Relevance Analysis of TPM2 and Clinicopathological Characteristics in Breast Cancer.

Background: The function of tropomyosin 2 (TPM2) in breast cancer is still far understudied. In this study, we aim to explore the roles of TPM2 in breast cancer progression. Methods: This research included 155 breast cancer tissues. The expression of TPM2 was analyzed by immunohistochemical staining and grading. The mRNA expression of TPM2 in pan-cancer was analyzed with The Cancer Genome Atlas (TCGA) data plate form. The differential expression of TPM2 protein and the differential promoter methylation level of TPM2 between breast cancer tissues and normal breast tissues were analyzed by the UALCAN online database. The relationship between TPM2 and signaling pathways was interpreted by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) pathway enrichment analyses. The survival curve of TPM2 was analyzed across the Kaplan-Meier plotter online database. Furthermore, the relationship between TPM2 expression and infiltrating macrophages was validated through in vitro co-culture experiments. Results: TPM2 expression was significantly down-regulated in breast cancer samples. In addition, TPM2 expression was correlated with lymph node metastasis and high-grade histopathological morphology. The receiver operating characteristic (ROC) curve indicated that TPM2 expression could well distinguish between normal breast tissue and breast cancer tissue. TPM2 may have potential value in breast cancer diagnosis. Bioinformatics analysis illustrated that TPM2 was mainly involved in extracellular matrix organization, collagen fibril organization, cell junction assembly, focal adhesion, cAMP signaling pathway, estrogen signaling pathway, Wnt signaling pathway, and adaptive immune system. TPM2 expression was correlated with immune infiltrating cells and immune checkpoint molecules. Our in vitro co-culture experiments showed that the M2 macrophages could upregulate the expression of TPM2. Conclusion: TPM2 may play key roles in breast cancer occurrence and development, especially in cancer metastasis. TPM2 may be a potential biomarker for breast cancer diagnosis.

Yixin-Fumai granules improve sick sinus syndrome in aging mice through Nrf-2/HO-1 pathway: A new target for sick sinus syndrome.

ETHNOPHARMACOLOGICAL RELEVANCE: Yixin-Fumai granules (YXFMs)-composed of Ginseng quinquefolium (L.) Alph. Wood, Ophiopogon japonicus (Thunb.) Ker Gawl, Schisandra arisanensis Hayata, Astragalus aaronsohnianus Eig, Salvia cryptantha Montbret & Aucher ex Benth, and Ligusticum striatum DC-are compound granules used in traditional Chinese medicine to increase heart rate and thus treat bradyarrhythmia. It may be effective in treating sick sinus syndrome (SSS). AIM: To observe the effect of YXFMs on aging-induced SSS in mice and explore whether this effect is related to the Nrf-2/HO-1 signaling pathway. MATERIALS AND METHODS: Mice with a significant decrease in the heart rate due to natural aging were selected to construct an SSS model. After the mice were administered YXFMs, the damage to their sinoartrial node (SAN) was assessed through electrocardiography, Masson's trichrome staining, and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL). Dihydroethidium staining and immunofluorescence staining were used to assay reactive oxygen species (ROS) content and HCN4, respectively. Moreover, to observe the effects of YXFMs in vitro, the HL-1 cell line, derived from mouse atrial myocytes, was used to simulate SAN pacemaker cells, with H2O2 used as the cellular oxidative stress (OS) inducer. 2,7-Dichlorodihydrofluorescein diacetate staining was used to assay ROS content, whereas immunofluorescence staining and Western blotting were used to elucidate the related protein expression. Finally, mice were injected the Nrf-2 inhibitor ML385 to reversely verify the effects of YXFMs. RESULTS: In our in vivo experiments, YXFMs significantly inhibited aging-induced SSS, shortened the R-R interval, increased heart rate, alleviated fibrosis, reduced apoptosis rate and ROS content, and promote HCN4 expression in the SAN. In our in vitro experiments, YXFMs significantly inhibited H2O2-induced cell peroxidation damage, promoted Nrf-2 activation and nuclear metastasis, increased HO-1 expression- thereby inhibiting ROS accumulation-and finally, upregulated HCN4 expression through the inhibition of histone deacetylase 4 (HDAC4) expression and its nuclear metastasis. Finally, injection of the Nrf-2 inhibitor ML385 after YXFMs administration inhibited their protective effect in the mice. CONCLUSION: Here, we elaborated on the relationship between aging-induced SSS and the Nrf-2/HO-1 pathway for the first time and proposed that YXFMs improve SSS via the Nrf-2/HO-1 axis. Specifically, YXFMs promoted Nrf-2 activation and plasma-nuclear transfer to enhance HO-1 expression via the Nrf-2/HO-1 axis. This inhibited OS and reduced ROS accumulation in the SAN, and then, through the ROS/HDAC4 axis, reduced HDAC4 expression and plasma-nuclear transfer. Thereby, the OS-induced HCN4 loss in the SAN was inhibited-improving the function of If channel and thus producing SAN protection effect against SSS and improving the heart rate and R-R interval. In the future, we plan to use bioinformatics analysis technology to execute the next step of our research, namely to determine the effect of isolated, purified components of YXFMs in SSS, to increase its efficiency and reduce the toxicity of YXFMs.

Nek9,a sensitive immunohistochemical marker for Schwannian, melanocytic and myogenic tumours.

AIMS: In our previous study, striking Nek9 staining was observed in peripheral nerves for the first time. Therefore, in the current study, we aimed to detect Nek9 expression in peripheral nerve sheath tumours, melanocytic tumours and their mimics. METHODS: The expression of Nek9 was analysed in 234 mesenchymal tumours including schwannoma, neurofibroma, malignant peripheral nerve sheath tumour (MPNST), melanoma and their mimics adopting immunohistochemistry. In addition, S-100 and SOX10 were detected in all tumours. RESULTS: The results revealed an intense and diffuse staining of Nek9 in all schwannomas (30/30) and melanomas (20/20). The neurofibromas (86%, 19/22) and MPNSTs (76%, 18/21) showed a high frequency of positive Nek9 staining. Nek9 showed a comparable sensitivity to S-100, and better sensitivity and less specificity than that of SOX10. Among the histological mimics, Nek9 was only strongly and diffusely expressed in rhabdomyosarcomas (RSs) (97%,37/38) while negatively stained in most of the other tumours. It was noted that Nek9 immunoresponse was more diffuse than that of MyoD1 and myogenin in RS. CONCLUSIONS: In summary, Nek9 has a good sensitivity in the diagnosis of tumours with Schwannian, melanocytic and skeletal muscle differentiations. The immunohistochemical analysis of Nek9 expression may be helpful in the diagnosis and differential diagnosis of the aforementioned tumours.

Differences in pathologic characteristics between ductal carcinoma in situ (DCIS), DCIS with microinvasion and DCIS with invasive ductal carcinoma.

In order to further our understanding of pathologic features in various ductal carcinoma in situ (DCIS) related breast ductal cancers, including DCIS, DCIS with microinvasion (DCIS-Mi) and DCIS with invasive ductal carcinoma (DCIS-IDC), a retrospective study including 453 cases of DCIS, 88 cases of DCIS-Mi, and 269 cases of DCIS-IDC was conducted. Statistical analysis showed significant pathological differences were found in DCIS, DCIS-Mi, and DCIS-IDC. Compared with DCIS, DCIS-IDC was significantly more associated with high nuclear grade, large tumor size, high Ki67 index, and lymph node metastasis (all P<0.05). Higher expression of steroid receptors was shown in DCIS-IDC than in DCIS (all P<0.05), but the status of HER2 between the two groups was similar (P=0.269). Compared with DCIS, DCIS-Mi was significantly more associated with high nuclear grade, large tumor size, comedonecrosis, absence of steroid receptors, HER2 overexpression, and high Ki67 index (all P<0.05). These features remain consistently even when compared with DCIS-IDC. According to the immunohistochemistry surrogate classification, the dominant types of DCIS and DCIS-IDC were luminal types (luminal A and luminal B, respectively), while the dominant type of DCIS-Mi was HER2 overexpression. These findings suggest that DCIS-Mi represents a distinct entity, and DCIS with features including high nuclear grade, large tumor size, comedonecrosis, steroid receptors negativity, HER2 positivity, and high Ki67 expression was more likely to have microinvasion than DCIS without these features.

MicroRNA-455-3p promotes invasion and migration in triple negative breast cancer by targeting tumor suppressor EI24.

Lacking of treatment methods for the patients with triple negative breast cancer (TNBC) underscores the pivotal needs to further understand its biology as well as to find better biomarkers and develop novel therapeutic strategies. Increasing evidences support that aberrantly expressed microRNAs (miRNAs) are involved in tumorigenesis and may serve as biomarkers for diagnostic and prognostic purposes of various cancers. In current study, we found that miR-455-3p and miR-196a-5p were intensively overexpressed in TNBC compared with the hormone receptor (HR) positive breast cancer whereas miR-425-5p was down-regulated by miRNA microarray analysis. qRT-PCR analysis confirmed that the expression of miR-455-3p in TNBC cell lines MDA-MB-231 and MDA-MB-468 was higher than that in HR positive breast cancer cell line MCF-7(p<0.01). Functional experiments in vitro showed that miR-455-3p enhanced cell proliferative, invasive and migrational abilities in TNBC cell lines. miRNA targets prediction showed SMAD2, LTBR and etoposide induced 2.4 (EI24) were potential target genes of miR-455-3p, and then it was confirmed by qRT-PCR assay. Dual luciferase reporter assay showed the specific binding of miR-455-3p to 3' UTR of EI24 in TNBC. Then we found miR-455-3p inhibited the EI24 expression at the levels of mRNA and protein. Through small interfering RNA (siRNA) targeting EI24 gene, there were strengthened capabilities of invasion and migration of TNBC cells, and increased expression of EI24 had the inverse effects. In conclusion, the data suggest that miRNA455-3p promotes invasion and migration by targeting tumor suppressor EI24 and might be a potential prognostic biomarker and therapeutic target in TNBC.

Evaluation of CCND1 amplification and CyclinD1 expression: diffuse and strong staining of CyclinD1 could have same predictive roles as CCND1 amplification in ER positive breast cancers.

CCND1 is amplified in around 10-20% of primary breast cancers and preferentially occurs in ER positive tumors. Though CCND1 amplification was reported predicting poor response of adjuvant tamoxifen treatment and poor prognosis in ER positive breast cancers, there were controversial data regarding the predicting value of CyclinD1 protein overexpression. In this study, we detected CyclinD1 expression using immunohistochemistry and CCND1 gene copy number using fluorescence in situ hybridization (FISH) in 355 invasive breast cancers with foci ductal carcinoma in situ (DCIS). CCND1 amplification was founded in 52 (14.6%) cases all of which showed moderate to strong CyclinD1 expression. However, majority of CCND1- tumors exhibited mild to moderate CyclinD1 staining. There were identical alterations in DCIS and the invasive lesions within the same tumor. CCND1 amplification was positively correlated with ER, PR and lymph node status (P<0.001) while negatively correlated with HER-2 amplification and p53 status (P<0.05). The majority of the CCND1 amplification/high CyclinD1 breast cancers were luminal B type while basal-like type often lost the expression of this protein. The ROC curve analysis showed that a cut-off point at which the immunostaining score of CyclinD1 is 6.5 could predict CCND1 gene amplification in breast cancer. This study indicated loss expression of CyclinD1 might be an important event in the tumorigenesis in basal-like breast cancers. Further, we confirmed an optimal cut-off point of immunostaining scores of CyclinD1 protein which could be used to predict the status of CCND1 gene and identify a subgroup of ER positive breast cancers with poor response to endocrine agents.

Breast Cancer Invasion and Metastasis by mPRα Through the PI3K/Akt Signaling Pathway.

Invasive breast cancer is the most common type of malignancy in women worldwide. However, the mechanism responsible for breast cancer metastasis is still unclear and needs further illustration. It has been proven that matrix metallopeptidase 9 (MMP-9) promotes metastasis of the cancer cells. However, the interaction between mPRα and MMP-9 has not been studied. Therefore, in the present research, the effect of MMP-9 on the malignant progression of invasive breast cancer promoted by membrane progesterone receptorα (mPRα) was investigated. The results showed that the protein expression of mPRα, p-Akt and MMP-9 increased in the cancerous tissues compared to that of the noncancerous breast tissue. Furthermore, a positive correlation was found between mPRα and C-erbB-2, as well as the number of involved local lymph nodes. On the other hand, a negative correlation was observed between mPRα and estrogen receptors (ER) along with progesterone receptors (PR). Similarly, a positive association was found between MMP-9 and the number of involved local lymph nodes. Besides, the high expression of MMP-9 also had a positive correlation with the tumor size. However, the high level of MMP-9 had a negative correlation with ER and PR. In addition, there was a positive correlation between mPRα and p-Akt together with MMP-9. The results confirm that mPRα was a major marker of harmful prognosis and it promoted the expression of MMP-9 during invasion to the local lymph nodes through the pathway of PI3K/Akt. The present study provided a novel therapeutic strategy to inhibit breast cancer growth by preventing mPRα signaling pathway.

Overexpression of Trps1 contributes to tumor angiogenesis and poor prognosis of human osteosarcoma.

BACKGROUND: Trichorhinophalangeal syndrome 1 (Trps1) gene is a member of GATA transcription factor family and has an important function in tumorigenesis and progression. However, there are rare studies on its roles in carcinogenesis and prognostic significance in human osteosarcoma. METHODS: The expression of Trps1 was detected by immunohistochemistry, and MVD was evaluated to determine the amounts of microvessels by counting CD31-positive endothelial cells. RESULTS: Of the 74 cases that underwent study, Trps1-positive cases were 24. And it was associated with MVD significantly (P = 0.008). The data also exhibited more cases of remote metastasis (P = 0.013) and higher Enneking stage (P = 0.017) in Trps1-positive group compared to Trps1-negative group. Univariate analysis revealed that distant metastasis, MVD and Trps1 expression were associated with a lower 3-year overall survival rate and disease-free survival rate (P = 0.003, and P = 0.012 respectively). Furthermore, Trps1 and distant metastasis retained their significant prognostic effects on patients survival rate by multivariate analysis (P < 0.05). CONCLUSIONS: Trps1 plays a crucial role in osteosarcoma angiogenesis, metastasis and clinical surgical stage. Trps1 can be a novel promising prognostic marker and therapeutic target, and antiangiogenic therapy which targets Trps1 molecule in patients with osteosarcoma may lead to improved prognosis and longer-term survival.

[Evaluation of c-myc and CCNE2 amplification in breast cancer with quantitative multi-gene fluorescence in-situ hybridization].

OBJECTIVE: To investigate c-myc and CCNE2 gene amplifications and their relationship in breast cancer. METHODS: Sixty-six infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ components collected from January 2005 to December 2007 were selected for tissue microarray and quantitative multi-gene FISH for c-myc and CCNE2 gene amplification, and the relationship with the clinicopathologic features was analyzed. RESULTS: Of the 66 cases, 18 (27.3%) showed c-myc amplification and 23 (34.8%) showed CCNE2 amplification. A strong correlation was found between c-myc and CCNE2 amplification (P < 0.01). The breast cancers showing c-myc and CCNE2 amplifications were all aneuploidy, and were HER2 positive (P < 0.05). Tumors with c-myc amplification also showed higher Ki-67 index (P < 0.05). CONCLUSIONS: C-myc and CCNE2 amplifications are common events in breast cancer, and they often coexist. C-myc and CCNE2 genes may play critical roles in the pathogenesis and development of breast cancer through unique and overlapping signaling pathways.

Amplification of Mdmx and overexpression of MDM2 contribute to mammary carcinogenesis by substituting for p53 mutations.

BACKGROUND: The p53 tumor suppressor gene is mutated or deleted in nearly half of human cancers. The murine double minute 2 (Mdm2) and Mdmx represent two important cellular regulators of p53. The aim of this study was to evaluate the abnormalities of p53, Mdmx and Mdm2 genes in archived breast cancers. METHODS: We assessed the genetic instability at p53, Mdmx and Mdm2 using high resolution multi-color fluorescent in situ hybridization (FISH) protocol and detected the expression status of the tumor protein p53 (TP53), MDMx and MDM2 by immunohistochemistry in 115 archived samples of infiltrating ductal breast carcinomas with foci of ductal carcinoma in situ (DCIS) components. RESULTS: The presence of p53 allelic loss and/or TP53 overexpression was observed in 38% out of all patients, and was significantly more often in larger, high grade, ER negative and high ki67 tumors. Mdmx amplification with low-level increase of gene copy number is at high frequency while Mdm2 amplification is rare in primary breast cancer. Mdmx amplification was seen in more invasive carcinomas than preinvasive lesions. MDMx and MDM2 overexpression were detected in 65% and 38% of all cases respectively. Moreover it was showed that most tumors contained either p53 dysfunction or Mdm2 alteration, but not both. This distribution was significant (P < 0.05). Inverse correlation between Mdmx amplification/overexpression and p53 disfunction was also observed (P < 0.05). CONCLUSIONS: Our results suggest the involvement of Mdm2 and Mdmx in p53-independent breast carcinogenesis and Mdmx may contribute to the regulation of p53 independently of Mdm2. VIRTUAL SLIDES: The virtual slides for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1450529994118798.