Whole-Exome Sequencing of Germline Variants in Non-BRCA Families with Hereditary Breast Cancer.

Breast cancer is the most prevalent malignancy among women worldwide and hereditary breast cancer (HBC) accounts for about 5−10% of the cases. Today, the most recurrent genes known are BRCA1 and BRCA2, accounting for around 25% of familial cases. Although thousands of loss-of-function variants in more than twenty predisposing genes have been found, the majority of familial cases of HBC remain unexplained. The aim of this study was to identify new predisposing genes for HBC in three non-BRCA families with autosomal dominant inheritance pattern using whole-exome sequencing and functional prediction tools. No pathogenic variants in known hereditary cancer-related genes could explain the breast cancer susceptibility in these families. Among 2122 exonic variants with maximum minor allele frequency (MMAF) < 0.1%, between 17−35 variants with combined annotation-dependent depletion (CADD) > 20 segregated with disease in the three analyzed families. Selected candidate genes, i.e., UBASH3A, MYH13, UTP11L, and PAX7, were further evaluated using protein expression analysis but no alterations of cancer-related pathways were observed. In conclusion, identification of new high-risk cancer genes using whole-exome sequencing has been more challenging than initially anticipated, in spite of selected families with pronounced family history of breast cancer. A combination of low- and intermediate-genetic-risk variants may instead contribute the breast cancer susceptibility in these families.

Association between Predicted Effects of TP53 Missense Variants on Protein Conformation and Their Phenotypic Presentation as Li-Fraumeni Syndrome or Hereditary Breast Cancer.

Rare germline pathogenic TP53 missense variants often predispose to a wide spectrum of tumors characterized by Li-Fraumeni syndrome (LFS) but a subset of variants is also seen in families with exclusively hereditary breast cancer (HBC) outcomes. We have developed a logistic regression model with the aim of predicting LFS and HBC outcomes, based on the predicted effects of individual TP53 variants on aspects of protein conformation. A total of 48 missense variants either unique for LFS (n = 24) or exclusively reported in HBC (n = 24) were included. LFS-variants were over-represented in residues tending to be buried in the core of the tertiary structure of TP53 (p = 0.0014). The favored logistic regression model describes disease outcome in terms of explanatory variables related to the surface or buried status of residues as well as their propensity to contribute to protein compactness or protein-protein interactions. Reduced, internally validated models discriminated well between LFS and HBC (C-statistic = 0.78-0.84; equivalent to the area under the ROC (receiver operating characteristic) curve), had a low risk for over-fitting and were well calibrated in relation to the known outcome risk. In conclusion, this study presents a phenotypic prediction model of LFS and HBC risk for germline TP53 missense variants, in an attempt to provide a complementary tool for future decision making and clinical handling.

Proteomics identifies neddylation as a potential therapy target in small intestinal neuroendocrine tumors.

Patients with small intestinal neuroendocrine tumors (SI-NETs) frequently develop spread disease; however, the underlying molecular mechanisms of disease progression are not known and effective preventive treatment strategies are lacking. Here, protein expression profiling was performed by HiRIEF-LC-MS in 14 primary SI-NETs from patients with and without liver metastases detected at the time of surgery and initial treatment. Among differentially expressed proteins, overexpression of the ubiquitin-like protein NEDD8 was identified in samples from patients with liver metastasis. Further, NEDD8 correlation analysis indicated co-expression with RBX1, a key component in cullin-RING ubiquitin ligases (CRLs). In vitro inhibition of neddylation with the therapeutic agent pevonedistat (MLN4924) resulted in a dramatic decrease of proliferation in SI-NET cell lines. Subsequent mass spectrometry-based proteomics analysis of pevonedistat effects and effects of the proteasome inhibitor bortezomib revealed stabilization of multiple targets of CRLs including p27, an established tumor suppressor in SI-NET. Silencing of NEDD8 and RBX1 using siRNA resulted in a stabilization of p27, suggesting that the cellular levels of NEDD8 and RBX1 affect CRL activity. Inhibition of CRL activity, by either NEDD8/RBX1 silencing or pevonedistat treatment of cells resulted in induction of apoptosis that could be partially rescued by siRNA-based silencing of p27. Differential expression of both p27 and NEDD8 was confirmed in a second cohort of SI-NET using immunohistochemistry. Collectively, these findings suggest a role for CRLs and the ubiquitin proteasome system in suppression of p27 in SI-NET, and inhibition of neddylation as a putative therapeutic strategy in SI-NET.

Functional characterization of novel germline TP53 variants in Swedish families.

Pathogenic germline TP53 variants predispose to a wide range of early onset cancers, often recognized as the Li-Fraumeni syndrome (LFS). They are also identified in 1% of families with hereditary breast cancer (HrBC) that do not fulfill the criteria for LFS. In this study, we present a total of 24 different TP53 variants identified in 31 Swedish families with LFS or HrBC. Ten of these variants, nine exonic and one splice, have previously not been described as germline pathogenic variants. The nine exonic variants were functionally characterized and demonstrated partial transactivation activity compared to wild-type p53. Some show nuclear localization similar to wild-type p53 while others possess cytoplasmic or perinuclear localization. The four frameshift variants (W91Gfs*32, L111 Wfs*12, S227 Lfs*20 and S240Kfs*25) had negligible, while F134 L and T231del had low level of p53 activity. The L111 Wfs*12 and T231del variants are also deficient for induction of apoptosis. The missense variant R110C retain p53 effects and the nonsense E349* shows at least partial transcription factor activity but has reduced ability to trigger apoptosis. This is the first functional characterization of novel germline TP53 pathogenic or likely pathogenic variants in the Swedish cohort as an attempt to understand its association with LFS and HrBC, respectively.

Tumor-derived exosomes: Implication in angiogenesis and antiangiogenesis cancer therapy.

Tumor cells utilize different strategies to communicate with neighboring tissues for facilitating tumor progression and invasion, one of these strategies has been shown to be the release of exosomes. Exosomes are small nanovesicles secreted by all kind of cells in the body, especially cancer cells, and mediate cell to cell communications. Exosomes play an important role in cancer invasiveness by harboring various cargoes that could accelerate angiogenesis. Here first, we will present an overview of exosomes, their biology, and their function in the body. Then, we will focus on exosomes derived from tumor cells as tumor angiogenesis mediators with a particular emphasis on the underlying mechanisms in various cancer origins. Also, exosomes derived from stem cells and tumor-associated macrophages will be discussed in this regard. Finally, we will discuss the novel therapeutic strategies of exosomes as drug delivery vehicles against angiogenesis.

Silencing FLI or targeting CD13/ANPEP lead to dephosphorylation of EPHA2, a mediator of BRAF inhibitor resistance, and induce growth arrest or apoptosis in melanoma cells.

A majority of patients with BRAF-mutated metastatic melanoma respond to therapy with BRAF inhibitors (BRAFi), but relapses are common owing to acquired resistance. To unravel BRAFi resistance mechanisms we have performed gene expression and mass spectrometry based proteome profiling of the sensitive parental A375 BRAF V600E-mutated human melanoma cell line and of daughter cell lines with induced BRAFi resistance. Increased expression of two novel resistance candidates, aminopeptidase-N (CD13/ANPEP) and ETS transcription factor FLI1 was observed in the BRAFi-resistant daughter cell lines. In addition, increased levels of the previously reported resistance mediators, receptor tyrosine kinase ephrine receptor A2 (EPHA2) and the hepatocyte growth factor receptor MET were also identified. The expression of these proteins was assessed in matched tumor samples from melanoma patients obtained before BRAFi and after disease progression. MET was overexpressed in all progression samples while the expression of the other candidates varied between the individual patients. Targeting CD13/ANPEP by a blocking antibody induced apoptosis in both parental A375- and BRAFi-resistant daughter cells as well as in melanoma cells with intrinsic BRAFi resistance and led to dephosphorylation of EPHA2 on S897, previously demonstrated to cause inhibition of the migratory capacity. AKT and RSK, both reported to induce EPHA2 S897 phosphorylation, were also dephosphorylated after inhibition of CD13/ANPEP. FLI1 silencing also caused decreases in EPHA2 S897 phosphorylation and in total MET protein expression. In addition, silencing of FLI1 sensitized the resistant cells to BRAFi. Furthermore, we show that BRAFi in combination with the multi kinase inhibitor dasatinib can abrogate BRAFi resistance and decrease both EPHA2 S897 phosphorylation and total FLI1 protein expression. This is the first report presenting CD13/ANPEP and FLI1 as important mediators of resistance to BRAF inhibition with potential as drug targets in BRAFi refractory melanoma.

Tumor acidosis enhances cytotoxic effects and autophagy inhibition by salinomycin on cancer cell lines and cancer stem cells.

Sustained autophagy contributes to the metabolic adaptation of cancer cells to hypoxic and acidic microenvironments. Since cells in such environments are resistant to conventional cytotoxic drugs, inhibition of autophagy represents a promising therapeutic strategy in clinical oncology. We previously reported that the efficacy of hydroxychloroquine (HCQ), an autophagy inhibitor under clinical investigation is strongly impaired in acidic tumor environments, due to poor uptake of the drug, a phenomenon widely associated with drug resistance towards many weak bases. In this study we identified salinomycin (SAL) as a potent inhibitor of autophagy and cytotoxic agent effective on several cancer cell lines under conditions of transient and chronic acidosis. Since SAL has been reported to specifically target cancer-stem cells (CSC), we used an established model of breast CSC and CSC derived from breast cancer patients to examine whether this specificity may be associated with autophagy inhibition. We indeed found that CSC-like cells are more sensitive to autophagy inhibition compared to cells not expressing CSC markers. We also report that the ability of SAL to inhibit mammosphere formation from CSC-like cells was dramatically enhanced in acidic conditions. We propose that the development and use of clinically suitable SAL derivatives may result in improved autophagy inhibition in cancer cells and CSC in the acidic tumor microenvironment and lead to clinical benefits.

Sorafenib-induced defective autophagy promotes cell death by necroptosis.

Autophagy is one of the main cytoprotective mechanisms that cancer cells deploy to withstand the cytotoxic stress and survive the lethal damage induced by anti-cancer drugs. However, under specific conditions, autophagy may, directly or indirectly, induce cell death. In our study, treatment of the Atg5-deficient DU145 prostate cancer cells, with the multi-tyrosine kinase inhibitor, sorafenib, induces mitochondrial damage, autophagy and cell death. Molecular inhibition of autophagy by silencing ULK1 and Beclin1 rescues DU145 cells from cell death indicating that, in this setting, autophagy promotes cell death. Re-expression of Atg5 restores the lipidation of LC3 and rescues DU145 and MEF atg5-/- cells from sorafenib-induced cell death. Despite the lack of Atg5 expression and LC3 lipidation, DU145 cells form autophagosomes as demonstrated by transmission and immuno-electron microscopy, and the formation of LC3 positive foci. However, the lack of cellular content in the autophagosomes, the accumulation of long-lived proteins, the presence of GFP-RFP-LC3 positive foci and the accumulated p62 protein levels indicate that these autophagosomes may not be fully functional. DU145 cells treated with sorafenib undergo a caspase-independent cell death that is inhibited by the RIPK1 inhibitor, necrostatin-1. Furthermore, treatment with sorafenib induces the interaction of RIPK1 with p62, as demonstrated by immunoprecipitation and a proximity ligation assay. Silencing of p62 decreases the RIPK1 protein levels and renders necrostatin-1 ineffective in blocking sorafenib-induced cell death. In summary, the formation of Atg5-deficient autophagosomes in response to sorafenib promotes the interaction of p62 with RIPK leading to cell death by necroptosis.

A standardized method for quantifying proliferation by Ki-67 and cyclin A immunohistochemistry in breast cancer.

Immunohistochemical analysis of proliferation markers such as Ki-67 and cyclin A is widely used in clinical evaluation as a prognostic factor in breast cancer. The proliferation status of tumors is guiding the decision of whether or not a patient should be treated with chemotherapy because low-proliferative tumors are less sensitive by such treatment. However, the lack of optimal cutoff points and selection of tumor areas hamper its use in clinical practice. This study was performed to compare the Ki-67 and cyclin A expression counted in hot-spot vs average counting based on 5 to 14 random tumor areas in 613 breast carcinomas. We correlated the findings with 10-year follow-up in order to standardize the evaluation of proliferation markers in clinical practice. A significant correlation was found between the percentage of positive cells estimated by Ki-67 and cyclin A both by hot-spot and by average counting. Both methods showed that high expression of Ki-67 and cyclin A is associated with more adverse tumor stage. The cutoff value for Ki-67 for distant metastases was set to 22% and to 15%, using hot-spot and average counting, respectively. For cyclin A, the values were set to 14% and 8% using the respective methods. Survival curves revealed that patients with a high hot-spot proliferation index had a significantly greater risk of shorter tumor-free survival. Our findings suggest that the determination of proliferation markers in breast cancer should be standardized to hot-spot counting and that specific cutoff values for proliferation could be useful as prognostic markers in clinical practice. Moreover, we suggest that expression levels of cyclin A could be used as a complementary marker to estimate the proliferation status in tumors, especially those with "borderline" expression levels of Ki-67, in order to more accurately estimate the proliferations status of the tumors.

Molecular profiling of prostate cancer derived exosomes may reveal a predictive signature for response to docetaxel.

Docetaxel is a cornerstone treatment for metastatic, castration resistant prostate cancer (CRPC) which remains a leading cause of cancer-related deaths, worldwide. The clinical usage of docetaxel has resulted in modest gains in survival, primarily due to the development of resistance. There are currently no clinical biomarkers available that predict whether a CRPC patient will respond or acquire resistance to this therapy. Comparative proteomics analysis of exosomes secreted from DU145 prostate cancer cells that are sensitive (DU145 Tax-Sen) or have acquired resistance (DU145 Tax-Res) to docetaxel, demonstrated significant differences in the amount of exosomes secreted and in their molecular composition. A panel of proteins was identified by proteomics to be differentially enriched in DU145 Tax-Res compared to DU145 Tax-Sen exosomes and was validated by western blotting. Importantly, we identified MDR-1, MDR-3, Endophilin-A2 and PABP4 that were enriched only in DU145 Tax-Res exosomes. We validated the presence of these proteins in the serum of a small cohort of patients. DU145 cells that have uptaken DU145 Tax-Res exosomes show properties of increased matrix degradation. In summary, exosomes derived from DU145 Tax-Res cells may be a valuable source of biomarkers for response to therapy.

Malignant cell-derived extracellular vesicles express different chromogranin epitopes compared to prostasomes.

BACKGROUND: Prostasomes are nanosized extracellular vesicles exocytosed by prostate epithelial cells. They have been assigned many roles propitious to sperm in favor of fertilization. Prostatic cancer cells can also produce and secrete extracellular vesicles. METHODS: We assessed using ELISA, the surface expression of chromogranin proproteins on prostasomes and malignant extracellular vesicles of four different prostate cancer cell-lines, two hormone sensitive and two hormone refractory. We used a panel of chromogranin A and chromogranin B antibodies against peptides in-between hypothetical cleavage sites along the proproteins. RESULTS: A diverging pattern of chromogranin peptides was apparent when comparing prostasomes and malignant extracellular vesicles indicating a phenotypical change. We also compared western blot patterns (prostasomes and malignant extracellular vesicles) for selected antibodies that displayed high absorbances in the ELISA. Western blot analyses revealed various cleavage patterns of those proproteins that were analyzed in prostasomes and extracellular vesicles. CONCLUSION: Chromogranins are constituents of not only prostasomes but also of malignant prostate cell-derived extracellular vesicles with different amino acid sequences exposed at the membrane surface giving rise to a mosaic pattern. These findings may be of relevance for designing new assays for detection or even possible treatment of prostate cancers.

Effect of acute exercise on prostate cancer cell growth.

Physical activity is associated with reduced risk of several cancers, including aggressive prostate cancer. The mechanisms mediating the effects are not yet understood; among the candidates are modifications of endogenous hormone levels. Long-term exercise is known to reduce serum levels of growth stimulating hormones. In contrast, the endocrine effects of acute endurance exercise include increased levels of mitogenic factors such as GH and IGF-1. It can be speculated that the elevation of serum growth factors may be detrimental to prostate cancer progression into malignancy. The incentive of the current study is to evaluate the effect of acute exercise serum on prostate cancer cell growth. We designed an exercise intervention where 10 male individuals performed 60 minutes of bicycle exercise at increasing intensity. Serum samples were obtained before (rest serum) and after completed exercise (exercise serum). The established prostate cancer cell line LNCaP was exposed to exercise or rest serum. Exercise serum from 9 out of 10 individuals had a growth inhibitory effect on LNCaP cells. Incubation with pooled exercise serum resulted in a 31% inhibition of LNCaP growth and pre-incubation before subcutaneous injection into SCID mice caused a delay in tumor formation. Serum analyses indicated two possible candidates for the effect; increased levels of IGFBP-1 and reduced levels of EGF. In conclusion, despite the fear of possible detrimental effects of acute exercise serum on tumor cell growth, we show that even the short-term effects seem to add to the overall beneficial influence of exercise on neoplasia.

Multitargeted therapies for multiple myeloma.

Multiple myeloma (MM) comprises 1% of all malignancies and 10% of all hematological malignancies. MM is a malignancy of plasma cells in the bone marrow where complex and dynamic interactions with the bone marrow microenvironment lead to tumor progression, skeletal destruction and angiogenesis. Despite the discovery of several novel treatments against MM, including the proteasome inhibitor bortezomib, it is considered to be an incurable disease with an average 4-5 years overall survival.

Sorafenib has potent antitumor activity against multiple myeloma in vitro, ex vivo, and in vivo in the 5T33MM mouse model.

Multiple myeloma (MM) is a B-cell malignancy characterized by the expansion of clonal plasma blasts/plasma cells within the bone marrow that relies on multiple signaling cascades, including tyrosine kinase activated pathways, to proliferate and evade cell death. Despite emerging new treatment strategies, multiple myeloma remains at present incurable. Thus, novel approaches targeting several signaling cascades by using the multi-tyrosine kinase inhibitor (TKI), sorafenib, seem a promising treatment approach for multiple myeloma. Here, we show that sorafenib induces cell death in multiple myeloma cell lines and in CD138(+)-enriched primary multiple myeloma patient samples in a caspase-dependent and -independent manner. Furthermore, sorafenib has a strong antitumoral and -angiogenic activity in the 5T33MM mouse model leading to increased overall survival. Multiple myeloma cells undergo autophagy in response to sorafenib, and inhibition of this cytoprotective pathway potentiated the efficacy of this TKI. Mcl-1, a survival factor in multiple myeloma, is downregulated at the protein level by sorafenib allowing for the execution of cell death, as ectopic overexpression of this protein protects multiple myeloma cells. Concomitant targeting of Mcl-1 by sorafenib and of Bcl-2/Bcl-xL by the antagonist ABT737 improves the efficacy of sorafenib in multiple myeloma cell lines and CD138(+)-enriched primary cells in the presence of bone marrow stromal cells. Altogether, our data support the use of sorafenib as a novel therapeutic modality against human multiple myeloma, and its efficacy may be potentiated in combination with ABT737.

Tumor cell-derived exosomes: a message in a bottle.

Exosomes constitute the newest mode of intercellular communication, transmitting information between cells. This exchange of molecular information is facilitated by their unique composition which is enriched with enzymes, structural proteins, adhesion molecules, lipid rafts and RNAs. Following the discovery that cancer cells secrete excessive amounts of exosomes compared to normal cells, it became evident that i) these vesicles can be used as diagnostic markers; ii) their active secretion has functional implications, albeit unknown whether they are tumor promoting or suppressing. Notably, the interplay via the exchange of exosomes between cancer cells and between cancer cells and the tumor stroma may promote the transfer of oncogenes (e.g. ß-catenin, CEA, HER2, Melan-A/Mart-1 and LMP-1) and onco-microRNAs (e.g. let7, miR1, miR15, miR16 and miR375) from one cell to another, leading to the reprogramming of the recipient cells. The molecular composition and functional role of tumor cell-derived exosomes in tumorigenesis, metastasis and response to therapy are slowly decrypted and the latest findings as well as potential therapeutic strategies are discussed in this review.

Sorafenib induces apoptosis and autophagy in prostate cancer cells in vitro.

The multiple tyrosine kinase inhibitor sorafenib has recently demonstrated clinical effects in patients with androgen-independent prostate cancer. These observations provided the rational for investigating the anti-tumoral properties of this compound on prostate cancer cell lines at the molecular level. Two hormone refractory (PC3 and DU145) and one hormone responsive cell line (22Rv1) were used. By use of a panel of cell biology techniques such as immunoblotting, flow cytometry and immunocytochemistry, effects on the MAPK pathway and induction of apoptosis and autophagy were evaluated. We demonstrate that sorafenib reduced cell viability in a dose-dependent manner, induced apoptosis and inactivated the MAPK pathway. Moreover, we show for the first time, that sorafenib treatment of prostate cancer cells also induces cellular autophagy. This feature is in accordance with the anticancer potential of sorafenib and adds another important effector mechanism of this compound. These observations may open potentially interesting treatment combinations that may augment the effect of sorafenib, either by drugs that promote autophagy such as the rapalogues, or by combining sorafenib with compounds that specifically inhibit the autophagic process.

Autophagy as the main means of cytotoxicity by glucocorticoids in hematological malignancies.

Glucocorticoids are fundamental drugs used in the treatment of lymphoid malignancies with apoptotic cell death as the hitherto proposed mechanism of action. We have recently shown that dexamethasone induces autophagy in lymphoid leukemia cells and in this particular setting this cell death modality is a prerequisite for the efficient killing of the leukemic cells by dexamethasone. Hence, inhibition of autophagy by siRNA-mediated silencing of Beclin 1, as well as chemical inhibition of type III PtdIns3K, inhibits apoptosis, demonstrating an important role of autophagy in dexamethasone-induced cell death. In this brief report, we review these findings and introduce the multiple myeloma cells as a novel system to study autophagy in response to dexamethasone.

Improvement of liver function in liver cirrhosis patients after autologous mesenchymal stem cell injection: a phase I-II clinical trial.

BACKGROUND: End-stage liver disease is a medical problem with high morbidity and mortality. We have investigated the feasibility, safety, and efficacy of using autologous mesenchymal stem cells (MSCs) as a treatment. METHODS: Eight patients (four hepatitis B, one hepatitis C, one alcoholic, and two cryptogenic) with end-stage liver disease having Model for End-Stage Liver Disease score > or =10 were included. Autologous MSCs were taken from iliac crest. Approximately, 30-50 million MSCs were proliferated and injected into peripheral or the portal vein. Liver function and clinical features were evaluated at baseline and 1, 2, 4, 8, and 24 weeks after injection. RESULTS: Treatment was well tolerated by all patients. Liver function improved as verified by the Model for End-Stage Liver Disease score, which decreased from 17.9+/-5.6 to 10.7+/-6.3 (P<0.05) and prothrombin complex from international normalized ratio 1.9+/-0.4 to 1.4+/-0.5 (P<0.05). Serum creatinine decreased from 114+/-35 to 80+/-18 micromol/l (P<0.05). Serum albumin changed from 30+/-5 to 33+/-5 g/l and bilirubin from 46+/-29 to 41+/-31 micromol/l. No adverse effects were noted. CONCLUSION: Our data show that MSCs injection can be used for the treatment of end-stage liver disease with satisfactory tolerability. Furthermore, this treatment may improve clinical indices of liver function in end-stage liver disease.