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

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

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

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

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

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

Anti-Tumor Effects of <em>Ganoderma lucidum</em> (Reishi) in Inflammatory Breast Cancer in <em>In Vivo</em> and <em>In Vitro</em> Models

<div><p>The medicinal mushroom <i>Ganoderma lucidum</i> (Reishi) was tested as a potential therapeutic for Inflammatory Breast Cancer (IBC) using <i>in vivo</i> and <i>in vitro</i> IBC models. IBC is a lethal and aggressive form of breast cancer that manifests itself without a typical tumor mass. Studies show that IBC tissue biopsies overexpress E-cadherin and the eukaryotic initiation factor 4GI (eIF4GI), two proteins that are partially responsible for the unique pathological properties of this disease. IBC is treated with a multimodal approach that includes non-targeted systemic chemotherapy, surgery, and radiation. Because of its non-toxic and selective anti-cancer activity, medicinal mushroom extracts have received attention for their use in cancer therapy. Our previous studies demonstrate these selective anti-cancer effects of Reishi, where IBC cell viability and invasion, as well as the expression of key IBC molecules, including eIF4G is compromised. Thus, herein we define the mechanistic effects of Reishi focusing on the phosphoinositide-3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway, a regulator of cell survival and growth. The present study demonstrates that Reishi treated IBC SUM-149 cells have reduced expression of mTOR downstream effectors at early treatment times, as we observe reduced eIF4G levels coupled with increased levels of eIF4E bound to 4E-BP, with consequential protein synthesis reduction. Severe combined immunodeficient mice injected with IBC cells treated with Reishi for 13 weeks show reduced tumor growth and weight by ∼50%, and Reishi treated tumors showed reduced expression of E-cadherin, mTOR, eIF4G, and p70S6K, and activity of extracellular regulated kinase (ERK1/2). Our results provide evidence that Reishi suppresses protein synthesis and tumor growth by affecting survival and proliferative signaling pathways that act on translation, suggesting that Reishi is a potential natural therapeutic for breast and other cancers.</p> </div

Reishi decreases EIF4F complex levels and protein synthesis in IBC cells.

<p>A. SUM-149 and MCF10A cells were treated with vehicle (0mg/mL, SV or MV) or 0.5mg/mL Reishi (SR or MR) for 24 hours before lysis. Western blot analyses were completed for total eIF4G, eIF4A, eIF4E, and 4E-BP1 obtained from m7GTP pull-down lysates and whole cell lysates. B. Graph represents quantification of eIF4F complex as in Dumstorf et al., 2010 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057431#pone.0057431-Dumstorf1" target="_blank">[25]</a>, where eIF4G normalized to eIF4E is divided by 4E-BP1 normalized to eIF4E [(eIF4G/eIF4E)/(4E-BP1/eIF4E)]. Number of biological replicates (n) varies among experiments (SUM-149; n = 3, MCF10A; n = 1). Columns show means ± SEM of integrated density units, shown relative to vehicle controls. Reishi significantly reduces eIF4F complex assembly at *<i>P</i><0.02 in IBC SUM-149 cells. C. 1×10⁵ cells (SUM-149 and MCF10A) were seeded per well in a six well plate and treated with vehicle or 0.5 mg/mL Reishi for 24 hours. The treatment was removed and the cells were re-incubated for 30 minutes in methionine/cysteine - free DMEM. L-[³⁵S] Methionine and L-[³⁵S] Cysteine (2 mCi/mL) +2% FBS was then added to the cultures. Total cell lysates prepared in NP-40 lysis buffer were analyzed for incorporated radioactivity in trichloroacetic acid precipitates. Data are expressed as means ± SEM of duplicate determinations. Experiment was repeated three times. Reishi significantly (*<i>P</i><0.05) reduces protein synthesis by 48%.</p

Reishi decreases the expression of PI3K/AKT signaling pathway genes and of mTORC1 effectors.

<p>A. Total SUM-149 cell RNA extraction was performed from three different experimental plates treated with 0 mg/mL (n = 3/vehicle) or 0.5 mg/mL Reishi (n = 3/treatment) for 3 hours. RT² PCR arrays designed to profile the expression of PI3K/AKT pathway-specific genes were used according to manufacturer’s instructions (SA Biosciences). Volcano plot shows the effects on gene expression analyzed at −1.4≥1.4 log₂-fold change (dashed lines). Down-regulated genes are to the left of the vertical black line while up-regulated genes are to the right. Statistically significant (<i>P</i><0.05) regulated genes are above the horizontal black line. B. SUM-149 cells were grown in 5% FBS media for 24 hours prior to treatment with vehicle (0 mg/mL) or Reishi extract (0.5 mg/mL) for 2, 4, and 6 hours before lysis. Equal amount of protein from each sample was used for Western blot analysis with antibodies against mTORC1 effector proteins. C. Columns represent means ±SEM of integrated density units of protein, normalized to β-actin levels and shown relative to vehicle controls (without Reishi treatment). Statistically significant differences are shown at *<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.0001.</p