Metabolic and anthropometric changes in early breast cancer patients receiving adjuvant therapy

Weight gain and metabolic changes have been related to survival of early breast cancer patients (EBC). ''However, factors influencing metabolism post-diagnosis are not fully understood. We measured anthropometric [body mass index (BMI), body weight, waist and hip circumferences, and waist-to-hip ratio] and metabolic (levels of insulin, glucose, H1Ac, total, HDL, and LDL cholesterol, triglycerides, and the homeostasis model assessment score [HOMA]) parameters in 433 pre- and post-menopausal women with EBC at diagnosis and 3, 6, 9, 12, and 24 months thereafter. At diagnosis, compared with post-menopausal women, pre-menopausal patients were more likely to be leaner and to have a lower BMI, smaller waist and hip circumferences, and waist-to-hip ratio. They had also lower glucose, HbA1c, and triglyceride levels and a lower HOMA score. Furthermore, they were more likely to have an estrogen- and/or progesterone-positive tumor and a higher proliferating breast cancer. During the first two post-diagnosis years, all women showed a significant increase of weight (+0.72 kg/year, P < 0.001), waist circumference (+1.53 cm/year, P < 0.001), and plasma levels of LDL cholesterol (+5.4 mg/dl per year, P = 0.045) and triglycerides (+10.73 mg/dl per year, P = 0.017). In patients receiving chemotherapy only, there was a significant increase in hip circumference (+3.16 cm/year, P < 0.001) and plasma cholesterol levels (+21.26 mg/dl per year, P < 0.001). We showed that weight, body fat distribution, and lipid profile changed in EBC patients receiving adjuvant therapy. These changes occurred during the first 2 years after diagnosis and were not specifically related to chemotherapy, menopausal status, or initial body weight

Cancer-associated CD43 glycoforms as target of immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy

Impairment of T cell development and acute inflammatory response in HIV-1 Tat transgenic mice

Immune activation and chronic inflammation are hallmark features of HIV infection causing T-cell depletion and cellular immune dysfunction in AIDS. Here, we addressed the issue whether HIV-1 Tat could affect T cell development and acute inflammatory response by generating a transgenic mouse expressing Tat in lymphoid tissue. Tat-Tg mice showed thymus atrophy and the maturation block from DN4 to DP thymic subpopulations, resulting in CD4(+) and CD8(+) T cells depletion in peripheral blood. In Tat-positive thymus, we observed the increased p65/NF-κB activity and deregulated expression of cytokines/chemokines and microRNA-181a-1, which are involved in T-lymphopoiesis. Upon LPS intraperitoneal injection, Tat-Tg mice developed an abnormal acute inflammatory response, which was characterized by enhanced lethality and production of inflammatory cytokines. Based on these findings, Tat-Tg mouse could represent an animal model for testing adjunctive therapies of HIV-1-associated inflammation and immune deregulation

Eukaryotic Initiation Factor 4H Is under Transcriptional Control of p65/NF-κB

<div><p>Protein synthesis is mainly regulated at the initiation step, allowing the fast, reversible and spatial control of gene expression. Initiation of protein synthesis requires at least 13 translation initiation factors to assemble the 80S ribosomal initiation complex. Loss of translation control may result in cell malignant transformation. Here, we asked whether translational initiation factors could be regulated by NF-κB transcription factor, a major regulator of genes involved in cell proliferation, survival, and inflammatory response. We show that the p65 subunit of NF-κB activates the transcription of eIF4H gene, which is the regulatory subunit of eIF4A, the most relevant RNA helicase in translation initiation. The p65-dependent transcriptional activation of eIF4H increased the eIF4H protein content augmenting the rate of global protein synthesis. In this context, our results provide novel insights into protein synthesis regulation in response to NF-κB activation signalling, suggesting a transcription-translation coupled mechanism of control.</p></div

Mass Spectrometry-Based Identification Of The Tumor Antigen UN1 as the Transmembrane CD43 Sialoglycoprotein*

The UN1 monoclonal antibody recognized the UN1 antigen as a heavily sialylated and O-glycosylated protein with the apparent molecular weight of 100–120 kDa; this antigen was peculiarly expressed in fetal tissues and several cancer tissues, including leukemic T cells, breast, and colon carcinomas. However, the lack of primary structure information has limited further investigation on the role of the UN1 antigen in neoplastic transformation

Cancer-associated CD43 glycoforms as target of immunotherapy

CD43 is a sialoglycosylated membrane protein that is involved in cell proliferation and differentiation. CD43 glycoforms that are recognized by the UN1 monoclonal antibody (mAb) were expressed in lymphoblastoid T-cell lines and solid tumors, such as breast, colon, gastric, and squamous cell lung carcinomas, while unexpressed in the normal counterparts. The cancer association of UN1/CD43 epitope suggested the possibility to use the UN1 mAb for tumor diagnosis and therapy. In this study, we show that the UN1 mAb was endowed with antitumor activity in vivo because its passive transfer inhibited the growth of UN1-positive HPB-ALL lymphoblastoid T cells in mice. Furthermore, we demonstrate that tumor inhibition was due to UN1 mAb-dependent natural killer-mediated cytotoxicity. By screening a phage-displayed random peptide library, we identified the phagotope 2/165 as a mimotope of the UN1 antigen, as it harbored a peptide sequence that was specifically recognized by the UN1 mAb and inhibited the binding of the UN1 mAb to UN1-positive tumor cells. On the basis of sequence homology with the extracellular region of CD43 (amino acids 64 to 83), the 2/165 peptide sequence was likely mimicking the protein core of the UN1/CD43 epitope. When used as vaccine in mice, the 2/165 phagotope raised antibodies against the UN1/CD43 antigen, indicating that the 2/165 phagotope mimicked the UN1 antigen structure, and could represent a novel immunogen for cancer immunotherapy. These findings support the feasibility of using monoclonal antibodies to identify cancer-associated mimotopes for immunotherapy

TNF-α induces the recruitment of p65 to eIF4H promoter.

<p>(A) HeLa cells (5×10⁶) were transfected with siRNA control or siRNA p65 (200 pmol). Forty-eight hours post-transfection, cells were 45 min-treated with TNF-α (20 ng/mL), or left untreated. Total RNA was extracted and analysed by qRT-PCR to measure the expression of eIF4H and eIF2S3. Values (mean ± SD, n = 3) are shown. Statistically significant differences between the samples are shown according to Student's <i>t</i>-test (p≤0.01). (B) HeLa cells (3×10⁷) were treated with TNF-α (20 ng/mL) for the indicated time, or left untreated. Chromatin was immunoprecipitated with anti-p65, anti-p50, anti-RelB, anti-c-Rel or IgG, and ChIP eluates were analysed by qRT-PCR.</p

p65-dependent transcriptional activation of eIF4H.

<p>(A) HeLa cells (5×10⁶) were transfected with pRc/CMV-p65 or pRc/CMV-empty vector (5 µg). Forty-eight hours post-transfection, total RNA was extracted and analysed by qRT-PCR to evaluate the expression of the indicated eIF genes. Values (mean ± SD, n = 5) are shown. The asterisk indicates a statistically significant difference between pRc/CMV-p65 and empty vector according to the Student's <i>t</i>-test (<i>p</i>≤0.01). (B) HeLa cells (5×10⁶) were transfected with siRNA control or siRNA p65 (200 pmol). Forty-eight hours post-transfection, total RNA was extracted and analysed by qRT-PCR for the expression of the indicated eIF genes. Values (mean ± SD, n = 5) are shown. The asterisk indicates a statistically significant difference between siRNA p65 and siRNA control according to the Student's <i>t</i>-test (<i>p</i> ≤0.01). (C) Wild type and p65^−/− MEFs (3×10⁵) were lysed, and total RNA was analysed by qRT-PCR for the expression of eIF4H gene. (D) Total cell extracts (20µg) of wild type and p65^−/− MEFs (3×10⁵) were separated by 12% SDS-PAGE and analysed by western blotting using anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the wild type taken as 1. (E) Nuclear extracts of wild type and p65^−/− MEFs (5×10⁶ cells) were analysed for the binding activity of the indicated NF-κB subunits to the NF-κB double-stranded oligonucleotide, as measured by ELISA EMSA using the NF-κB Transcription Factor ELISA assay kit (Cayman). (F) Total RNA from tumour cell lines (MDA-MB-231, MCF-7, SH-SY5Y, U251, D54, MC3, DeFew) (3×10⁵ cells) was analysed by qRT-PCR for the expression of eIF4H gene. (G) Whole protein cell extracts (20µg) of the indicated tumour cell lines were separated by 12% SDS–PAGE and analysed by western blotting using anti-eIF4H, or anti-γ-Tubulin antibodies. Densitometry values (D) of the bands were expressed as fold increase above the MDA-MB-231 cells taken as 1. (H) Nuclear extracts of the indicated tumor cell lines (5×10⁶ cells) were analysed for the p65 binding to the NF-κB double-stranded oligonucleotide, using the NF-κB Transcription Factor ELISA assay kit (Cayman).</p