but identification of relevant stromal signals has been hampered by the lack of models allowing characterization of the metastatic niche. Here, we describe an implantable bioengineered scaffold, amenable to in vivo imaging, ex vivo manipulation, and serial transplantation for the continuous study of human metastasis in mice. Orthotopic or systemic inoculation of tagged human cancer cells into the mouse leads to the release of circulating tumor cells into the vasculature, which seed the scaffold, initiating a metastatic tumor focus. Mouse stromal cells can be readily recovered and profiled, revealing differential expression of cytokines, such as IL1ß, from tumor-bearing versus unseeded scaffolds. Finally, this platform can be used to test the effect of drugs on suppressing initiation of metastatic lesions. This generalizable model to study cancer metastasis may thus identify key stromal-derived factors with important implications for basic and translational cancer research.

Single-cell RNA sequencing identifies extracellular matrix gene expression by pancreatic circulating tumor cells.

Circulating tumor cells (CTCs) are shed from primary tumors into the bloodstream, mediating the hematogenous spread of cancer to distant organs. To define their composition, we compared genome-wide expression profiles of CTCs with matched primary tumors in a mouse model of pancreatic cancer, isolating individual CTCs using epitope-independent microfluidic capture, followed by single-cell RNA sequencing. CTCs clustered separately from primary tumors and tumor-derived cell lines, showing low-proliferative signatures, enrichment for the stem-cell-associated gene Aldh1a2, biphenotypic expression of epithelial and mesenchymal markers, and expression of Igfbp5, a gene transcript enriched at the epithelial-stromal interface. Mouse as well as human pancreatic CTCs exhibit a very high expression of stromal-derived extracellular matrix (ECM) proteins, including SPARC, whose knockdown in cancer cells suppresses cell migration and invasiveness. The aberrant expression by CTCs of stromal ECM genes points to their contribution of microenvironmental signals for the spread of cancer to distant organs.

Cancer therapy. Ex vivo culture of circulating breast tumor cells for individualized testing of drug susceptibility.

Circulating tumor cells (CTCs) are present at low concentrations in the peripheral blood of patients with solid tumors. It has been proposed that the isolation, ex vivo culture, and characterization of CTCs may provide an opportunity to noninvasively monitor the changing patterns of drug susceptibility in individual patients as their tumors acquire new mutations. In a proof-of-concept study, we established CTC cultures from six patients with estrogen receptor-positive breast cancer. Three of five CTC lines tested were tumorigenic in mice. Genome sequencing of the CTC lines revealed preexisting mutations in the PIK3CA gene and newly acquired mutations in the estrogen receptor gene (ESR1), PIK3CA gene, and fibroblast growth factor receptor gene (FGFR2), among others. Drug sensitivity testing of CTC lines with multiple mutations revealed potential new therapeutic targets. With optimization of CTC culture conditions, this strategy may help identify the best therapies for individual cancer patients over the course of their disease.

The IGF2 intronic miR-483 selectively enhances transcription from IGF2 fetal promoters and enhances tumorigenesis.

Insulin-like growth factor 2 (IGF2), a developmentally regulated and maternally imprinted gene, is frequently overexpressed in pediatric cancers. Although loss of imprinting (LOI) at fetal promoters contributes to increased IGF2 in tumors, the magnitude of IGF2 expression suggests the involvement of additional regulatory mechanisms. A microRNA (miRNA) screen of primary Wilms' tumors identified specific overexpression of miR-483-5p, which is embedded within the IGF2 gene. Unexpectedly, the IGF2 mRNA itself is transcriptionally up-regulated by miR-483-5p. A nuclear pool of miR-483-5p binds directly to the 5' untranslated region (UTR) of fetal IGF2 mRNA, enhancing the association of the RNA helicase DHX9 to the IGF2 transcript and promoting IGF2 transcription. Ectopic expression of miR-483-5p in IGF2-dependent sarcoma cells is correlated with increased tumorigenesis in vivo. Together, these observations suggest a functional positive feedback loop of an intronic miRNA on transcription of its host gene.

Capture and printing of fixed stromal cell membranes for bioactive display on PDMS surfaces.

Poly(dimethylsiloxane) (PDMS) has emerged as an extremely useful polymer for various biological applications. The conjugation of PDMS with bioactive molecules to create functional surfaces is feasible yet limited to a single-molecule display with imprecise localization of the molecules on PDMS. Here we report a robust technique that can transfer and print the membrane surface of glutaraldehyde-fixed stromal cells intact onto a PDMS substrate using an intermediate polyvinylalcohol (PVA) film as a transporter system. The cell-PVA film capturing the entirety of surface molecules can be peeled off and subsequently printed onto PDMS while maintaining the spatial display of the original cell surface molecules. Proof-of-concept studies are described using human bone marrow stromal cell membranes including a demonstration of the bioactivity of transferred membranes to capture and adhere hematopoietic cells. The presented process is applicable to virtually any adherent cell and can broaden the functional display of biomolecules on PDMS for biotechnology applications.

Met receptor acts uniquely for survival and morphogenesis of EGFR-dependent normal mammary epithelial and cancer cells.

Mammary gland development and breast cancer growth require multiple factors both of endocrine and paracrine origin. We analyzed the roles of Epidermal Growth Factor Receptor (EGFR) and Hepatocyte Growth Factor Receptor (Met) in mammary epithelial cells and mammary tumor cells derived from a mutated-ErbB2 transgenic mice. By using highly specific tyrosine kinase inhibitors we found that MCF-10A and NMuMG mammary epithelial cell lines are totally dependent on EGFR activation for their growth and survival. Proliferation and 3D-morphogenesis assays showed that HGF had no role in maintaining mammary cell viability, but was the only cytokine able to rescue EGFR-inhibited mammary cells. Insulin-Like Growth Factor-I (IGF-I), basic-Fibroblast Growth Factor (b-FGF) and Neuregulin, which are well known mammary morphogenic factors, did not rescue proliferation or morphogenesis in these cell lines, following EGFR inhibition. Similarly, ErbB2-driven tumor cells are EGFR-dependent and also display HGF-mediated rescue. Western-blot analysis of the signaling pathways involved in rescue after EGFR inhibition indicated that concomitant ERK1/2 and AKT activation was exclusively driven by Met, but not by IGF-I or b-FGF. These results describe a unique role for EGFR and Met in mammary epithelial cells by showing that similar pathways can be used by tumorigenic cells to sustain growth and resist to EGFR-directed anti-tumorigenic drugs.