Human Cytomegalovirus Infection Enhances NF-κB/p65 Signaling in Inflammatory Breast Cancer Patients

<div><p>Human Cytomegalovirus (HCMV) is an endemic herpes virus that re-emerges in cancer patients enhancing oncogenic potential. Recent studies have shown that HCMV infection is associated with certain types of cancer morbidity such as glioblastoma. Although HCMV has been detected in breast cancer tissues, its role, if any, in the etiology of specific forms of breast cancer has not been investigated. In the present study we investigated the presence of HCMV infection in inflammatory breast cancer (IBC), a rapidly progressing form of breast cancer characterized by specific molecular signature. We screened for anti-CMV IgG antibodies in peripheral blood of 49 non-IBC invasive ductal carcinoma (IDC) and 28 IBC patients. In addition, we screened for HCMV-DNA in postsurgical cancer and non-cancer breast tissues of non-IBC and IBC patients. We also tested whether HCMV infection can modulate the expression and activation of transcriptional factor NF-κB/p65, a hallmark of IBC. Our results reveal that IBC patients are characterized by a statistically significant increase in HCMV IgG antibody titers compared to non-IBC patients. HCMV-DNA was significantly detected in cancer tissues than in the adjacent non-carcinoma tissues of IBC and IDC, and IBC cancer tissues were significantly more infected with HCMV-DNA compared to IDC. Further, HCMV sequence analysis detected different HCMV strains in IBC patients tissues, but not in the IDC specimens. Moreover, HCMV-infected IBC cancer tissues were found to be enhanced in NF-κB/p65 signaling compared to non-IBC patients. The present results demonstrated a correlation between HCMV infection and IBC. Etiology and causality of HCMV infection with IBC now needs to be rigorously examined.</p> </div

Comparison of detected of HCMV-DNA in breast carcinoma tissues of non-IBC and IBC patients.

<p>Significant p value calculated Fisher’s exact test.</p><p>n: number of patients.</p

Clinical and pathological characterization of non-IBC versus IBC patients.

<p>Data are reported as means± SD</p>a<p>Student’s t-test.</p>b<p>Fisher’s exact test.</p><p>NA =  not available</p>*<p>Significant p value calculated by:</p>†<p>P value was calculated between <4 including 0 and ≥4.</p

Agarose gel electrophoresis for nested PCR analysis of HCMV-DNA.

<p>Samples were considered positive when a band of 293bp was detected. (<b>A</b>) Representatives of non-IBC carcinoma tissue samples: lanes 1–7 display positive-HCMV; lanes 8–10 represent negative samples. (<b>B</b>) Representatives of IBC carcinoma tissue samples: lanes from 1–4, 6–7, 9–10 display positive HCMV; lanes 5 and 8 represent negative samples. Lanes +C represent HCMV positive control; lanes -C represent HCMV negative control. (<b>C</b>) Nucleotide sequence alignment from the purified nested PCR products. *represents nucleotide identity.</p

HCMV infection in non-IBC (n = 49) and IBC (n = 28) patients.

<p>(<b>A</b>) Bars represents percentage of serological diagnosis of HCMV-IgG within non-IBC (<i>p = </i>0.001) and IBC (<i>p</i> = 0.001) patient groups. *Indicates a significant <i>p</i> value as determined by Fisher’s exact test. (<b>B</b>) Serum titers of HCMV-IgG in non-IBC versus IBC in HCMV infected patients. Each dot represents one patient. Antibody titer of HCMV was significantly higher (<i>p</i> = 0.04) in IBC than in non-IBC. *Indicates a significant <i>p</i> value as determined by Student’s t-test.</p

Expression of phospho-NF-κB p65 (pNF-κB p65 ^{(Ser 276)}) in randomly selected non-IBC and IBC cancer tissue.

<p>(<b>A</b>) Immunoblot analysis showing equal level of expression of pNF-κB p65 ^{(Ser 276)} (65 kDa) in tissue lysates of non-infected and HCMV-infected non-IBC patients; β-actin (45 kDa) was used as loading control. (<b>B</b>) Immunoblot analysis showing low expression level of phospho-NF-κB p65 (Ser²⁷⁶) in the non-infected tissue lysates of IBC patients and greater of expression of pNF-κB p65 ^{(Ser 276)} in the tissue lyastes of HCMV-infected IBC patients. Immunoblot results are representative of at least 3 independent experiments. (<b>C</b>) Bars represent relative densities (mean ± S.D.) of pNF-κB p65 ^{(Ser 276)} as quantified by imageJ software. Statistical analysis revealed no significant differences in the expression of pNF-κB p65 ^{(Ser 276)} in tissue lysates of non-infected compared with HCMV-infected patients in non-IBC and IBC patient groups. (<b>D</b>) Photomicrographs are representative of immunohistochemical staining of pNF-κB p65 ^{(Ser 276)} in non-IBC and IBC paraffin tissue sections of patients not infected and infected with HCMV. There was low expression of pNF-κB p65 ^{(Ser 276)} in non-infected tissues of non-IBC and IBC. But pNF-κB p65 ^{(Ser 276)} was moderately expressed in non-IBC and IBC HCMV-infected patients (original magnifications 400X). *Indicates significant <i>p</i> value as determined by Student’s t-test.</p

Expression of total NF-κB/p65 in randomly selected non-IBC and IBC cancer tissues.

<p>(<b>A</b>) Immunoblot analysis showing the expression of NF-κB/p65 (65 kDa) in tissue lysates of non-infected and HCMV-infected non-IBC patients; β-actin (45 kDa) was used as loading control. (<b>B</b>) Immunoblot analysis showing low level of expression of NF-κB/p65 in non-infected tissue lysates of IBC patients and high level of expression of NF-κB/p65 in the tissue lyastes of HCMV-infected IBC patients. Immunoblot results are representative of 3 independent experiments. (<b>C</b>) Bars represent relative densities of total NF-κB/p65 (mean ± S.D.) as quantified by imageJ software, showing significant increases in the level of expression of total NF-κB/p65 in tissue lysates of HCMV-infected IBC patients compared with tissue lysates of non-infected IBC patients. (<b>D</b>) Photomicrographs representative of immunohistochemistry staining of NF-κB/p65 (brown color) in non-IBC and IBC paraffin tissue sections with and without HCMV infection. Non-IBC intraductal carcinoma tissues showed moderate level of expression of NF-κB/p65 by non-infected and HCMV-infected tissues. On the other hand, NF-κB/p65 was moderately expressed by non-infected IBC tissue sections and over-expressed by IBC tissues infected with HCMV, (original magnification 400X). *Indicates significant <i>p</i> value as determined by Student’s t-test.</p

Assessing the Awareness of Egyptian Medical Students about Responsible Conduct of Research and Research Ethics: Impact of an Educational Campaign

<p>This is a quasi-experimental pre-post assessment study utilizing an anonymous self-administered questionnaire to assess Egyptian medical students’ awareness about responsible conduct of research (RCR) and research ethics. Students’ were assessed before and after an RCR awareness campaign. Our results showed that most of the pre-campaign respondents were not familiar with the basic principles and terms of RCR. An increase in the awareness about RCR across all discussed topics was noted following the campaign. We concluded that an educational awareness campaign is effective in increasing medical students’ awareness about RCR and should be incorporated into current medical school curricula in Egypt.</p