Biological and targeting differences between the rare KRAS A146T and canonical KRAS mutants in gastric cancer models.

BACKGROUND: Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with a poor prognosis for patients with advanced disease. Since the oncogenic role of KRAS mutants has been poorly investigated in GC, this study aims to biochemically and biologically characterize different KRAS-mutated models and unravel differences among KRAS mutants in response to therapy. METHODS: Taking advantage of a proprietary, molecularly annotated platform of more than 200 GC PDXs (patient-derived xenografts), we identified KRAS-mutated PDXs, from which primary cell lines were established. The different mutants were challenged with KRAS downstream inhibitors in in vitro and in vivo experiments. RESULTS: Cells expressing the rare KRAS A146T mutant showed lower RAS-GTP levels compared to those bearing the canonical G12/13D mutations. Nevertheless, all the KRAS-mutated cells displayed KRAS addiction. Surprisingly, even if the GEF SOS1 is considered critical for the activation of KRAS A146T mutants, its abrogation did not significantly affect cell viability. From the pharmacologic point of view, Trametinib monotherapy was more effective in A146T than in G12D-mutated models, suggesting a vulnerability to MEK inhibition. However, in the presence of mutations in the PI3K pathway, more frequently co-occurrent in A146T models, the association of Trametinib and the AKT inhibitor MK-2206 was required to optimize the response. CONCLUSION: A deeper genomic and biological characterization of KRAS mutants might sustain the development of more efficient and long-lasting therapeutic options for patients harbouring KRAS-driven GC.

Inhibition of Mitochondrial Redox Signaling with MitoQ Prevents Metastasis of Human Pancreatic Cancer in Mice.

At diagnosis, about 35% of pancreatic cancers are at the locally invasive yet premetastatic stage. Surgical resection is not a treatment option, leaving patients with a largely incurable disease that often evolves to the polymetastatic stage despite chemotherapeutic interventions. In this preclinical study, we hypothesized that pancreatic cancer metastasis can be prevented by inhibiting mitochondrial redox signaling with MitoQ, a mitochondria-targeted antioxidant. Using four different cancer cell lines, we report that, at clinically relevant concentrations (100-500 nM), MitoQ selectively repressed mesenchymal pancreatic cancer cell respiration, which involved the inhibition of the expression of PGC-1α, NRF1 and a reduced expression of electron-transfer-chain complexes I to III. MitoQ consequently decreased the mitochondrial membrane potential and mitochondrial superoxide production by these cells. Phenotypically, MitoQ further inhibited pancreatic cancer cell migration, invasion, clonogenicity and the expression of stem cell markers. It reduced by ~50% the metastatic homing of human MIA PaCa-2 cells in the lungs of mice. We further show that combination treatments with chemotherapy are conceivable. Collectively, this study indicates that the inhibition of mitochondrial redox signaling is a possible therapeutic option to inhibit the metastatic progression of pancreatic cancer.

Mitochondrial Protein Cox7b Is a Metabolic Sensor Driving Brain-Specific Metastasis of Human Breast Cancer Cells.

Distant metastases are detrimental for cancer patients, but the increasingly early detection of tumors offers a chance for metastasis prevention. Importantly, cancers do not metastasize randomly: depending on the type of cancer, metastatic progenitor cells have a predilection for well-defined organs. This has been theorized by Stephen Paget, who proposed the "seed-and-soil hypothesis", according to which metastatic colonization occurs only when the needs of a given metastatic progenitor cell (the seed) match with the resources provided by a given organ (the soil). Here, we propose to explore the seed-and-soil hypothesis in the context of cancer metabolism, thus hypothesizing that metastatic progenitor cells must be capable of detecting the availability of metabolic resources in order to home in a secondary organ. If true, it would imply the existence of metabolic sensors. Using human triple-negative MDA-MB-231 breast cancer cells and two independent brain-seeking variants as models, we report that cyclooxygenase 7b (Cox7b), a structural component of Complex IV of the mitochondrial electron transport chain, belongs to a probably larger family of proteins responsible for breast cancer brain tropism in mice. For metastasis prevention therapy, this proof-of-principle study opens a quest for the identification of therapeutically targetable metabolic sensors that drive cancer organotropism.

Ketogenic diets slow melanoma growth in vivo regardless of tumor genetics and metabolic plasticity.

BACKGROUND: Growing evidence supports the use of low-carbohydrate/high-fat ketogenic diets as an adjunctive cancer therapy. However, it is unclear which genetic, metabolic, or immunological factors contribute to the beneficial effect of ketogenic diets. Therefore, we investigated the effect of ketogenic diets on the progression and metabolism of genetically and metabolically heterogeneous melanoma xenografts, as well as on the development of melanoma metastases in mice with a functional immune system. METHODS: Mice bearing BRAF mutant, NRAS mutant, and wild-type melanoma xenografts as well as mice bearing highly metastatic melanoma allografts were fed with a control diet or ketogenic diets, differing in their triglyceride composition, to evaluate the effect of ketogenic diets on tumor growth and metastasis. We performed an in-depth targeted metabolomics analysis in plasma and xenografts to elucidate potential antitumor mechanisms in vivo. RESULTS: We show that ketogenic diets effectively reduced tumor growth in immunocompromised mice bearing genetically and metabolically heterogeneous human melanoma xenografts. Furthermore, the ketogenic diets exerted a metastasis-reducing effect in the immunocompetent syngeneic melanoma mouse model. Targeted analysis of plasma and tumor metabolomes revealed that ketogenic diets induced distinct changes in amino acid metabolism. Interestingly, ketogenic diets reduced the levels of alpha-amino adipic acid, a biomarker of cancer, in circulation to levels observed in tumor-free mice. Additionally, alpha-amino adipic acid was reduced in xenografts by ketogenic diets. Moreover, the ketogenic diets increased sphingomyelin levels in plasma and the hydroxylation of sphingomyelins and acylcarnitines in tumors. CONCLUSIONS: Ketogenic diets induced antitumor effects toward melanoma regardless of the tumors´ genetic background, its metabolic signature, and the host immune status. Moreover, ketogenic diets simultaneously affected multiple metabolic pathways to create an unfavorable environment for melanoma cell proliferation, supporting their potential as a complementary nutritional approach to melanoma therapy.

MitoQ Prevents Human Breast Cancer Recurrence and Lung Metastasis in Mice.

In oncology, the occurrence of distant metastases often marks the transition from curative to palliative care. Such outcome is highly predictable for breast cancer patients, even if tumors are detected early, and there is no specific treatment to prevent metastasis. Previous observations indicated that cancer cell mitochondria are bioenergetic sensors of the tumor microenvironment that produce superoxide to promote evasion. Here, we tested whether mitochondria-targeted antioxidant MitoQ is capable to prevent metastasis in the MDA-MB-231 model of triple-negative human breast cancer in mice and in the MMTV-PyMT model of spontaneously metastatic mouse breast cancer. At clinically relevant doses, we report that MitoQ not only prevented metastatic take and dissemination, but also local recurrence after surgery. We further provide in vitro evidence that MitoQ does not interfere with conventional chemotherapies used to treat breast cancer patients. Since MitoQ already successfully passed Phase I safety clinical trials, our preclinical data collectively provide a strong incentive to test this drug for the prevention of cancer dissemination and relapse in clinical trials with breast cancer patients.

MitoQ Inhibits Human Breast Cancer Cell Migration, Invasion and Clonogenicity.

To successfully generate distant metastases, metastatic progenitor cells must simultaneously possess mesenchymal characteristics, resist to anoïkis, migrate and invade directionally, resist to redox and shear stresses in the systemic circulation, and possess stem cell characteristics. These cells primarily originate from metabolically hostile areas of the primary tumor, where oxygen and nutrient deprivation, together with metabolic waste accumulation, exert a strong selection pressure promoting evasion. Here, we followed the hypothesis according to which metastasis as a whole implies the existence of metabolic sensors. Among others, mitochondria are singled out as a major source of superoxide that supports the metastatic phenotype. Molecularly, stressed cancer cells increase mitochondrial superoxide production, which activates the transforming growth factor-ß pathway through src directly within mitochondria, ultimately activating focal adhesion kinase Pyk2. The existence of mitochondria-targeted antioxidants constitutes an opportunity to interfere with the metastatic process. Here, using aggressive triple-negative and HER2-positive human breast cancer cell lines as models, we report that MitoQ inhibits all the metastatic traits that we tested in vitro. Compared to other mitochondria-targeted antioxidants, MitoQ already successfully passed Phase I safety clinical trials, which provides an important incentive for future preclinical and clinical evaluations of this drug for the prevention of breast cancer metastasis.

Personalized therapeutic strategies in HER2-driven gastric cancer.

BACKGROUND: Trastuzumab is the only approved targeted therapy in patients with HER2-amplified metastatic gastric cancer (GC). Regrettably, in clinical practice, only a fraction of them achieves long-term benefit from trastuzumab-based upfront strategy. To advance precision oncology, we investigated the therapeutic efficacy of different HER2-targeted strategies, in HER2 "hyper"-amplified (≥ 8 copies) tumors. METHODS: We undertook a prospective evaluation of HER2 targeting with monoclonal antibodies, tyrosine kinase inhibitors and antibody-drug conjugates, in a selected subgroup of HER2 "hyper"-amplified gastric patient-derived xenografts (PDXs), through the design of ad hoc preclinical trials. RESULTS: Despite the high level of HER2 amplification, trastuzumab elicited a partial response only in 2 out of 8 PDX models. The dual-HER2 blockade with trastuzumab plus either pertuzumab or lapatinib led to complete and durable responses in 5 (62.5%) out of 8 models, including one tumor bearing a concomitant HER2 mutation. In a resistant PDX harboring KRAS amplification, the novel antibody-drug conjugate trastuzumab deruxtecan (but not trastuzumab emtansine) overcame KRAS-mediated resistance. We also identified a HGF-mediated non-cell-autonomous mechanism of secondary resistance to anti-HER2 drugs, responsive to MET co-targeting. CONCLUSION: These preclinical randomized trials clearly indicate that in HER2-driven gastric tumors, a boosted HER2 therapeutic blockade is required for optimal efficacy, leading to complete and durable responses in most of the cases. Our results suggest that a selected subpopulation of HER2-"hyper"-amplified GC patients could strongly benefit from this strategy. Despite the negative results of clinical trials, the dual blockade should be reconsidered for patients with clearly HER2-addicted cancers.

In Vitro and In Vivo Characterization of MCT1 Inhibitor AZD3965 Confirms Preclinical Safety Compatible with Breast Cancer Treatment.

To survive and proliferate in solid tumors, cancer cells adapt and evolve rapidly in microenvironments where oxygen and substrate bioavailability fluctuates over time and space. This creates metabolic heterogeneity. Cancer cells can further cooperate metabolically, for example by swapping glycolytic end-product lactate for blood-borne glucose. This type of cooperation can be targeted therapeutically, since transmembrane lactate exchanges are facilitated by lactate-proton symporters of the monocarboxylate (MCT) family. Among new drugs, AZD3965 is a first-in-class selective MCT1 inhibitor currently tested in Phase I/II clinical trials for patients with different types of cancers. Because MCT1 can function bidirectionally, we tested here whether and how malignant and nonmalignant cells adapt their metabolism and MCT repertoire when AZD3965 inhibits either lactate import or export. Using breast-associated malignant and nonmalignant cell lines as models, we report that AZD3965 is not directly cytotoxic. In the presence of glucose and glutamine, oxidative cells can survive when lactate uptake is blocked, and proliferating cells compensate MCT1 inhibition by overexpressing MCT4, a specialized facilitator of lactate export. Phenotypic characterization of mice focusing on metabolism, muscle and brain physiology found partial and transient memory retention defect as sole consequence of MCT1 inhibition by AZD3965. We therefore conclude that AZD3965 is compatible with anticancer therapy.

An EPR Study Using Cyclic Hydroxylamines To Assess The Level of Mitochondrial ROS in Superinvasive Cancer Cells.

It has been proposed that a mitochondrial switch involving a high mitochondrial superoxide production is associated with cancer metastasis. We here report an EPR analysis of ROS production using cyclic hydroxylamines in superinvasive SiHa-F3 compared with less invasive SiHa wild-type human cervix cancer cells. Using the CMH probe, no significant difference was observed in the overall level of ROS between SiHa and SiHa-F3 cells. However, using mitochondria-targeted cyclic hydroxylamine probe mitoTEMPO-H, we detected a significantly higher mitochondrial ROS content in SiHa-F3 compared with the wild-type SiHa cells. To investigate the nature of mitochondrial ROS, we overexpressed superoxide dismutase 2, a SOD isoform exclusively localized in mitochondria, in SiHa-F3 superinvasive cells. A significantly lower signal was detected in SiHa-F3 cells overexpressing SOD2 compared with SiHa-F3. Despite some limitations discussed in the paper, our EPR results suggest that mitochondrial ROS (at least partly superoxide) are produced to a larger extent in superinvasive cancer cells compared with less invasive wild-type cancer cells.

Mitochondria in cancer.

The rediscovery and reinterpretation of the Warburg effect in the year 2000 occulted for almost a decade the key functions exerted by mitochondria in cancer cells. Until recent times, the scientific community indeed focused on constitutive glycolysis as a hallmark of cancer cells, which it is not, largely ignoring the contribution of mitochondria to the malignancy of oxidative and glycolytic cancer cells, being Warburgian or merely adapted to hypoxia. In this review, we highlight that mitochondria are not only powerhouses in some cancer cells, but also dynamic regulators of life, death, proliferation, motion and stemness in other types of cancer cells. Similar to the cells that host them, mitochondria are capable to adapt to tumoral conditions, and probably to evolve to 'oncogenic mitochondria' capable of transferring malignant capacities to recipient cells. In the wider quest of metabolic modulators of cancer, treatments have already been identified targeting mitochondria in cancer cells, but the field is still in infancy.

Metabolic and non-metabolic pathways that control cancer resistance to anthracyclines.

Anthracyclines Doxorubicin, Epirubicin, Daunorubicin and Idarubicin are used to treat a variety of tumor types in the clinics, either alone or, most often, in combination therapies. While their cardiotoxicity is well known, the emergence of chemoresistance is also a major issue accounting for treatment discontinuation. Resistance to anthracyclines is associated to the acquisition of multidrug resistance conferred by overexpression of permeability glycoprotein-1 or other efflux pumps, by altered DNA repair, changes in topoisomerase II activity, cancer stemness and metabolic adaptations. This review further details the metabolic aspects of resistance to anthracyclines, emphasizing the contributions of glycolysis, the pentose phosphate pathway and nucleotide biosynthesis, glutathione, lipid metabolism and autophagy to the chemoresistant phenotype.

A Comprehensive PDX Gastric Cancer Collection Captures Cancer Cell-Intrinsic Transcriptional MSI Traits.

Gastric cancer is the world's third leading cause of cancer mortality. In spite of significant therapeutic improvements, the clinical outcome for patients with advanced gastric cancer is poor; thus, the identification and validation of novel targets is extremely important from a clinical point of view. We generated a wide, multilevel platform of gastric cancer models, comprising 100 patient-derived xenografts (PDX), primary cell lines, and organoids. Samples were classified according to their histology, microsatellite stability, Epstein-Barr virus status, and molecular profile. This PDX platform is the widest in an academic institution, and it includes all the gastric cancer histologic and molecular types identified by The Cancer Genome Atlas. PDX histopathologic features were consistent with those of patients' primary tumors and were maintained throughout passages in mice. Factors modulating grafting rate were histology, TNM stage, copy number gain of tyrosine kinases/KRAS genes, and microsatellite stability status. PDX and PDX-derived cells/organoids demonstrated potential usefulness to study targeted therapy response. Finally, PDX transcriptomic analysis identified a cancer cell-intrinsic microsatellite instability (MSI) signature, which was efficiently exported to gastric cancer, allowing the identification, among microsatellite stable (MSS) patients, of a subset of MSI-like tumors with common molecular aspects and significant better prognosis. In conclusion, we generated a wide gastric cancer PDX platform, whose exploitation will help identify and validate novel "druggable" targets and optimize therapeutic strategies. Moreover, transcriptomic analysis of gastric cancer PDXs allowed the identification of a cancer cell-intrinsic MSI signature, recognizing a subset of MSS patients with MSI transcriptional traits, endowed with better prognosis. SIGNIFICANCE: This study reports a multilevel platform of gastric cancer PDXs and identifies a MSI gastric signature that could contribute to the advancement of precision medicine in gastric cancer.

Biodegradable microparticles designed to efficiently reach and act on cystic fibrosis mucus barrier.

Cystic fibrosis (CF) is a progressive genetic disease caused by mutations in the gene that produces the CF transmembrane conductance regulator (CFTR) protein. The malfunction of the CFTR protein causes a thick buildup of mucus in the lungs that clogs the airways and traps bacteria, thus leading to infections, extensive lung damage and respiratory failure. Micro-delivery systems are currently being investigated as an efficient way to cross the viscous and complex architecture of the CF mucus. In this study, we produced synthetic and natural microparticles (MPs) based on poly(dl­lactide­co­glycolide) (PLGA) or gellan gum through tailored water/oil emulsion procedures. Morphological and physico-chemical characterizations were carried out on both classes of MPs showing particles having diameters within suitable ranges to reach the CF airways. In vitro biocompatibility tests were also performed on both MPs using a human lung cancer cell line (A549) demonstrating that treatment with MPs induces no cytotoxic effects. Both classes of MPs were loaded with a mucolytic agent (N­acetyl cysteine, NAC) and their release kinetics evaluated using high performance liquid chromatography (HPLC). The analysis pointed out that the amount of NAC released from MPs resulted in a dose-dependent increment, with a rapid release kinetic to satisfy the requirement for inducing an early mucus degradation. Finally, mucolytic action of NAC-loaded MPs was evaluated in an artificial sputum model through its rheological analysis obtaining the lowest viscosity profile after the addition of drug-loaded MPs. Taken together, gained results allowed us to select suitable MPs as potential drug targeting platforms having a mucolytic action for CF treatment.

Rituximab Treatment Prevents Lymphoma Onset in Gastric Cancer Patient-Derived Xenografts.

Patient-Derived Xenografts (PDXs), entailing implantation of cancer specimens in immunocompromised mice, are emerging as a valuable translational model that could help validate biologically relevant targets and assist the clinical development of novel therapeutic strategies for gastric cancer. More than 30% of PDXs generated from gastric carcinoma samples developed human B-cell lymphomas instead of gastric cancer. These lymphomas were monoclonal, Epstein Barr Virus (EBV) positive, originated tumorigenic cell cultures and displayed a mutational burden and an expression profile distinct from gastric adenocarcinomas. The ability of grafted samples to develop lymphomas did not correlate with patient outcome, nor with the histotype, the lymphocyte infiltration level, or the EBV status of the original gastric tumor, impeding from foreseeing lymphoma onset. Interestingly, lymphoma development was significantly more frequent when primary rather than metastatic samples were grafted. Notably, the development of such lympho-proliferative disease could be prevented by a short rituximab treatment upon mice implant, without negatively affecting gastric carcinoma engraftment. Due to the high frequency of human lymphoma onset, our data show that a careful histologic analysis is mandatory when generating gastric cancer PDXs. Such care would avoid misleading results that could occur if testing of putative gastric cancer therapies is performed in lymphoma PDXs. We propose rituximab treatment of mice to prevent lymphoma development in PDX models, averting the loss of human-derived samples.

Human mesenchymal stem cells labelled with dye-loaded amorphous silica nanoparticles: long-term biosafety, stemness preservation and traceability in the beating heart.

BACKGROUND: Treatment of myocardial infarction with mesenchymal stem cells (MSCs) has proven beneficial effects in both animal and clinical studies. Engineered silica nanoparticles (SiO2-NPs) have been extensively used as contrast agents in regenerative medicine, due to their resistance to degradation and ease of functionalization. However, there are still controversies on their effective biosafety on cellular systems. In this perspective, the aims of the present study are: 1) to deeply investigate the impact of amorphous 50 nm SiO2-NPs on viability and function of human bone marrow-derived MSCs (hMSCs); 2) to optimize a protocol of harmless hMSCs labelling and test its feasibility in a beating heart model. RESULTS: Optimal cell labelling is obtained after 16 h exposure of hMSCs to fluorescent 50 nm SiO2-NPs (50 µg mL(-1)); interestingly, lysosomal activation consequent to NPs storage is not associated to oxidative stress. During prolonged culture hMSCs do not undergo cyto- or genotoxicity, preserve their proliferative potential and their stemness/differentiation properties. Finally, the bright fluorescence emitted by internalized SiO2-NPs allows both clear visualization of hMSCs in normal and infarcted rat hearts and ultrastructural analysis of cell engraftment inside myocardial tissue. CONCLUSIONS: Overall, 50 nm SiO2-NPs display elevated compatibility with hMSCs in terms of lack of cyto- and genotoxicity and maintenance of important features of these cells. The demonstrated biosafety, combined with proper cell labelling and visualization in histological sections, make these SiO2-NPs optimal candidates for the purpose of stem cell tracking inside heart tissue.

Role of methamphetamine on glioblastoma cytotoxicity induced by doxorubicin and methotrexate.

Glioblastoma multiforme (GBM) is the most common and malignant primary brain tumor with a high mortality rate. Doxorubicin (DOX) and methotrexate (MTX) showed to be effective against a wide range of tumors, but its use in GBM treatment is limited in part due to the inability to cross the blood-brain barrier (BBB). Based on recent studies demonstrating that methamphetamine (METH) increases BBB permeability, we hypothesized that it could be used as a pharmacological tool to allow the entry of potential therapeutic drugs into the brain. Nevertheless, before attempting this approach it is crucial to understand the cytotoxicity of such drug combinations. Herein, we evaluated the effects of METH on cell viability, migration, chemotaxis, and cell cycle, as well as its modulator effects on DOX or MTX-induced cytotoxicity in a human U118 GBM cell line. Our results demonstrated that both chemotherapeutic drugs DOX and MTX induced a pronounced decrease in cell viability, migration, and chemotaxis, and led to a cell cycle arrest at G2 and S phases, respectively. Additionally, METH (1 µM) neither interfered with U-118 cell viability, migration, or cell cycle nor modified DOX- or MTX-induced cytotoxicity. Noteworthy, METH by itself impaired cell chemotaxis with a similar effect to that induced by DOX or MTX alone. Overall, we can conclude that both DOX and MTX are highly cytotoxic against GBM cells and that METH, at a concentration previously shown to increase endothelial cell permeability without leading to cell death, does not interfere with the cytotoxicity of both chemotherapeutic drugs.