Everolimus restrains the paracrine pro-osteoclast activity of breast cancer cells.

BACKGROUND: Breast cancer (BC) cells secrete soluble factors that accelerate osteoclast (OC) differentiation, leading to the formation of osteolytic bone metastases. In the BOLERO-2 trial, BC patients with bone involvement who received Everolimus had a delayed tumor progression in the skeleton as a result of direct OC suppression through the inhibition of mTOR, in addition to the general suppressor effect on the cancer cells. Here, we explored the effect of Everolimus, as mTOR inhibitor, on the pro-OC paracrine activity of BC cells. METHODS: Both MDA-MB-231 and MCF-7 BC cell lines were incubated with sub-lethal amounts of Everolimus, and their conditioned supernatants were assessed for their capacity to differentiate OCs from PBMC from healthy donors, as well as to interfere with their bone resorbing activity shown on calcium phosphate slices. We also measured the mRNA levels of major pro-OC factors in Everolimus-treated BC cells and their secreted levels by ELISA, and evaluated by immunoblotting the phosphorylation of transcription factors enrolled by pathways cooperating with the mTOR inhibition. Finally, the in vivo pro-OC activity of these cells was assessed in SCID mice after intra-tibial injections. RESULTS: We found that Everolimus significantly inhibited the differentiation of OCs and their in vitro bone-resorbing activity, and also found decreases of both mRNA and secreted pro-OC factors such as M-CSF, IL-6, and IL-1ß, whose lower ELISA levels paralleled the defective phosphorylation of NFkB pathway effectors. Moreover, when intra-tibially injected in SCID mice, Everolimus-treated BC cells produced smaller bone metastases than the untreated cells. CONCLUSIONS: mTOR inhibition in BC cells leads to a suppression of their paracrine pro-OC activity by interfering with the NFkB pathway; this effect may also account for the delayed progression of bone metastatic disease observed in the BOLERO-2 trial.

A peculiar molecular profile of umbilical cord-mesenchymal stromal cells drives their inhibitory effects on multiple myeloma cell growth and tumor progression.

Bone marrow-derived mesenchymal stromal cells (BM-MSCs) are under intensive investigation in preclinical models of cytotherapies against cancer, including multiple myeloma (MM). However, the therapeutic use of stromal progenitors holds critical safety concerns due to their potential MM-supporting activity in vivo. Here, we explored whether MSCs from sources other than BM, such as adipose tissue (AD-MSCs) and umbilical cord (UC-MSCs), affect MM cell growth in comparison to either normal (nBM-MSCs) or myelomatous marrow MSCs (MM-BM-MSCs). Results from both proliferation and clonogenic assays indicated that, in contrast to nBM- and MM-BM-MSCs, both AD and particularly UC-MSCs significantly inhibit MM cell clonogenicity and growth in vitro. Furthermore, when co-injected with UC-MSCs into mice, RPMI-8226 MM cells formed smaller subcutaneous tumor masses, while peritumoral injections of the same MSC subtype significantly delayed the tumor burden growing in subcutaneous plasmocytoma-bearing mice. Finally, both microarrays and ELISA revealed different expression of several genes and soluble factors in UC-MSCs as compared with other MSCs. Our data suggest that UC-MSCs have a distinct molecular profile that correlates with their intrinsic anti-MM activity and emphasize the UCs as ideal sources of MSCs for future cell-based therapies against MM.

An imbalance between Beclin-1 and p62 expression promotes the proliferation of myeloma cells through autophagy regulation.

Autophagy occurs in tumor cells acquiring cytotoxic drug resistance and its activation may impair their susceptibility to apoptosis in response to apoptogen agents. We investigated the pro-apoptotic effect of dexamethasone (Dex) on MM cell lines (U266, INA-6, LR5-8226, LIG, and MCC2) and primary malignant plasma cells from naïve and refractory/relapsed patients. We evaluated the transcriptional and ultrastructural events leading to autophagy by measuring Beclin-1 and p62 levels and transmission electronic microscopy. Autophagy was inhibited by hydroxychloroquine (HCQ), whereas the ability of Dex-resistant MM cells to recover the susceptibility to apoptosis was measured. A direct relationship between autophagy and Beclin-1 or LC3/Atg8 levels was observed, whereas their mRNAs were inversely correlated to p62 expression. Starvation strongly activated autophagy by inducing cellular, transcriptional, and ultrastructural modifications that were reversed by HCQ. Taken together, these data suggest that autophagy is a potential mechanism leading to drug resistance in MM, and suggest Beclin-1 and p62 as early markers of cell susceptibility to apoptosis. The combination of HCQ with novel agents may thus be considered to improve the therapeutic response in relapsed/resistant MM patients.

PTHrP produced by myeloma plasma cells regulates their survival and pro-osteoclast activity for bone disease progression.

To promote their survival and progression in the skeleton, osteotropic malignancies of breast, lung, and prostate produce parathyroid hormone-related protein (PTHrP), which induces hypercalcemia. PTHrP serum elevations have also been described in multiple myeloma (MM), although their role is not well defined. When we investigated MM cells from patients and cell lines, we found that PTHrP and its receptor (PTH-R1) are highly expressed, and that PTHrP is secreted both as a full-length molecule and as small subunits. Among these subunits, the mid-region, including the nuclear localization sequence (NLS), exerted a proliferative effect because it was accumulated in nuclei of MM cells surviving in starvation conditions. This was confirmed by increased transcription of several genes enrolled in proliferation and apoptosis control. PTHrP was also found to stimulate PTH-R1 in MM cells. PTH-R1's selective activation by the full-length PTHrP molecule or the NH2 -terminal fragment resulted in a significant increase of intracellular Ca(2+) influx, cyclic adenosine monophosphate (cAMP) content, and expression of receptor activator of NF-κB ligand (RANKL) and monocyte chemoattractant protein-1 (MCP-1). Our data definitely clarify the role of PTHrP in MM. The PTHrP peptide is functionally secreted by malignant plasma cells and contributes to MM tumor biology and progression, both by intracrine maintenance of cell proliferation in stress conditions and by autocrine or paracrine stimulation of PTH-R1, which in turn reinforces the production of osteoclastogenic factors. © 2014 American Society for Bone and Mineral Research.

Cytotherapies in multiple myeloma: a complementary approach to current treatments?

INTRODUCTION: Based on their tumor tropism, mesenchymal stem cells (MSCs) have been proposed as carriers of cytotoxic molecules in pioneering strategies of anti-cancer gene therapy. Similar to solid tumors, MSCs, genetically modified to stably express the TNF-related apoptosis-inducing ligand (TRAIL), have been applied to counter-attack multiple myeloma (MM) in vitro and envisioned as a promising strategy for future anti-MM treatments. AREAS COVERED: Accumulating evidence based on the detection of genetic and functional abnormalities in MSCs from MM patients points to the supportive function of MSCs in both the development and progression of MM, driven by chronic interplays with malignant cells within the marrow milieu. In this review, we revisit the function of MSCs in the pathophysiology of MM and explore the pivotal mechanisms of their interaction with myeloma cells. We also discuss the therapeutic significance of novel strategies using TRAIL-engineered MSCs in this cancer model, dissecting their role as new tools for future treatments against MM. EXPERT OPINION: A cytotherapy based on TRAIL-engineered MSCs against MM may be successfully combined with either conventional approaches of autologous stem cell transplantation or with novel anti-MM drugs. Intensive preclinical investigations are required to identify the best sources as well as modalities of MSC administration, thus defining the translational suitability of this strategy in the clinical setting.

Immature dendritic cells in multiple myeloma are prone to osteoclast-like differentiation through interleukin-17A stimulation.

Interleukin 17A (IL17A), a cytokine involved in allergy, inflammation and osteoclastogenesis, was investigated in multiple myeloma (MM) to assess its role in the osteoclast (OC)-like activity of marrow immature dendritic cells (iDCs). Comparing nine MM patients with control subjects affected by monoclonal gammopathy of undetermined significance, we found high IL17A expression in the marrow plasma of MM patients in parallel with its deposits within the stromal matrix. Increased expression of the IL17A receptor (IL17RA) was also found in primary myeloma iDCs, which underwent OC-like transdifferentiation after IL17A stimulation. To assess the role of IL17A, we measured the activity of the IL17/IL17RA pathway in IL17A-transdifferentiated iDCs and the expression of functional OC genes by Western blotting and real-time polymerase chain reaction. These cells showed increased RNA transcription of genes enrolled in the maturation of OCs, while NFATC1 and FOS were induced by IL17A, independently of NFKB1 phosphorylation. Moreover, the concurrent phosphorylation of the Lip isoform of CEBPB and the down-regulation of MAFB supported the activation of IL17RA pathway in OC-like transdifferentiated iDCs that was apparently unrelated to TNFRSF11A signalling. These data emphasize the involvement of iDCs in MM hyperactive osteoclastogenesis and suggest that their bone resorption activity is also regulated, at least in vitro, by IL17RA.

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM).

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).
Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.

PAI-1 4G/5G repeat is a target in gastric carcinomas with microsatellite instability.

BACKGROUND: The plasminogen activator inhibitor-1 (PAI-1) plays an important role in the pathogenesis of cancer. The 4G/5G promoter polymorphism of PAI-1 is potentially involved in regulating gene transcription. AIMS: To explore the role of the PAI-1 4G/5G repeat as target of microsatellite instability (MSI), 50 gastric carcinomas (GCs), characterized for MSI, were screened. METHODS: PAI-1 4G/5G was analysed by direct sequencing. RESULTS: Allelic imbalance was observed in 5 of the 50 (10%) GCs. Specifically, 2 cases (40%) harboured a G deletion and 3 (60%) a G insertion in tumour compared to normal DNA. These five cases were included in the subgroup of 20 GCs (25%) with high level of MSI (MSI-H). A statistically significant association emerged between PAI-1 mutations and MSI-H status (p=0.0073). The frequency of PAI-1 mutations was also evaluated, together with other known target genes, by analysing MSI-H GCs for mutations at selected coding repeats within genes controlling cell growth, apoptosis and DNA repair. Overall, mutation frequency ranged from 56.3% to 5.3%. CONCLUSION: The frequency of PAI-1 mutations here reported in MSI-H GCs is, accordingly, comparable with values obtained for real targets. The relatively high incidence of PAI-1 mutations is suggestive of a positive pressure towards selection in MSI-H GCs.

Preanalytical Procedures for DNA Studies: The Experience of the Interinstitutional Multidisciplinary BioBank (BioBIM).

Preanalytical variables, including the anticoagulants and stabilizing agents, time, storage temperature, and methods of DNA extraction applied to blood samples, may affect quality and quantity of isolated nucleic acids for future genomic applications. Considering the large number of collected samples, standard operating procedure optimization for whole blood preservation before DNA extraction is a crucial step in a biological repository. Moreover, the future application of the biological material may not be known subsequent to its extraction. To define standard operating procedures for whole blood preservation before DNA extraction, we aimed to determine whether different combinations of anticoagulants, blood storage temperatures, and time intervals before storage at -80°C might have an impact on quality and quantity of subsequent extracted DNA. After spectrophotometer quantification, the quality and integrity of DNA were assessed by agarose gel electrophoresis, polymerase chain reaction, and real-time polymerase chain reaction methods. We observed that decrease in DNA recovery during blood storage time was more pronounced at room temperature than at 4°C. Based on our experience, we recommend as anticoagulants of choice sodium citrate and ethylenediaminetetraacetate, whereas sodium citrate theophylline adenosine dipyridamole could represent an alternative choice, excluding a priori lithium heparin and Fluoride-Oxalate. Based on the overall evaluation criteria, we conclude that the procedures necessary to preserve the whole blood before the DNA extraction may have a significant impact on downstream molecular biological applications.

Predictive value of VEGF gene polymorphisms for metastatic colorectal cancer patients receiving first-line treatment including fluorouracil, irinotecan, and bevacizumab.

PURPOSE: The aim of this study is to evaluate the influence of germline vascular endothelial growth factor (VEGF) gene polymorphisms (VGPs) on the efficacy of the anti-VEGF antibody bevacizumab (Bev) in metastatic colorectal cancer (MCRC) patients. METHODS: Forty MCRC patients eligible for a first-line therapy were enrolled in this prospective trial and treated with FOLinate/Fluorouracil/Irinotecan (FOLFIRI) + Bev (male/female = 22:18, age (median) = 61 years). Eight VGPs within the promoter/5'UTR region were evaluated in patient blood samples. Primary endpoint was association between VGPs and median progression-free survival (mPFS). Overall radiological response rate (ORR), overall survival (OS), and toxicity were assessed as secondary outcomes. RESULTS: VGPs -2578, -1512, -1451, -1411, and -460 were in complete linkage disequilibrium and therefore analyzed as haplotype (two variants: Haplo1: A-18 bp insertion-T-4G-C and Haplo2: C-18 bp deletion-C-5G-T, respectively). Seventeen patients Haplo2/Haplo2 had significantly shorter mPFS compared to 23 patients Haplo1/Haplo1 or Haplo1/Haplo2 (mPFS, 9 vs. 15.4 months, respectively, p = 0.02; hazard ratio (HR), 2.64). Also, VGPs -152 (G/G vs. G/A + A/A) and -1154 (G/G vs. G/A + A/A) were significantly associated with PFS (mPFS, 8.9 vs. 15.4 months, p = 0.007; HR, 3.53 and 9.8 vs. 16 months, p = 0.03, HR, 2.32, respectively). In the multivariate analysis including also biochemical variables known to influence prognosis, VGP -1154 retained an independent predictive value for mPFS (G/G over G/A + A/A = HR, 4.43; p = 0.02). With regard to ORR, only VGP -634 was significantly associated with response (G/G vs. G/C + C/C = 64% vs. 14%, p = 0.03). No significant influence on OS and toxicity by the investigated VGPs was observed. CONCLUSIONS: Although these data need to be confirmed in larger trials, investigation of germline VGPs may help identify patients who are more sensitive to anti-VEGF agents.

Concurrent mutation in exons 1 and 2 of the K-ras oncogene in colorectal cancer.

The K-ras gene is frequently mutated in colorectal cancer and has been associated with tumor initiation and progression; approximately 90% of the activating mutations are found in codons 12 and 13 of exon 1 and just under 5% in codon 61 located in exon 2. These mutations determine single aminoacidic substitutions in the GTPase pocket leading to a block of the GTP hydrolytic activity of the K-ras p21 protein, and therefore to its constitutive activation. Point mutations in sites of the K-ras gene, other than codons 12, 13 and 61, and other types of genetic alterations, may occur in a minority of cases, such as in the less frequent cases of double mutations in the K-ras gene. However, all mutations in this gene, even those which occur in non-canonical sites or double mutations, are relevant oncogenic alterations in colorectal cancer and may underlie K-ras pathway hyperactivation. In the present study, we report the case of a patient with colorectal cancer presenting a concurrent point mutation in exons 1 and 2 of the K-ras gene, a GGT to TGT substitution (Glycine to Cysteine) at codon 12, and a GAC to AAC substitution (Aspartic Acid to Asparagine) at codon 57. In addition, we found in the same patient's sample a silent polymorphism at codon 11 (Ala11Ala) of exon 1.

VEGF-A gene promoter polymorphisms and microvascular complications in patients with essential hypertension.

OBJECTIVES: We investigated the possible involvement of vascular endothelial growth factor (VEGF-A) gene promoter polymorphisms in essential hypertension (EH). DESIGN AND METHODS: 1225bp of the VEGF-A gene promoter were screened for polymorphisms using PCR amplification and direct DNA sequence analysis in 62 EH and 62 normotensive (HS) individuals. Circulating VEGF-A levels were determined by immunoassay. RESULTS: -152G/A (p=0.009) and -116G/A (p=0.016) polymorphisms were correlated to hypertension (p<0.05). Median platelet VEGF-A load in EH was 2.10fg/plt. Patients with microvascular complications (MC) had higher platelet VEGF-A load than those without (p=0.005). Multivariate analyses showed that -116 A allele was an independent predictor of microalbuminuria (p=0.014) and increased platelet VEGF-A load (p=0.009) in EH. Platelet VEGF-A load independently predicted MC (p=0.049) in addition to -116G/A polymorphism (p=0.035). CONCLUSIONS: Abnormal regulation of VEGF-A due to polymorphism at position -116 might represent a genetic factor for increased VEGF-A production and MC in EH.

Association between Birt Hogg Dube syndrome and cancer predisposition.

The Birt Hogg Dubé syndrome (BHD) is a rare autosomal dominant genodermatosis predisposing patients to developing fibrofolliculoma, trichodiscoma and acrochordon. The syndrome is caused by germline mutations in the folliculin (FLCN) gene, encoding the folliculin tumor-suppressor protein. Numerous mutations have been described in the FLCN gene, the most frequent occurring within a C8 tract of exon 11. This hypermutability is probably due to a slippage in DNA polymerase during replication, resulting in gains/losses of repeat units, causing cancer predisposition. The main phenotypic manifestations related to this disease are lung cysts, leading to pneumothorax, and a 7-fold increased risk for renal neoplasia, although other neoplastic manifestations have been described in BHD-affected individuals. Of particular interest is the often reported genotype/phenotype correlation between FLCN mutations and risk of colon or breast cancer. This paper describes our current knowledge on the association between BHD and cancer predisposition and briefly summarizes our experience in this field.

TNFA gene promoter polymorphisms and susceptibility to recurrent pregnancy loss in Italian women.

The aim of this study was to investigate the relationship between serum tumor necrosis factor alpha (TNF-alpha) levels and single nucleotide polymorphisms (SNPs) of the TNFA gene promoter (-376G/A, -308G/A, and -238G/A) in 100 Italian Caucasian women with reproductive failure and 100 fertile controls. Molecular analysis of TNFA SNPs showed higher frequencies of -238G allele (P = .028) as well as the presence of a 3-loci haplotype (-376G/-308A/-238G; P = .020) in fertile controls compared to women with reproductive failure. Serum TNF-alpha levels were higher in study women compared to controls ( P = .001). Of interest, the TNFA -376G/-308A/-238G haplotype was an independent predictor of low TNF-alpha levels (P = .021) and miscarriage (P = .023) in multivariate analyses. In conclusion, these findings support the concept of an association of TNFA polymorphisms and recurrent pregnancy loss (RPL). In particular, the TNFA -238GG variant and the TNFA -376G/-308A/-238G haplotype might represent protective factors, probably through reduced TNF-alpha production and/or mediated responses.

A novel K-ras mutation in colorectal cancer. A case report and literature review.

BACKGROUND: Activating mutations in the K-ras oncogene mainly occur in codons 12 and 13 and may be predictive of response to drugs directly linked to the K-ras signaling pathway, such as panitumumab and cetuximab. MATERIALS AND METHODS: K-ras analysis was carried out on DNA extracted from paraffin-embedded tumor samples after microdissection. Exons 1 and 2 were amplified by PCR and then sequenced. RESULTS: A never-reported K-ras mutation (CAG>TAG) determining a premature stop signal at codon 22 (Gln22Stop) was found in a patient with metastatic colorectal cancer. BRAF and p53 were not found to be modified and microsatellite instability was not present. The patient, however, was found to be unresponsive to an anti-EGFR MAb treatment. CONCLUSION: This study is the first report of a novel K-ras truncating mutation in a patient with metastatic colorectal cancer and is also suggestive for the evaluation of alternative pathways to better identify individuals who are likely to benefit from targeted therapies.

Prognostic value of pre-surgical plasma PAI-1 (plasminogen activator inhibitor-1) levels in breast cancer.

INTRODUCTION: Plasminogen activator inhibitor (PAI-1) may have an independent prognostic value in breast cancer (BC). PAI-1 4G/5G polymorphism may have significance for antigen expression. Thus, we analyzed the possible associations between PAI-1 4G/5G polymorphism, plasma PAI-1 levels, and clinicopathological features of breast cancer (BC) patients. PATIENTS AND METHODS: PAI-1 4G/5G polymorphism (both on germinal and tumor DNA) and plasma PAI-1 levels were investigated in 99 BC patients and 50 unrelated healthy women similar for age and menopausal status. RESULTS: No association was found between allele frequencies and clinicopathological features of BC or plasma antigen levels. Plasma PAI-1 levels were higher in BC compared to controls (p=0.002), particularly in patients with large tumors (p<0.001). 5-year follow-up was achieved in 79 patients: 30% had relapsing disease, 63% with positive compared to 37% with negative PAI-1 levels (p<0.05). 5-year relapse-free survival rate of positive PAI-1 was 46% vs., 77% of negative patients (p=0.02). CONCLUSIONS: We may conclude that plasma PAI-1 levels in BC patients could represent a useful prognostic variable for relapse, although PAI-1 polymorphism might not represent a genetic susceptibility factor.

Birt-Hogg-Dubé (BHD) syndrome: report of two novel germline mutations in the folliculin (FLCN) gene.

Molecular analysis of the folliculin (FLCN) gene was performed in four consenting patients from two families with Birt-Hogg-Dubé (BHD) syndrome, showing the occurrence of two frameshift mutations located respectively in exons 5 (802insA) and 9 (1345delAAAG) of the FLCN gene. A novel homozygous sequence variant in the intron 9 (IVS9 +5C>T) was also found. 1345delAAAG was associated with a wide variety of tumors, including stomach, colon, breast and parotid cancer. Conversely, the family carrying 802insA only had clinical evidence of dermatological lesions. These findings further suggest the relevance of exon 9 mutations in cancer predisposition for BHD.

c-erbB-2 protein level in tissue and sera of breast cancer patients: a possibly useful clinical correlation.

AIMS AND BACKGROUND: The aims of this study were to assess the clinical utility of circulating preoperative HER-2 extracellular domain p105 detected by enzyme immunoassay (ELISA), to compare the tissue expression of HER-2/neu determined by immunohistochemistry (IHC), to correlate prognostic factors including tumor size, nodal involvement, and hormone receptor status, and to analyze the prognostic significance of the marker in relation to clinical outcome as measured by disease-free and overall survival. METHODS: In this study, we enrolled 108 consecutive patients with breast carcinoma, and obtained serum samples and frozen tumor tissues. We compared them with 57 women with fibroadenoma and 63 healthy women as controls. RESULTS: Univariate ANOVA analysis showed no relationship between HER-2/neu in tissue and serum. Preoperative serum levels of p105 were significantly higher in breast cancer patients than in women with benign disease or healthy women. Concerning the correlation between p105, HER-2/neu tissue expression, and the other prognostic factors, a statistically significant correlation between high serum p105 levels and ER-negative status in breast cancer patients was found. Kaplan-Meier analysis confirmed that patients with positive HER-2/neu tissue expression had a significantly shorter survival than those with negative expression. Analysis with the Cox model demonstrated that tumor size was the only significant independent prognostic factor. CONCLUSIONS: This research failed to demonstrate a relationship between preoperative tissue overexpression and circulating HER-2/neu, suggesting that p105 does not represent a valid alternative to predict a worsened prognosis in breast cancer, but it could be a diagnostic marker to discriminate healthy subjects from breast cancer patients.