Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines.

BACKGROUND: Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy. RESULTS: In this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression. CONCLUSIONS: Our results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients' outcome.

Development of High-Loading Trastuzumab PLGA Nanoparticles: A Powerful Tool Against HER2 Positive Breast Cancer Cells.

Background: Trastuzumab, a therapeutic monoclonal antibody directed against HER2, is routinely used to treat HER2-positive breast cancer with a good response rate. However, concerns have arisen in the clinical practice due to adverse side effects. One way to overcome these limitations is to encapsulate trastuzumab in nanoparticles to improve cytotoxic activity, increase intracellular drug concentrations, escape the immune system and avoid systemic degradation of the drug in vivo. Methods: A double emulsion method was used to encapsulate trastuzumab into poly(lactic-co-glycolic) nanoparticles, effective for their biocompatibility and biodegradability. These nanocarriers, hereafter referred to as TZPs, were characterised in terms of size, homogeneity, zeta potential and tested for their stability and drug release kinetics. Finally, the TZPs cytotoxicity was assessed in vitro on the HER2 positive SKBR3 breast cancer cell line and compared to free trastuzumab. Results: The TZPs were stable, homogeneous in size, with a reduced zeta potential. They showed higher encapsulation efficiency and drug loading, a prolonged trastuzumab release kinetics that retained its physicochemical properties and functionality. TZPs showed a stronger cytotoxicity and increased apoptosis than similar doses of free trastuzumab in the cell line analysed. Confocal microscopy and flow cytometry assessed TZPs and trastuzumab cellular uptake while Western blot evaluated downstream signalling, overall HER2 content and shedding. Conclusion: TZPs exert more robust effects than free trastuzumab via a dual mode of action: TZPs are taken up by cells through an endocytosis mechanism and release the drug intracellularly for longer time. Additionally, the TZPs that remain in the extracellular space release trastuzumab which binds to the cognate receptor and impairs downstream signalling. This is the sole modality used by free trastuzumab. Remarkably, half dose of TZPs is as efficacious as the highest dose of free drug supporting their possible use for drug delivery in vivo.

Quercetin's Dual Mode of Action to Counteract the Sp1-miR-27a Axis in Colorectal Cancer Cells.

Quercetin (Qc) inhibits cell proliferation and induces apoptosis in a variety of cancer cells. The molecular mechanism of action has not been fully elucidated; however, interplay with some miRNAs has been reported, specifically with miR-27a, an onco-miRNA overexpressed in several malignancies. Here, we show that Qc reduces cell viability and induces apoptosis in HCT116 and HT-29 colon cancer cells, by upregulating negative modulators of proliferation pathways such as Sprouty2, PTEN and SFRP1. These are targets of miR-27a whose high expression is reduced by Qc. Moreover, miR-23a, and miR-24-2, the two other components of the unique gene cluster, and the pri-miRNA transcript are reduced, evoking a transcriptional regulation of the entire cluster by Sp1. Mechanistically, we show that Qc is rapidly internalized and localizes in the nucleus, where it likely interacts with Sp1, inducing its proteasomal degradation. Sp1 is further repressed by ZBTB10, an Sp1 competitor for DNA binding that is an miR-27a target and whose levels increase following Qc. SP1 mRNA is also reduced, supporting the regulation of its own gene transcription. Finally, Sp1 knockdown elicits the impaired transcription of the entire cluster and the upregulation of the miR-27a targets, phenocopying the effects of Qc. Through this dual mode of action, Qc counteracts the protumoral Sp1-miR-27a axis, opening the way for novel therapies based on its association as neoadjuvant with known anticancer treatments.

Transcriptomic Analysis of Colorectal Cancer Cells Treated with Oil Production Waste Products (OPWPs) Reveals Enrichment of Pathways of Mitochondrial Functionality.

Oil production waste products (OPWPs) derive from olive mill and represent a crucial environmental problem due to their high polyphenolic content able to pollute the ground. One option to reduce the OPWPs' environmental impact is to exploit polyphenols' biological properties. We sought to analyze the transcriptomic variations of colorectal cancer cells exposed to the OPWPs extracts and hydroxytyrosol, the major component, to recognize unknown and ill-defined characteristics. Among the top affected pathways identified by GSEA, we focused on oxidative phosphorylation in an in vitro system. Colorectal cancer HCT116 and LoVo cells treated with hydroxytyrosol or OPWPs extracts showed enhancement of the respiratory chain complexes' protein levels, ATP production and membrane potential, suggesting stimulation of mitochondrial functions. The major proteins involved in mitochondrial biogenesis and fusion events of mitochondrial dynamics were positively affected, as by Western blot, fostering increase of the mitochondrial mass organized in a network of elongated organelles. Mechanistically, we proved that PPARγ mediates the effects as they are mimicked by a specific ligand and impaired by a specific inhibitor. OPWP extracts and hydroxytyrosol, thus, promote mitochondrial functionality via a feed-forward regulatory loop involving the PPARγ/PGC-1α axis. These results support their use in functional foods and as adjuvants in cancer therapy.

Targeting CXCR4 and CD47 Receptors: An Overview of New and Old Molecules for a Biological Personalized Anticancer Therapy.

Biological therapy, with its multifaceted applications, has revolutionized the treatment of tumors, mainly due to its ability to exclusively target cancer cells and reduce the adverse effects on normal tissues. This review focuses on the therapies targeting the CXCR4 and CD47 receptors. We surveyed the results of early clinical trials testing compounds classified as nonpeptides, small peptides, CXCR4 antagonists or specific antibodies whose activity reduces or completely blocks the intracellular signaling pathways and cell proliferation. We then examined antibodies and fusion proteins against CD47, the receptor that acts as a "do not eat me" signal to phagocytes escaping immune surveillance. Despite these molecules being tested in early clinical trials, some drawbacks are emerging that impair their use in practice. Finally, we examined the ImmunoGenic Surrender mechanism that involves crosstalk and co-internalization of CXCR4 and CD47 upon engagement of CXCR4 by ligands or other molecules. The favorable effect of such compounds is dual as CD47 surface reduction impact on the immune response adds to the block of CXCR4 proliferative potential. These results suggest that a combination of different therapeutic approaches has more beneficial effects on patients' survival and may pave the way for new accomplishments in personalized anticancer therapy.

SHMT2-mediated mitochondrial serine metabolism drives 5-FU resistance by fueling nucleotide biosynthesis.

5-Fluorouracil (5-FU) is a key component of chemotherapy for colorectal cancer (CRC). 5-FU efficacy is established by intracellular levels of folate cofactors and DNA damage repair strategies. However, drug resistance still represents a major challenge. Here, we report that alterations in serine metabolism affect 5-FU sensitivity in in vitro and in vivo CRC models. In particular, 5-FU-resistant CRC cells display a strong serine dependency achieved either by upregulating endogenous serine synthesis or increasing exogenous serine uptake. Importantly, regardless of the serine feeder strategy, serine hydroxymethyltransferase-2 (SHMT2)-driven compartmentalization of one-carbon metabolism inside the mitochondria represents a specific adaptation of resistant cells to support purine biosynthesis and potentiate DNA damage response. Interfering with serine availability or affecting its mitochondrial metabolism revert 5-FU resistance. These data disclose a relevant mechanism of mitochondrial serine use supporting 5-FU resistance in CRC and provide perspectives for therapeutic approaches.

CXCR4/CXCL12 Activities in the Tumor Microenvironment and Implications for Tumor Immunotherapy.

CXCR4 is a G-Protein coupled receptor that is expressed nearly ubiquitously and is known to control cell migration via its interaction with CXCL12, the most ancient chemokine. The functions of CXCR4/CXCL12 extend beyond cell migration and involve the recognition and disposal of unhealthy or tumor cells. The CXCR4/CXCL12 axis plays a relevant role in shaping the tumor microenvironment (TME), mainly towards dampening immune responses. Notably, CXCR4/CXCL12 cross-signal via the T and B cell receptors (TCR and BCR) and co-internalize with CD47, promoting tumor cell phagocytosis by macrophages in an anti-tumor immune process called ImmunoGenic Surrender (IGS). These specific activities in shaping the immune response might be exploited to improve current immunotherapies.

Polyphenols Extracts from Oil Production Waste Products (OPWPs) Reduce Cell Viability and Exert Anti-Inflammatory Activity via PPARγ Induction in Colorectal Cancer Cells.

Olive oil production is associated with the generation of oil production waste products (OPWPs) rich in water-soluble polyphenols that represent serious environmental problems. Yet OPWPs can offer new opportunities by exploiting their bioactive properties. In this study, we chemically characterized OPWPs polyphenolic extracts and investigated their biological activities in normal and colorectal cancer cells. Hydroxytyrosol (HTyr), the major constituent of these extracts, was used as the control. We show that both HTyr and the extracts affect cell viability by inducing apoptosis and cell cycle arrest. They downregulate inflammation by impairing NF-κB phosphorylation and expression of responsive cytokine genes, as TNF-α and IL-8, at both mRNA and protein levels, and prevent any further increase elicited by external challenges. Mechanistically, HTyr and the extracts activate PPARγ while hampering pro-inflammatory genes expression, acting as a specific agonist, likely through a trans-repression process. Altogether, OPWPs polyphenolic extracts show stronger effects than HTyr, conceivably due to additive or synergistic effects of all polyphenols contained. They display anti-inflammatory properties and these results may pave the way for improving OPWPs extraction and enrichment methods to reduce the environmental impact and support their use to ameliorate the inflammation associated with diseases and tumors.

Loss of circadian gene Timeless induces EMT and tumor progression in colorectal cancer via Zeb1-dependent mechanism.

The circadian gene Timeless (TIM) provides a molecular bridge between circadian and cell cycle/DNA replication regulatory systems and has been recently involved in human cancer development and progression. However, its functional role in colorectal cancer (CRC), the third leading cause of cancer-related deaths worldwide, has not been fully clarified yet. Here, the analysis of two independent CRC patient cohorts (total 1159 samples) reveals that loss of TIM expression is an unfavorable prognostic factor significantly correlated with advanced tumor stage, metastatic spreading, and microsatellite stability status. Genome-wide expression profiling, in vitro and in vivo experiments, revealed that TIM knockdown induces the activation of the epithelial-to-mesenchymal transition (EMT) program. Accordingly, the analysis of a large set of human samples showed that TIM expression inversely correlated with a previously established gene signature of canonical EMT markers (EMT score), and its ectopic silencing promotes migration, invasion, and acquisition of stem-like phenotype in CRC cells. Mechanistically, we found that loss of TIM expression unleashes ZEB1 expression that in turn drives the EMT program and enhances the aggressive behavior of CRC cells. Besides, the deranged TIM-ZEB1 axis sets off the accumulation of DNA damage and delays DNA damage recovery. Furthermore, we show that the aggressive and genetically unstable 'CMS4 colorectal cancer molecular subtype' is characterized by a lower expression of TIM and that patients with the combination of low-TIM/high-ZEB1 expression have a poorer outcome. In conclusion, our results as a whole suggest the engagement of an unedited TIM-ZEB1 axis in key pathological processes driving malignant phenotype acquisition in colorectal carcinogenesis. Thus, TIM-ZEB1 expression profiling could provide a robust prognostic biomarker in CRC patients, supporting targeted therapeutic strategies with better treatment selection and patients' outcomes.

The miR-27a/FOXJ3 Axis Dysregulates Mitochondrial Homeostasis in Colorectal Cancer Cells.

miR-27a plays a driver role in rewiring tumor cell metabolism. We searched for new miR-27a targets that could affect mitochondria and identified FOXJ3, an apical factor of mitochondrial biogenesis. We analyzed FOXJ3 levels in an in vitro cell model system that was genetically modified for miR-27a expression and validated it as an miR-27a target. We showed that the miR-27a/FOXJ3 axis down-modulates mitochondrial biogenesis and other key members of the pathway, implying multiple levels of control. As assessed by specific markers, the miR-27a/FOXJ3 axis also dysregulates mitochondrial dynamics, resulting in fewer, short, and punctate organelles. Consistently, in high miR-27a-/low FOXJ3-expressing cells, mitochondria are functionally characterized by lower superoxide production, respiration capacity, and membrane potential, as evaluated by OCR assays and confocal microscopy. The analysis of a mouse xenograft model confirmed FOXJ3 as a target and suggested that the miR-27a/FOXJ3 axis affects mitochondrial abundance in vivo. A survey of the TCGA-COADREAD dataset supported the inverse relationship of FOXJ3 with miR-27a and reinforced cellular component organization or biogenesis as the most affected pathway. The miR-27a/FOXJ3 axis acts as a central hub in regulating mitochondrial homeostasis. Its discovery paves the way for new therapeutic strategies aimed at restraining tumor growth by targeting mitochondrial activities.

DNMT3A epigenetically regulates key microRNAs involved in epithelial-to-mesenchymal transition in prostate cancer.

Epithelial-to-mesenchymal transition (EMT) is involved in prostate cancer (PCa) metastatic progression, and its plasticity suggests epigenetic implications. Deregulation of DNA methyltransferases (DNMTs) and several microRNAs (miRNAs) plays a relevant role in EMT, but their interplay has not been clarified yet. In this study, we provide evidence that DNMT3A interaction with several miRNAs has a central role in an ex vivo EMT PCa model obtained via exposure of PC3 cells to conditioned media from cancer-associated fibroblasts. The analysis of the alterations of the miRNA profile shows that miR-200 family (miR-200a/200b/429, miR-200c/141), miR-205 and miR-203, known to modulate key EMT factors, are down-regulated and hyper-methylated at their promoters. DNMT3A (mainly isoform a) is recruited onto these miRNA promoters, coupled with the increase of H3K27me3/H3K9me3 and/or the decrease of H3K4me3/H3K36me3. Most interestingly, our results reveal the differential expression of two DNMT3A isoforms (a and b) during ex vivo EMT and a regulatory feedback loop between miR-429 and DNMT3A that can promote and sustain the transition towards a more mesenchymal phenotype. We demonstrate the ability of miR-429 to target DNMT3A 3'UTR and modulate the expression of EMT factors, in particular ZEB1. Survey of the PRAD-TCGA dataset shows that patients expressing an EMT-like signature are indeed characterized by down-regulation of the same miRNAs with a diffused hyper-methylation at miR-200c/141 and miR-200a/200b/429 promoters. Finally, we show that miR-1260a also targets DNMT3A, although it does not seem to be involved in EMT in PCa.

Sample-Pooling Strategy for SARS-CoV-2 Detection among Students and Staff of the University of Sannio.

Since the beginning of the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) pandemic, it has been clear that testing large groups of the population was the key to stem infection and prevent the effects of the coronavirus disease of 2019, mostly among sensitive patients. On the other hand, time and cost-sustainability of virus detection by molecular analysis such as reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) may be a major issue if testing is extended to large communities, mainly asymptomatic large communities. In this context, sample-pooling and test grouping could offer an effective solution. Here we report the screening on 1195 oral-nasopharyngeal swabs collected from students and staff of the Università degli Studi del Sannio (University of Sannio, Benevento, Campania, Italy) and analyzed by an in-house developed multiplex RT-qPCR for SARS-CoV-2 detection through a simple monodimensional sample pooling strategy. Overall, 400 distinct pools were generated and, within 24 h after swab collection, five positive samples were identified. Out of them, four were confirmed by using a commercially available kit suitable for in vitro diagnostic use (IVD). High accuracy, sensitivity and specificity were also determined by comparing our results with a reference IVD assay for all deconvoluted samples. Overall, we conducted 463 analyses instead of 1195, reducing testing resources by more than 60% without lengthening diagnosis time and without significant losses in sensitivity, suggesting that our strategy was successful in recognizing positive cases in a community of asymptomatic individuals with minor requirements of reagents and time when compared to normal testing procedures.

Geometric Features of the Pial Arteriolar Networks in Spontaneous Hypertensive Rats: A Crucial Aspect Underlying the Blood Flow Regulation.

BACKGROUND: Several studies indicate that hypertension causes major changes in the structure of the vessel wall by affecting the regulation of blood supply to the tissues. Recently, it has been observed that capillary blood flow is also considerably influenced by the structural arrangement of the microvascular networks that undergo rarefaction (reduction of the perfused vessel number). Therefore, this study aimed to assess the geometric arrangements of the pial arteriolar networks and the arteriolar rhythmic diameter changes in spontaneously hypertensive rats (SHRs). METHODS: Fluorescence microscopy was utilized to observe in vivo the pial microcirculation through a closed cranial window. Pial arterioles were classified according to Strahler's method. The arteriolar rhythmic diameter changes were evaluated by a generalization short-time Fourier transform. RESULT: Young SHRs showed four orders of vessels while the adult ones only three orders. The diameter, length, and branching number obeyed Horton's law; therefore, the vessels were distributed in a fractal manner. Larger arterioles showed more asymmetrical branches than did the smaller ones in young SHRs, while in adult SHRs smaller vessels presented asymmetrical branchings. In adult SHRs, there was a significant reduction in the cross-sectional area compared with the young SHRs: this implies an increase in peripheral resistance. Young and adult age-matched normotensive rats did not show significant alterations in the geometric arteriolar arrangement with advancing age, both had four orders of arteriolar vessels, and the peripheral resistance did not change significantly. Conversely, the frequency components evaluated in arteriolar rhythmic diameter changes of young and adult SHRs showed significant differences because of a reduction in the frequency components related to endothelial activity detected in adult SHRs. CONCLUSION: In conclusion, hypertension progressively causes changes in the microarchitecture of the arteriolar networks with a smaller number of vessels and consequent reduced conductivity, characteristic of rarefaction. This was accompanied by a reduction in the formation and release of independent and dependent - endothelial nitric oxide components regulating arterial vasomotion.

CXCR4 engagement triggers CD47 internalization and antitumor immunization in a mouse model of mesothelioma.

Boosting antitumor immunity has emerged as a powerful strategy in cancer treatment. While releasing T-cell brakes has received most attention, tumor recognition by T cells is a pre-requisite. Radiotherapy and certain cytotoxic drugs induce the release of damage-associated molecular patterns, which promote tumor antigen cross-presentation and T-cell priming. Antibodies against the "do not eat me" signal CD47 cause macrophage phagocytosis of live tumor cells and drive the emergence of antitumor T cells. Here we show that CXCR4 activation, so far associated only with tumor progression and metastasis, also flags tumor cells to immune recognition. Both CXCL12, the natural CXCR4 ligand, and BoxA, a fragment of HMGB1, promote the release of DAMPs and the internalization of CD47, leading to protective antitumor immunity. We designate as Immunogenic Surrender the process by which CXCR4 turns in tumor cells to macrophages, thereby subjecting a rapidly growing tissue to immunological scrutiny. Importantly, while CXCL12 promotes tumor cell proliferation, BoxA reduces it, and might be exploited for the treatment of malignant mesothelioma and a variety of other tumors.

Correction: miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

miR-27a is a master regulator of metabolic reprogramming and chemoresistance in colorectal cancer.

BACKGROUND: Metabolic reprogramming towards aerobic glycolysis in cancer supports unrestricted cell proliferation, survival and chemoresistance. The molecular bases of these processes are still undefined. Recent reports suggest crucial roles for microRNAs. Here, we provide new evidence of the implication of miR-27a in modulating colorectal cancer (CRC) metabolism and chemoresistance. METHODS: A survey of miR-27a expression profile in TCGA-COAD dataset revealed that miR-27a-overexpressing CRCs are enriched in gene signatures of mitochondrial dysfunction, deregulated oxidative phosphorylation, mTOR activation and reduced chemosensitivity. The same pathways were analysed in cell lines in which we modified miR-27a levels. The response to chemotherapy was investigated in an independent cohort and cell lines. RESULTS: miR-27a upregulation in vitro associated with impaired oxidative phosphorylation, overall mitochondrial activities and slight influence on glycolysis. miR-27a hampered AMPK, enhanced mTOR signalling and acted in concert with oncogenes and tumour cell metabolic regulators to force an aerobic glycolytic metabolism supporting biomass production, unrestricted growth and chemoresistance. This latter association was confirmed in our cohort of patients and cell lines. CONCLUSIONS: We disclose an unprecedented role for miR-27a as a master regulator of cancer metabolism reprogramming that impinges on CRC response to chemotherapy, underscoring its theragnostic properties.

The Pomace Extract Taurisolo Protects Rat Brain From Ischemia-Reperfusion Injury.

Taurisolo® is a pomace extract from Aglianico Grapes, a wine cultivar native to Campania (Southern Italy). It exhibits a very high polyphenolic content and, consumed as a nutraceutical, is effective in reducing the level of Trimethylamine N-oxide (TMAO), a cardiovascular disease risk factor marker. We here show the effects of Taurisolo® on rat brain microvascular alterations induced by a diminution in cerebral blood flow (CBFD) for 30 min, due to bilateral common carotid artery occlusion, and subsequent blood flow restoration (CBFR) for 60 min. The rat pial microcirculation was investigated by intravital fluorescence microscopy through a parietal closed window implanted into the skull bone. The rat pial arterioles were classified according to Strahler's ordering scheme, from smaller penetrating arterioles up to the larger ones. Western blotting analysis and mass spectrometry (MS)-based metabolomics were used to investigate the expression of endothelial nitric oxide synthase (eNOS) or the presence of peroxidized cardiolipin and several inflammatory mediators, respectively. Radical Oxygen Species (ROS) formation and neuronal loss were assessed. In rats CBFD and CBFR caused a decrease in arteriolar diameter, increase in fluorescent leakage and in adhesion of leukocytes to venular walls, reduction in the length of perfused capillaries and increment of ROS formation with large infarct size. Taurisolo®, intravenously or orally administered, induced pial arteriolar dilation (up to >30% of baseline), prevented fluorescent leakage, adhesion of leukocytes, ROS formation, while facilitated capillary perfusion and significantly reduced infarct size. These effects were accompanied by an increase in eNOS expression. Mass-spectrometry metabolomics analysis detected a marked decrease in the amount of peroxidized cardiolipin and pronounced reduction in pro-inflammatory prostaglandins and thromboxane Txb2. Altogether, these results extend the nutraceutical potential of Taurisolo® and suggest their eligibility for preventing brain damage due to ischemia-reperfusion injury.

Molecular and environmental analysis of Campania (Italy) sweet cherry (Prunus avium L.) cultivars for biocultural refugia identification and conservation.

Conservation of agrobiodiversity is a major concern worldwide. Several strategies have been designed and programmed to reduce biodiversity erosion due to anthropic and non-anthropic causes. To this end, we set up a multidisciplinary approach based on the genetic analysis of selected cultivars and recognition of the environmental parameters. We genotyped the sweet cherry cultivars of Campania region in southern Italy by using simple sequence repeats and further investigated them by cluster analysis, disclosing a homogeneous genetic constitution, different from that of commercial accessions. By structure analysis we identified three distinct genetic clusters, each characterized by common and distinct alleles. Survey of the cultivars' geographical distribution by quartic kernel function identified four preferred districts further characterized for soil origin, pedologic, agronomic features and urbanization impact. We correlated these environmental parameters, typical of the identified areas, with the three genetic pools and found a statistically significant association for each cluster. When we overlaid the cultivation traditions and cultural heritage, we found they have a dominant role; on these premises, we generated new territorial maps. In conclusion, we propose a novel methodological approach based on molecular, geo-pedological and cultural parameters with the aim to recognize biocultural refugia and preserve endangered or valuable cultivars.

Chiral phenoxyacetic acid analogues inhibit colon cancer cell proliferation acting as PPARγ partial agonists.

Peroxisome Proliferator-Activated Receptor γ (PPARγ) is an important sensor at the crossroad of diabetes, obesity, immunity and cancer as it regulates adipogenesis, metabolism, inflammation and proliferation. PPARγ exerts its pleiotropic functions upon binding of natural or synthetic ligands. The molecular mechanisms through which PPARγ controls cancer initiation/progression depend on the different mode of binding of distinctive ligands. Here, we analyzed a series of chiral phenoxyacetic acid analogues for their ability to inhibit colorectal cancer (CRC) cells growth by binding PPARγ as partial agonists as assessed in transactivation assays of a PPARG-reporter gene. We further investigated compounds (R,S)-3, (S)-3 and (R,S)-7 because they combine the best antiproliferative activity and a limited transactivation potential and found that they induce cell cycle arrest mainly via upregulation of p21waf1/cip1. Interestingly, they also counteract the ß-catenin/TCF pathway by repressing c-Myc and cyclin D1, supporting their antiproliferative effect. Docking experiments provided insight into the binding mode of the most active compound (S)-3, suggesting that its partial agonism could be related to a better stabilization of H3 rather than H11 and H12. In conclusion, we identified a series of PPARγ partial agonists affecting distinct pathways all leading to strong antiproliferative effects. These findings may pave the way for novel therapeutic strategies in CRC.

Centrosome Linker-induced Tetraploid Segregation Errors Link Rhabdoid Phenotypes and Lethal Colorectal Cancers.

Centrosome anomalies contribute to tumorigenesis, but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAFV600E-mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosome linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAFV600E-mutant and microsatellite stable (MSS) rhabdoid colorectal cancers, but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAFV600E-mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors, resulting in a highly aggressive rhabdoid-like phenotype in vitro Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability.Implications: Mis-segregation of chromosomes is a prominent feature of chromosome instability and intratumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. This study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Mol Cancer Res; 16(9); 1385-95. ©2018 AACR.