HGF/c-Met Signalling in the Tumor Microenvironment.

Recently, it has become clearer that tumor plasticity increases the chance that cancer cells could acquire new mechanisms to escape immune surveillance, become resistant to conventional drugs, and spread to distant sites.Effectively, tumor plasticity drives adaptive response of cancer cells to hypoxia and nutrient deprivation leading to stimulation of neoangionesis or tumor escape. Therefore, tumor plasticity is believed to be a great contributor in recurrence and metastatic dissemination of cancer cells. Importantly, it could be an Achilles' heel of cancer if we could identify molecular mechanisms dictating this phenotype.The reactivation of stem-like signalling pathways is considered a great determinant of tumor plasticity; in addition, a key role has been also attributed to tumor microenvironment (TME). Indeed, it has been proved that cancer cells interact with different cells in the surrounding extracellular matrix (ECM). Interestingly, well-established communication represents a potential allied in maintenance of a plastic phenotype in cancer cells supporting tumor growth and spread. An important signalling pathway mediating cancer cell-TME crosstalk is represented by the HGF/c-Met signalling.Here, we review the role of the HGF/c-Met signalling in tumor-stroma crosstalk focusing on novel findings underlying its role in tumor plasticity, immune escape, and development of adaptive mechanisms.

Alternative splicing in Alzheimer's disease.

Alzheimer's disease (AD) is the most frequent neurodegenerative disorder in the elderly, occurring in approximately 20% of people older than 80. The molecular causes of AD are still poorly understood. However, recent studies have shown that Alternative Splicing (AS) is involved in the gene expression reprogramming associated with the functional changes observed in AD patients. In particular, mutations in cis-acting regulatory sequences as well as alterations in the activity and sub-cellular localization of trans-acting splicing factors and components of the spliceosome machinery are associated with splicing abnormalities in AD tissues, which may influence the onset and progression of the disease. In this review, we discuss the current molecular understanding of how alterations in the AS process contribute to AD pathogenesis. Finally, recent therapeutic approaches targeting aberrant AS regulation in AD are also reviewed.

NOTCH3 expression is linked to breast cancer seeding and distant metastasis.

BACKGROUND: Development of distant metastases involves a complex multistep biological process termed the invasion-metastasis cascade, which includes dissemination of cancer cells from the primary tumor to secondary organs. NOTCH developmental signaling plays a critical role in promoting epithelial-to-mesenchymal transition, tumor stemness, and metastasis. Although all four NOTCH receptors show oncogenic properties, the unique role of each of these receptors in the sequential stepwise events that typify the invasion-metastasis cascade remains elusive. METHODS: We have established metastatic xenografts expressing high endogenous levels of NOTCH3 using estrogen receptor alpha-positive (ERα+) MCF-7 breast cancer cells with constitutive active Raf-1/mitogen-associated protein kinase (MAPK) signaling (vMCF-7Raf-1) and MDA-MB-231 triple-negative breast cancer (TNBC) cells. The critical role of NOTCH3 in inducing an invasive phenotype and poor outcome was corroborated in unique TNBC cells resulting from a patient-derived brain metastasis (TNBC-M25) and in publicly available claudin-low breast tumor specimens collected from participants in the Molecular Taxonomy of Breast Cancer International Consortium database. RESULTS: In this study, we identified an association between NOTCH3 expression and development of metastases in ERα+ and TNBC models. ERα+ breast tumor xenografts with a constitutive active Raf-1/MAPK signaling developed spontaneous lung metastases through the clonal expansion of cancer cells expressing a NOTCH3 reprogramming network. Abrogation of NOTCH3 expression significantly reduced the self-renewal and invasive capacity of ex vivo breast cancer cells, restoring a luminal CD44low/CD24high/ERαhigh phenotype. Forced expression of the mitotic Aurora kinase A (AURKA), which promotes breast cancer metastases, failed to restore the invasive capacity of NOTCH3-null cells, demonstrating that NOTCH3 expression is required for an invasive phenotype. Likewise, pharmacologic inhibition of NOTCH signaling also impaired TNBC cell seeding and metastatic growth. Significantly, the role of aberrant NOTCH3 expression in promoting tumor self-renewal, invasiveness, and poor outcome was corroborated in unique TNBC cells from a patient-derived brain metastasis and in publicly available claudin-low breast tumor specimens. CONCLUSIONS: These findings demonstrate the key role of NOTCH3 oncogenic signaling in the genesis of breast cancer metastasis and provide a compelling preclinical rationale for the design of novel therapeutic strategies that will selectively target NOTCH3 to halt metastatic seeding and to improve the clinical outcomes of patients with breast cancer.

Translating Proteomic Into Functional Data: An High Mobility Group A1 (HMGA1) Proteomic Signature Has Prognostic Value in Breast Cancer.

Cancer is a very heterogeneous disease, and biological variability adds a further level of complexity, thus limiting the ability to identify new genes involved in cancer development. Oncogenes whose expression levels control cell aggressiveness are very useful for developing cellular models that permit differential expression screenings in isogenic contexts. HMGA1 protein has this unique property because it is a master regulator in breast cancer cells that control the transition from a nontumorigenic epithelial-like phenotype toward a highly aggressive mesenchymal-like one. The proteins extracted from HMGA1-silenced and control MDA-MB-231 cells were analyzed using label-free shotgun mass spectrometry. The differentially expressed proteins were cross-referenced with DNA microarray data obtained using the same cellular model and the overlapping genes were filtered for factors linked to poor prognosis in breast cancer gene expression meta-data sets, resulting in an HMGA1 protein signature composed of 21 members (HRS, HMGA1 reduced signature). This signature had a prognostic value (overall survival, relapse-free survival, and distant metastasis-free survival) in breast cancer. qRT-PCR, Western blot, and immunohistochemistry analyses validated the link of three members of this signature (KIFC1, LRRC59, and TRIP13) with HMGA1 expression levels both in vitro and in vivo and wound healing assays demonstrated that these three proteins are involved in modulating tumor cell motility. Combining proteomic and genomic data with the aid of bioinformatic tools, our results highlight the potential involvement in neoplastic transformation of a restricted list of factors with an as-yet-unexplored role in cancer. These factors are druggable targets that could be exploited for the development of new, targeted therapeutic approaches in triple-negative breast cancer.

Alternative Splicing in Adhesion- and Motility-Related Genes in Breast Cancer.

Breast cancer is the most common tumor and the second leading cause of cancer death among woman, mainly caused by the metastatic spread. Tumor invasiveness is due to an altered expression of adhesion molecules. Among them, semaphorins are of peculiar interest. Cancer cells can manipulate alternative splicing patterns to modulate the expression of adhesion- and motility-related molecules, also at the isoform level. In this study, combining RNA-Sequencing on MCF-7 to targeted experimental validations-in human breast cell lines and breast tumor biopsies-we identified 12 new alternative splicing transcripts in genes encoding adhesion- and motility-related molecules, including semaphorins, their receptors and co-receptors. Among them, a new SEMA3F transcript is expressed in all breast cell lines and breast cancer biopsies, and is translated into a new semaphorin 3F isoform. In silico analysis predicted that most of the new putative proteins lack functional domains, potentially missing some functions and acquiring new ones. Our findings better describe the extent of alternative splicing in breast cancer and highlight the need to further investigate adhesion- and motility-related molecules to gain insights into breast cancer progression.

Clinical and cytologic characteristics of allergic rhinitis in elderly patients.

BACKGROUND: The constant increase in the elderly population worldwide has led to a greater interest in immunologic responses during aging. Thus, special attention to allergic diseases in elderly people has begun to emerge, but little is known about the effect and features of allergic rhinitis in elderly people. OBJECTIVE: To evaluate the clinical and cytologic characteristics of respiratory allergy and its impact on the quality of life in elderly people. METHODS: Elderly patients with rhinitis referred to our allergy unit during a 3-month period underwent clinical evaluation and responded to the Rhinasthma Questionnaire. All patients also underwent skin prick testing, measurement of total IgE level, and nasal cytologic analysis. The data were compared with a control group of young adults. RESULTS: Fifty-four patients older than 65 years (mean age, 69.3 years) and 89 young adults (mean age, 26.3 years) with allergic rhinitis were studied. The elderly patients had a less positive family history of atopy (P=.02) and had rhinitis plus conjunctivitis more frequently (P=.002) than young adults, whereas the difference between groups in total IgE level was not statistically significant. On nasal cytologic analysis, the differential count of inflammatory cells did not differ between groups, but in the elderly patients the epithelial-goblet cell ratio was decreased. The quality of life in elderly people was more impaired than in young adults (P=.01). CONCLUSION: In elderly people with allergic rhinitis, the clinical characteristics are different and quality of life is more heavily impaired compared with young adults.

Carbonic anhydrase IX and pathological features as predictors of outcome in patients with metastatic clear-cell renal cell carcinoma receiving vascular endothelial growth factor-targeted therapy.

OBJECTIVE: To investigate the utility of tumour carbonic anhydrase IX (CAIX) expression and histological features for predicting the outcome in patients with metastatic clear-cell renal cell carcinoma (mRCC) treated with vascular endothelial growth factor (VEGF)-targeted therapy. PATIENTS AND METHODS: We identified 118 patients with mRCC initiating first-line VEGF-targeted therapy, including 94 with clinical and histological data, and available tissue. The primary endpoint was to detect an interaction between sorafenib vs sunitinib treatment and CAIX status on tumour shrinkage. Other treatment outcomes were also assessed. RESULTS: There was heterogeneity in tumour responsiveness to sunitinib or sorafenib according to CAIX status; the mean shrinkage was -17% vs -25% for sunitinib-treated patients with high vs low tumour CAIX expression, compared to -13% vs +9% for sorafenib-treated patients (P interaction, 0.05). A higher tumour clear-cell component was independently associated with greater tumour shrinkage (P= 0.02), response (P= 0.02) and treatment duration (P= 0.02). CONCLUSIONS: Although CAIX expression had no prognostic value in patients with clear-cell mRCC treated with VEGF-targeted therapy, it might be a predictive biomarker for response to sorafenib treatment. Patients with a higher clear-cell component in their tumours are likely to have a superior clinical benefit from VEGF-targeted therapy.

LDH-A inhibition, a therapeutic strategy for treatment of hereditary leiomyomatosis and renal cell cancer.

The genetic basis for the hereditary leiomyomatosis and renal cell cancer syndrome is germ-line inactivating mutation in the gene for the Krebs/tricarboxylic acid cycle enzyme, fumarate hydratase (FH), the enzyme that converts fumarate to malate. These individuals are predisposed to development of leiomyomas of the skin and uterus as well as highly aggressive kidney cancers. Inhibition of FH should result in significant decrease in oxidative phosphorylation necessitating that glycolysis followed by fermentation of pyruvate to lactate will be required to provide adequate ATP as well as to regenerate NAD+. Moreover, FH deficiency is known to up-regulate expression of hypoxia-inducible factor (HIF)-1alpha by enhancing the stability of HIF transcript. This leads to activation of various HIF-regulated genes including vascular endothelial growth factor and glucose transporter GLUT1 and increased expression of several glycolytic enzymes. Because lactate dehydrogenase-A (LDH-A), also a HIF-1alpha target, promotes fermentative glycolysis (conversion of pyruvate to lactate), a step essential for regenerating NAD+, we asked whether FH-deficient cells would be exquisitely sensitive to LDH-A blockade. Here, we report that hereditary leiomyomatosis and renal cell cancer tumors indeed overexpress LDH-A, that LDH-A inhibition results in increased apoptosis in a cell with FH deficiency and that this effect is reactive oxygen species mediated, and that LDH-A knockdown in the background of FH knockdown results in significant reduction in tumor growth in a xenograft mouse model.

RNAi mediated acute depletion of retinoblastoma protein (pRb) promotes aneuploidy in human primary cells via micronuclei formation.

BACKGROUND: Changes in chromosome number or structure as well as supernumerary centrosomes and multipolar mitoses are commonly observed in human tumors. Thus, centrosome amplification and mitotic checkpoint dysfunctions are believed possible causes of chromosomal instability. The Retinoblastoma tumor suppressor (RB) participates in the regulation of synchrony between DNA synthesis and centrosome duplication and it is involved in transcription regulation of some mitotic genes. Primary human fibroblasts were transfected transiently with short interfering RNA (siRNA) specific for human pRb to investigate the effects of pRb acute loss on chromosomal stability. RESULTS: Acutely pRb-depleted fibroblasts showed altered expression of genes necessary for cell cycle progression, centrosome homeostasis, kinetochore and mitotic checkpoint proteins. Despite altered expression of genes involved in the Spindle Assembly Checkpoint (SAC) the checkpoint seemed to function properly in pRb-depleted fibroblasts. In particular AURORA-A and PLK1 overexpression suggested that these two genes might have a role in the observed genomic instability. However, when they were post-transcriptionally silenced in pRb-depleted fibroblasts we did not observe reduction in the number of aneuploid cells. This finding suggests that overexpression of these two genes did not contribute to genomic instability triggered by RB acute loss although it affected cell proliferation. Acutely pRb-depleted human fibroblasts showed the presence of micronuclei containing whole chromosomes besides the presence of supernumerary centrosomes and aneuploidy. CONCLUSION: Here we show for the first time that RB acute loss triggers centrosome amplification and aneuploidy in human primary fibroblasts. Altogether, our results suggest that pRb-depleted primary human fibroblasts possess an intact spindle checkpoint and that micronuclei, likely caused by mis-attached kinetochores that in turn trigger chromosome segregation errors, are responsible for aneuploidy in primary human fibroblasts where pRb is acutely depleted.

CENPA overexpression promotes genome instability in pRb-depleted human cells.

BACKGROUND: Aneuploidy is a hallmark of most human cancers that arises as a consequence of chromosomal instability and it is frequently associated with centrosome amplification. Functional inactivation of the Retinoblastoma protein (pRb) has been indicated as a cause promoting chromosomal instability as well centrosome amplification. However, the underlying molecular mechanism still remains to be clarified. RESULTS: Here we show that pRb depletion both in wild type and p53 knockout HCT116 cells was associated with the presence of multipolar spindles, anaphase bridges, lagging chromosomes and micronuclei harbouring whole chromosomes. In addition aneuploidy caused by pRb acute loss was not affected by p53 loss.Quantitative real-time RT-PCR showed that pRB depletion altered expression of genes involved in centrosome duplication, kinetochore assembly and in the Spindle Assembly Checkpoint (SAC). However, despite MAD2 up-regulation pRb-depleted cells seemed to have a functional SAC since they arrested in mitosis after treatments with mitotic poisons. Moreover pRb-depleted HCT116 cells showed BRCA1 overexpression that seemed responsible for MAD2 up-regulation.Post-transcriptional silencing of CENPA by RNA interference, resulting in CENP-A protein levels similar to those present in control cells greatly reduced aneuploid cell numbers in pRb-depleted cells. CONCLUSION: Altogether our findings indicate a novel aspect of pRb acute loss that promotes aneuploidy mainly by inducing CENPA overexpression that in turn might induce micronuclei by affecting the correct attachment of spindle microtubules to kinetochores.

An Intricate Connection between Alternative Splicing and Phenotypic Plasticity in Development and Cancer.

During tumor progression, hypoxia, nutrient deprivation or changes in the extracellular environment (i.e., induced by anti-cancer drugs) elicit adaptive responses in cancer cells. Cellular plasticity increases the chance that tumor cells may survive in a challenging microenvironment, acquire new mechanisms of resistance to conventional drugs, and spread to distant sites. Re-activation of stem pathways appears as a significant cause of cellular plasticity because it promotes the acquisition of stem-like properties through a profound phenotypic reprogramming of cancer cells. In addition, it is a major contributor to tumor heterogeneity, depending on the coexistence of phenotypically distinct subpopulations in the same tumor bulk. Several cellular mechanisms may drive this fundamental change, in particular, high-throughput sequencing technologies revealed a key role for alternative splicing (AS). Effectively, AS is one of the most important pre-mRNA processes that increases the diversity of transcriptome and proteome in a tissue- and development-dependent manner. Moreover, defective AS has been associated with several human diseases. However, its role in cancer cell plasticity and tumor heterogeneity remains unclear. Therefore, unravelling the intricate relationship between AS and the maintenance of a stem-like phenotype may explain molecular mechanisms underlying cancer cell plasticity and improve cancer diagnosis and treatment.

Aurora-A transcriptional silencing and vincristine treatment show a synergistic effect in human tumor cells.

Aurora-A is a centrosome-associated serine/threonine kinase that is overexpressed in multiple types of human tumors. Primarily, Aurora-A functions in centrosome maturation and mitotic spindle assembly. Overexpression of Aurora-A induces centrosome amplification and G2/M cell cycle progression. Recently, it was observed that overexpression of Aurora-A renders cells resistant to cisplatin (CDDP)-, etoposide-, and paclitaxel-induced apoptosis. Our results indicate that already in initial stages of cancer progression Aurora-A overexpression could have a major role in inducing supernumerary centrosomes and aneuploidy, as shown by immunohistochemistry on tissue sections from various stages of human colon cancer. Aneuploidy was also observed after Aurora-A ectopic overexpression in colon cancer cells with MIN phenotype. Silencing of Aurora-A by RNA interference in tumor cell lines triggered arrest of the cell cycle associated to apoptosis/ mitotic catastrophe. Finally, Aurora-A transcriptional silencing seems to confer cancer cells a greater sensitivity to chemotherapy by vincristine, indicating Aurora-A as a possible gene target in cancer therapy.

Simultaneous Aurora-A/STK15 overexpression and centrosome amplification induce chromosomal instability in tumour cells with a MIN phenotype.

BACKGROUND: Genetic instability is a hallmark of tumours and preneoplastic lesions. The predominant form of genome instability in human cancer is chromosome instability (CIN). CIN is characterized by chromosomal aberrations, gains or losses of whole chromosomes (aneuploidy), and it is often associated with centrosome amplification. Centrosomes control cell division by forming a bipolar mitotic spindle and play an essential role in the maintenance of chromosomal stability.However, whether centrosome amplification could directly cause aneuploidy is not fully established. Also, alterations in genes required for mitotic progression could be involved in CIN.A major candidate is represented by Aurora-A/STK15 that associates with centrosomes and is overexpressed in several types of human tumour. METHODS: Centrosome amplification were induced by hydroxyurea treatment and visualized by immunofluorescence microscopy. Aurora-A/STK15 ectopic expression was achieved by retroviral infection and puromycin selection in HCT116 tumour cells. Effects of Aurora-A/STK15 depletion on centrosome status and ploidy were determined by Aurora-A/STK15 transcriptional silencing by RNA interference. Changes in the expression levels of some mitotic genes were determined by Real time RT-PCR. RESULTS: We investigated whether amplification of centrosomes and overexpression of Aurora-A/STK15 induce CIN using as a model system a colon carcinoma cell line (HCT116). We found that in HCT116 cells, chromosomally stable and near diploid cells harbouring a MIN phenotype, centrosome amplification induced by hydroxyurea treatment is neither maintained nor induces aneuploidy. On the contrary, ectopic overexpression of Aurora-A/STK15 induced supernumerary centrosomes and aneuploidy. Aurora-A/STK15 transcriptional silencing by RNA interference in cells ectopically overexpressing this kinase promptly decreased cell numbers with supernumerary centrosomes and aneuploidy. CONCLUSION: Our results show that centrosome amplification alone is not sufficient to induce chromosomal instability in colon cancer cells with a MIN phenotype. Alternatively, centrosome amplification has to be associated with alterations in genes regulating mitosis progression such as Aurora-A/STK15 to trigger CIN.

Correction: A Network-Based Data Integration Approach to Support Drug Repurposing and Multi-Target Therapies in Triple Negative Breast Cancer.

[This corrects the article DOI: 10.1371/journal.pone.0162407.].

Reversible Cognitive Frailty, Dementia, and All-Cause Mortality. The Italian Longitudinal Study on Aging.

OBJECTIVES: Cognitive frailty, a condition describing the simultaneous presence of physical frailty and mild cognitive impairment, has been recently defined by an international consensus group. We estimated the predictive role of a "reversible" cognitive frailty model on incident dementia, its subtypes, and all-cause mortality in nondemented older individuals. We verified if vascular risk factors or depressive symptoms could modify this predictive role. DESIGN: Longitudinal population-based study with 3.5- and 7-year of median follow-up. SETTING: Eight Italian municipalities included in the Italian Longitudinal Study on Aging. PARTICIPANTS: In 2150 older individuals from the Italian Longitudinal Study on Aging, we operationalized reversible cognitive frailty with the presence of physical frailty and pre-mild cognitive impairment subjective cognitive decline, diagnosed with a self-report measure based on item 14 of the Geriatric Depression Scale. MEASUREMENTS: Incidence of dementia, its subtypes, and all-cause mortality. RESULTS: Over a 3.5-year follow-up, participants with reversible cognitive frailty showed an increased risk of overall dementia [hazard ratio (HR) 2.30, 95% confidence interval (CI) 1.02-5.18], particularly vascular dementia (VaD), and all-cause mortality (HR 1.74, 95% CI 1.07-2.83). Over a 7-year follow-up, participants with reversible cognitive frailty showed an increased risk of overall dementia (HR 2.12, 95% CI 1.12-4.03), particularly VaD, and all-cause mortality (HR 1.39, 95% CI 1.03-2.00). Vascular risk factors and depressive symptoms did not have any effect modifier on the relationship between reversible cognitive frailty and incident dementia and all-cause mortality. CONCLUSIONS: A model of reversible cognitive frailty was a short- and long-term predictor of all-cause mortality and overall dementia, particularly VaD. The absence of vascular risk factors and depressive symptoms did not modify the predictive role of reversible cognitive frailty on these outcomes.

RB acute loss induces centrosome amplification and aneuploidy in murine primary fibroblasts.

BACKGROUND: Incorrect segregation of whole chromosomes or parts of chromosome leads to aneuploidy commonly observed in cancer. The correct centrosome duplication, assuring assembly of a bipolar mitotic spindle, is essential for chromosome segregation fidelity and preventing aneuploidy. Alteration of p53 and pRb functions by expression of HPV16-E6 and E7 oncoproteins has been associated with centrosome amplification. However, these last findings could be the result of targeting cellular proteins in addition to pRb by HPV16-E7 oncoprotein. To get a more detailed picture on the role of pRb in chromosomal instability and centrosome amplification, we analyzed the effects of the acute loss of retinoblastoma gene function in primary conditional Rb deficient mouse embryonic fibroblasts (MEFs). Moreover, since pRb is a transcriptional repressor, microarray analysis was done on pRb-competent and pRb-deficient MEFs to evaluate changes in expression of genes for centrosome homeostasis and for correct mitosis. RESULTS: Acute loss of pRb induces centrosome amplification and aneuploidy in the vast majority of cells analyzed. A time course analysis shows a decrease of cells with amplified centrosomes after 40 days from the adenoviral infection. At this time only 12% of cells still show amplified centrosomes. Interestingly, cells with pRb constitutive loss show a similar percentage of cells with amplified centrosomes. DNA-Chip analyses in MEFs wt (mock infected) and pRb depleted (Ad-Cre infected) cells reveal differential expression of genes controlling both centrosome duplication and mitotic progression. CONCLUSION: Our findings suggest a direct link between pRb status, centrosome amplification and chromosomal instability, and define specific mitotic genes as targets whose gene expression has to be altered to achieve or maintain aneuploidy.

Centrosome amplification induced by hydroxyurea leads to aneuploidy in pRB deficient human and mouse fibroblasts.

Alterations in the number and/or morphology of centrosomes are frequently observed in human tumours. However, it is still debated if a direct link between supernumerary centrosomes and tumorigenesis exists and if centrosome amplification could directly cause aneuploidy. Here, we report that hydroxyurea treatment induced centrosome amplification in both human fibroblasts expressing the HPV16 -E6-E7 oncoproteins, which act principally by targeting p53 and pRB, respectively, and in conditional pRB deficient mouse fibroblasts. Following hydroxyurea removal both normal and p53 deficient human fibroblasts arrested. On the contrary pRB deficient fibroblasts entered the cell cycle generating aneuploid cells. Also the majority of conditional Rb deficient MEFs showed supernumerary centrosomes and aneuploid cells which increased over time. Finally, our results suggest that pRB dysfunction both in human and murine fibroblasts transiently arrested in G1/S by hydroxyurea allows centrosomes amplification, in the absence of DNA synthesis, that in turn could drive aneuploidy.

A Network-Based Data Integration Approach to Support Drug Repurposing and Multi-Target Therapies in Triple Negative Breast Cancer.

The integration of data and knowledge from heterogeneous sources can be a key success factor in drug design, drug repurposing and multi-target therapies. In this context, biological networks provide a useful instrument to highlight the relationships and to model the phenomena underlying therapeutic action in cancer. In our work, we applied network-based modeling within a novel bioinformatics pipeline to identify promising multi-target drugs. Given a certain tumor type/subtype, we derive a disease-specific Protein-Protein Interaction (PPI) network by combining different data-bases and knowledge repositories. Next, the application of suitable graph-based algorithms allows selecting a set of potentially interesting combinations of drug targets. A list of drug candidates is then extracted by applying a recent data fusion approach based on matrix tri-factorization. Available knowledge about selected drugs mechanisms of action is finally exploited to identify the most promising candidates for planning in vitro studies. We applied this approach to the case of Triple Negative Breast Cancer (TNBC), a subtype of breast cancer whose biology is poorly understood and that lacks of specific molecular targets. Our "in-silico" findings have been confirmed by a number of in vitro experiments, whose results demonstrated the ability of the method to select candidates for drug repurposing.

Proteasome machinery is instrumental in a common gain-of-function program of the p53 missense mutants in cancer.

In cancer, the tumour suppressor gene TP53 undergoes frequent missense mutations that endow mutant p53 proteins with oncogenic properties. Until now, a universal mutant p53 gain-of-function program has not been defined. By means of multi-omics: proteome, DNA interactome (chromatin immunoprecipitation followed by sequencing) and transcriptome (RNA sequencing/microarray) analyses, we identified the proteasome machinery as a common target of p53 missense mutants. The mutant p53-proteasome axis globally affects protein homeostasis, inhibiting multiple tumour-suppressive pathways, including the anti-oncogenic KSRP-microRNA pathway. In cancer cells, p53 missense mutants cooperate with Nrf2 (NFE2L2) to activate proteasome gene transcription, resulting in resistance to the proteasome inhibitor carfilzomib. Combining the mutant p53-inactivating agent APR-246 (PRIMA-1MET) with the proteasome inhibitor carfilzomib is effective in overcoming chemoresistance in triple-negative breast cancer cells, creating a therapeutic opportunity for treatment of solid tumours and metastasis with mutant p53.

Adipose-derived Mesenchymal Stem Cells (ASCs) may favour breast cancer recurrence via HGF/c-Met signaling.

Adipose tissue is a reservoir of Mesenchymal Stem Cells (Adipose-derived Mesenchymal Stem Cells, ASCs), endowed with regenerative properties. Fat graft was proposed for breast reconstruction in post-surgery cancer patients achieving good aesthetic results and tissues regeneration. However, recent findings highlight a potential tumorigenic role that ASCs may have in cancer recurrence, raising some concerns about their safety in clinical application. To address this issue, we established a model where autologous ASCs were combined with primary normal or cancer cells from breast of human donors, in order to evaluate potential effects of their interactions, in vitro and in vivo. Surprisingly, we found that ASCs are not tumorigenic per sè, as they are not able to induce a neoplastic transformation of normal mammary cells, however they could exhacerbate tumorigenic behaviour of c-Met-expressing breast cancer cells, creating an inflammatory microenvironment which sustained tumor growth and angiogenesis. Pharmacological c-Met inhibition showed that a HGF/c-Met crosstalk between ASCs and breast cancer cells enhanced tumor cells migration, acquiring a metastatic signature, and sustained tumor self-renewal. The master role of HGF/c-Met pathway in cancer recurrence was further confirmed by c-Met immunostaining in primary breast cancer from human donors, revealing a strong positivity in patients displaying a recurrent pathology after fat grafts and a weak/moderate staining in patients without signs of recurrence. Altogether our findings, for the first time, suggest c-Met expression, as predictive to evaluate risk of cancer recurrence after autologous fat graft in post-surgery breast cancer patients, increasing the safety of fat graft in clinical application.