Multiomics Landscape Uncovers the Molecular Mechanism of the Malignant Evolution of Lung Adenocarcinoma Cells to Chronic Low Dose Cadmium Exposure.

Cadmium (Cd) from cigarette smoke and polluted air can lead to lung adenocarcinoma after long-term inhalation. However, most studies are based on short-term exposure to this toxic metal at high concentrations. Here, we investigate the effects of long-term exposure of A549 cells (lung adenocarcinoma) to cadmium at low concentrations using morphological and multiomics analyses. First, we treated A549 cells continuously with CdCl2 at 1µM for 8 months and found that CdCl2 promoted cellular migration and invasion. After that, we applied transmission electron and fluorescence microscopies and did not observe significant morphological changes in Golgi apparatus, endoplasmic reticulum, lysosomes, or mitochondria on Cd treated cells; microfilaments, in contrast, accumulated in lamellipodium and adhesion plaques, which suggested that Cd enhanced cellular activity. Second, by using whole-exome sequencing (WES) we detected 4222 unique SNPs in Cd-treated cells, which included 382 unique non-synonymous mutation sites. The corresponding mutated genes, after GO and KEGG enrichments, were involved mainly in cell adhesion, movement, and metabolic pathways. Third, by RNA-seq analysis, we showed that 1250 genes (784 up and 466 down), 1623 mRNAs (1023 up and 591 down), and 679 lncRNAs (375 up and 304 down) were expressed differently. Furthermore, GO enrichment of these RNA-seq results suggested that most differentially expressed genes were related to cell adhesion and organization of the extracellular matrix in biological process terms; KEGG enrichment revealed that the differentially expressed genes took part in 26 pathways, among which the metabolic pathway was the most significant. These findings could be important for unveiling mechanisms of Cd-related cancers and for developing cancer therapies in the future.

Clinical comparison of tenosynovial giant cell tumors, synovial chondromatosis, and synovial sarcoma: analysis and report of 53 cases.

BACKGROUND: Tenosynovial giant cell tumors (TGCTs), synovial chondromatosis (SC), and synovial sarcoma (SS) exhibit similarities in clinical features and histochemical characteristics, and differential diagnosis remains challenging in clinical practice. METHODS: Data were collected from the pathology database of Shanghai Ninth People's Hospital regarding patients who underwent surgery from 2010 to 2019 with histologically confirmed TGCTs, SC, and SS. Demographic and clinicopathological data of these patients were reviewed. Immunohistochemistry staining of 14 different markers was performed. Correlation analyses of the prognoses were evaluated. RESULTS: A total of 26 patients with TGCTs (8 diffuse TGCTs and 18 localized TGCTs), 16 with SC, and 11 with SS were identified. Pain was the main symptom of patients with both TGCTs and SC, while a palpable mass was the most common symptom for patients with SS. In addition to clinical features, we identified vital risk factors for disease recurrence. The mean follow-up periods were 51, 39, and 14 months for TGCTs, SC, and SS, respectively. Younger patients with diffuse TGCTs or patients with a higher neutrophil/lymphocyte ratio (NLR) displayed a significantly higher frequency of recurrence. We also plotted receiver operating characteristic (ROC) curve analysis for age and NLR. The area under the ROC curve (AUC) was calculated and demonstrated the ability to distinguish recurrent from nonrecurrent cases. In addition, higher CD163 expression was linked to recurrent diffuse TGCT cases. CONCLUSIONS: These data indicated possible characteristics of different aspects of TGCTs, SC, and SS. Further clarification and understanding of these factors will help with differential clinical diagnosis and recurrent risk assessment.

Neurocytoma arising from a mature ovary teratoma: a case report.

Central neurocytoma/extraventricular neurocytoma is a central nervous system (CNS) tumor composed of uniform round cells with neuronal differentiation. The typical lesions of central neurocytoma/extraventricular neurocytoma are at the interventricular foramen of the lateral ventricles (central neurocytoma) or brain parenchyma (extraventricular neurocytoma). Mature teratoma is a benign germ cell tumor commonly found in young women. Herein, we report a 24-year-old female with neurocytoma in a mature teratoma of the right ovary. The histological examinations showed mature epidermis, skin appendages, adipose and bone tissues in the tumor; microscopic foci of immature cartilage tissues were also found in some parts. In addition, massive solid sheets and uniform round tumor cells were found in the neuroectodermal tissues, with the formation of neuropil-like islands. Immunohistochemical examinations showed that the tumor cells were synaptophysin- and NeuN-positive but GFAP-negative. Based on these findings, the woman was diagnosed with neurocytoma arising from mature ovary teratoma, with microscopic foci of immature cartilage tissues. This is the fourth case report of neurocytoma outside the CNS to date.

Overexpression of δ-catenin is associated with a malignant phenotype and poor prognosis in colorectal cancer.

Little is known regarding the expression or clinical significance of δ-catenin, a member of the catenin family, in colorectal cancer (CRC). The present study examined the expression of δ-catenin using immunohistochemistry in 110 cases of CRC, including 70 cases with complete follow­up records and 40 cases with paired lymph node metastases. In addition, δ­catenin mRNA and protein expression were compared in 30 pairs of matched CRC and normal colorectal tissues by reverse transcription quantitative polymerase chain reaction and western blot analysis. δ­Catenin was weakly expressed or absent in the cytoplasm of normal intestinal epithelial cells, whereas positive δ­catenin expression localized to the cytoplasm was observed in CRC cells. The rate of positive δ­catenin expression in CRC (68.18%; 75/110) was significantly higher than that in normal colorectal tissues (36.7%; 11/30; P<0.001). In addition, δ­catenin mRNA and protein expression were significantly increased in CRC tissues compared to those in their matched normal tissues (all P<0.05). The expression of δ­catenin in stage III­IV CRC was higher than that in stage I­II CRC, and the expression of δ­catenin in the tumors of patients with lymph node metastases was higher than that in patients without lymph node metastases. Kaplan­Meier survival curves demonstrated that the survival time of patients with positive δ­catenin expression was shorter than that of patients with negative δ­catenin expression (P=0.005). Furthermore, Cox multivariate analysis indicated that the tumor, nodes and metastasis stage (P=0.02) and positive δ-catenin expression (P=0.033) were independent prognostic factors in CRC. The present study therefore indicated that δ-catenin may be a suitable independent prognostic factor for CRC.

Co-expression of delta-catenin and RhoA is significantly associated with a malignant lung cancer phenotype.

Delta-catenin, a member of the p120-catenin subfamily, and the Rho GTPase RhoA both have roles in the regulation of the cytoskeleton. In this study, we found that delta-catenin positive expression and RhoA over-expression is consistently found in non-small cell lung cancer, but not in normal lung tissue, and that their co-expression was significantly associated with histological type, differentiation, pTNM stage, lymphatic metastasis and a poor prognosis. We also demonstrate that delta-catenin can directly interact with RhoA and regulate its activity, which in turn mediates tumor invasion and metastasis.

Malignant solitary fibrous tumor of breast: a rare case report.

Solitary fibrous tumor (SFT) is rare mesenchymal neoplasm that has been originally and most often documented in the pleura. Recently, the ubiquitous nature of the SFT has been recognized with reports of involvement of numerous sites all over the body such as: upper respiratory tract, somatic tissue, mediastinum, head, and neck. Less than 10 cases SFT of breast have been reported. Herein, we presented a 52-year-old Asian female with SFT of breast, this tumor showed predominant malignant features. To our knowledge, SFT of breast with such malignant evidence is extremely rare.

Delta-catenin promotes the proliferation and invasion of colorectal cancer cells by binding to E-cadherin in a competitive manner with p120 catenin.

δ-Catenin is the only member of the p120 catenin (p120ctn) subfamily whose normal pattern of expression is restricted to the brain. Similar to p120ctn, δ-catenin can bind to the juxtamembrane domain of E-cadherin. We examined the expression of δ-catenin, p120ctn, and E-cadherin using immunohistochemistry in 95 cases of colorectal cancer (CRC) and 15 normal colon tissues. Co-immunoprecipitation was used to examine whether δ-catenin competed with p120ctn to bind E-cadherin in CRC cells. The effects of δ-catenin overexpression or siRNA-mediated knockdown on the proliferation and invasive ability of CRC cells were investigated using the MTT and Matrigel invasion assays. The results showed that positive δ-catenin expression was significantly more frequent in CRC compared to normal colon tissues and associated with poor differentiation, stage III-IV disease, and lymph node metastasis in CRC (all P < 0.05). In two CRC cell lines, δ-catenin bound to E-cadherin in competition with p120ctn. Overexpression of δ-catenin promoted the proliferation and invasion of CRC cells; knockdown of δ-catenin reduced CRC cell proliferation and invasion. In conclusion, we speculate that overexpression of δ-catenin reduces the expression of E-cadherin and alters the balance between E-cadherin and p120ctn, which in turn affects the formation of intercellular adhesions and promotes invasion and metastasis in CRC.

Variable Ki67 proliferative index in 65 cases of nodular fasciitis, compared with fibrosarcoma and fibromatosis.

Nodular fasciitis is the most common pseudosarcomatous lesion of soft tissue. Ki67 was considered as a useful marker for distinguishing some benign and malignant lesions. To study the usefulness of Ki67 in diagnosis of nodular fasciitis, the expression of Ki67 was examined by using immunostaining in 65 nodular fasciitis specimens, 15 desmoid fibromatosis specimens and 20 fibrosarcoma specimens. The results showed that there was a variable Ki67 index in all 65 cases of nodular fasciitis, and the mean labeling index was 23.71±15.01%. In majority (70.77%) of all cases,the index was ranged from 10% to 50%, in 6.15% (4/65) of cases the higher Ki67 index (over 50%) could be seen. The Ki67 proliferative index was closely related to duration of lesion, but not to age distribution, lesion size, sites of lesions and gender. Moreover, the mean proliferative index in desmoid fibromatosis and fibrosarcoma was 3.20±1.26% and 26.15±3.30% respectively. The mean Ki67 index of nodular fasciitis was not significantly lower than fibrosarcoma, but higher than desmoid fibromatosis. The variable and high Ki67 index in nodular fasciitis may pose a diagnostic challenge. We should not misdiagnose nodular fasciitis as a sarcoma because of its high Ki67 index. The recurrence of nodular fasciitis is rare; and the utility of Ki67 immunostaining may be not suitable for recurrence assessment in nodular fasciitis. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/4782335818876666.

Atonal homolog 1 expression in lung cancer correlates with inhibitors of the Wnt pathway as well as the differentiation and primary tumor stage.

Atonal homolog 1 (Atoh1) is crucial to the differentiation of many cell types and participates in tumorigenesis and progression. This study investigated the role of Atoh1 in lung cancer development and its correlation with key members of the Wnt pathway. We used immunohistochemistry to examine the expressions of Atoh1, ß-catenin, Axin, chibby, and Disabled-2 (Dab2) in 118 samples of lung cancer. We also detected the cytoplasmic and nuclear expression of Atoh1 in lung cancer tissues using western blot. Atoh1 nuclear expression was negatively correlated with differentiation level (p = 0.004) and primary tumor stage (p = 0.044) of lung cancer. Nuclear Atoh1 expression was positively correlated with nuclear expression of chibby (p < 0.001) and Dab2 (p < 0.001). Cytoplasmic Atoh1 expression was positively correlated with the cytoplasmic expression of Axin (p = 0.028), chibby (p < 0.001), and Dab2 (p < 0.001). We conclude that the nuclear expression of Atoh1 was inversely correlated with the differentiation and primary tumor stage of lung cancers. The expression and localization of Atoh1 correlated with Axin, chibby, or Dab2. Atoh1 may be a potential therapeutic target for the inhibition of growth and progression of lung cancers.

Pulmonary sclerosing hemangioma presenting with dense spindle stroma cells: a potential diagnostic pitfall.

Pulmonary sclerosing hemangioma (PSH) is an uncommon pulmonary tumor. Histologically, PSH typically consists of two types of cells, surface cuboidal cells and polygonal cells, four architectural patterns including papillary, sclerotic, solid, and hemorrhagic. Herein, we present a case of PSH in a 59-year-old Chinese female. The tumor was predominantly composed of solid area presenting with diffuse spindle cells rather than polygonal cells. Focally, classical papillary and sclerotic area could be seen. Immunohistochemical staining showed that the spindle cells were positive for TTF-1, EMA, Actin(SM) and Vimentin, and negative for cytokeratin, cytokeratin7, cytokeratin5/6, surfactant apoprotein A, surfactant apoprotein B, CD34, CD99, S-100, HMB45, Desmin, Synaptophysin, CD56, ALK and Calretinin. The immunophenotype of the dense spindle cells in this case was similar to that of the polygonal cells, and thus the spindle cells may be the variants of polygonal cells. Based on morphologic features and the immunohistochemical profile, the tumor was diagnosed as a PSH. The significance of spindle cells change is unclear for us. To our knowledge, this is the first reported case of PSH showing dense spindle cells in solid area. This case represents a potential diagnostic pitfall, as it may be misdiagnosed as a mesenchymal tumor such as inflammatory myofibroblastic tumor, synovial sarcoma, solitary fibrous tumor, leiomyoma, or even mesothelioma, especially if the specimen is limited or from fine- needle aspiration. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1235401622806126.

X-radiation inhibits histone deacetylase 1 and 2, upregulates Axin expression and induces apoptosis in non-small cell lung cancer.

BACKGROUND: Histone deacetylase (HDAC) plays an important role in the deacetylation of histone, which can alter gene expression patterns and affect cell behavior associated with malignant transformation. The aims of this study were to investigate the relationships between HDAC1, HDAC2, clinicopathologic characteristics, patient prognosis and apoptosis, to clarify the mechanism of upregulation of the Axis inhibitor Axin (an important regulator of the Wnt pathway) by X-radiation and to elucidate the effect of siRNA on radiation therapy of non-small cell lung cancer (NSCLC). METHODS: HDAC1 and HDAC2 expression levels were measured by immunohistochemistry and reverse transcription PCR. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated dUTP-nick end labeling and fluorescence activated cell sorting. BE1 cells expressing Axin were exposed to 2 Gy of X-radiation. RESULTS: Expression of HDAC1 and that of HDAC2 were correlated, and significantly higher in NSCLC tissues than in normal lung tissues (P < 0.05). HDAC1 and HDAC2 expression was correlated with pTNM stage and negatively correlated with differentiation of NSCLC and apoptotic index (P < 0.05). The prognosis of patients with low expression of HDAC1 and HDAC2 was better than that of those with high expression. X-radiation and siRNA inhibited HDAC1 and HDAC2 expression in NSCLC cells and Axin levels were significantly higher in BE1 cells. CONCLUSIONS: X-radiation and siRNA inhibit expression of HDAC1 and HDAC2, weaken the inhibitory effect of HDAC on Axin, upregulate Axin expression and induce apoptosis of lung cancer cells. Inhibition of HDAC1 and HDAC2 is a means of enhancing the radiosensitivity of NSCLC.

Expression and significance of stem cell markers in pulmonary sclerosing haemangioma.

AIMS: The two major types of cells of pulmonary sclerosing haemangioma (PSH) with the same origin show significant differences in morphological phenotype. Whether these differences are caused by their different differentiation status is still uncertain. The aim of this study was to analyse their differentiation status by detecting the expression of several stem cell markers in PSH. METHODS AND RESULTS: The expression of stem cell markers was examined by using streptavidin peroxidase (SP) immunohistochemisty in 45 PSH specimens. Also, the two types of cells were, respectively, captured by laser capture microdissection (LCM) from 28 PSH specimens, and total RNA was then extracted followed by reverse transcription-polymerase chain reaction (RT-PCR). The results demonstrated that the expression rates of ABCG2, Notch1 and Notch3 in polygonal cells were significantly higher than those in cuboidal cells (P < 0.05), and the expression levels of ABCG2, Notch3 and Jagged1 in polygonal cells were clearly higher than those in cuboidal cells (P < 0.05). CONCLUSION: The data obtained provided evidence that the two types of cells in PSH may be different in differentiation status. The differentiation difference between the two types of cells might lead to variation in their morphological phenotype.

In pulmonary sclerosing hemangioma expression of ß-catenin, Axin, and C-myc differs between the two cell types.

Previous studies have shown that pulmonary sclerosing hemangioma is a tumor derived from primitive respiratory epithelium, but its character and the differentiation status of the two cell types (polygonal and cuboidal) composing the lesion are still controversial. We hypothesize that the polygonal cells are immature compared with cuboidal cells and have higher proliferative activity. To further study this question, we examined the expression of ß-catenin, Axin, and C-myc by immunostaining in 45 primary sclerosing hemangioma (PSH) specimens. The two cell types were captured by laser capture microdissection from 28 PSH specimens, and total RNA was extracted. Messenger RNA (mRNA) expression of Axin and C-myc was examined by reverse transcription polymerase chain reaction (RT-PCR). By immunostaining, ß-catenin was predominantly strongly expressed on the cell membrane of cuboidal cells, while in polygonal cells, ß-catenin was predominantly expressed in the cytoplasm and significantly decreased on cell membranes. Axin was expressed in cuboidal cells in 93 % of our 45 cases, but only expressed in 18 % of these in polygonal cells. C-myc expression in polygonal cells was significantly stronger than in cuboidal cells (P < 0.05). RT-PCR showed that the expression level of Axin mRNA in cuboidal cells was significantly higher than in polygonal cells (P < 0.05), and expression level of C-myc mRNA in polygonal cells was significantly higher than in cuboidal cells (P < 0.05).The two PHS cell types have distinct expression of ß-catenin, Axin, and C-myc, suggesting that their differentiation status may be different. The higher expression of C-myc in polygonal cells suggests that these cells might have higher proliferative activity.

Expression of metastasis-associated protein 2 (MTA2) might predict proliferation in non-small cell lung cancer.

Metastatic tumor antigen 2 (MTA2) is a member of the MTA family that is closely associated with tumor progression and metastasis. In this study, the expression profile of MTA2 in 223 cases of non-small cell lung cancer (NSCLC) tissues and two lung cancer cell lines was investigated. Interestingly, we found MTA2, which was believed to have nuclear distribution only, was distributed in both nucleus and cytoplasm in normal and cancer cells. Nuclear MTA2 expression was detected in 148 cases of NSCLC (66.4%), and was correlated with advanced TNM stages (p=0.023), tumor size (p=0.036), and lymph node metastasis (p=0.004). Besides, the Ki-67 proliferation index was significantly higher in nuclear MTA2-positive tumors than in nuclear MTA2-negative tumors (r=0.538, p=0.006). However, there was no significant difference in cytoplasmic MTA2 status by age, gender, tumor stage, histology, grade, lymph node metastasis, and Ki-67 proliferation index. Univariate analysis revealed nuclear MTA2 expression was correlated with poor overall survival (p=0.035), whereas there was a nonsignificant trend in the same direction for cytoplasmic MTA2 (p=0.134). Multivariate Cox regression analysis revealed the overexpression of nuclear and cytoplasmic MTA2 not to be independent factors predictive of poor disease outcome. Our data suggested that MTA2 might play roles in both the nucleus and cytoplasm in the progression of NSCLC.

PI3K/AKT/mTOR signaling pathway inhibitors in proliferation of retinal pigment epithelial cells.

AIM: To determine whether the PI3K/AKT/mTOR pathway is activated in proliferative vitreoretinopathy (PVR) in homo-sapiens. METHODS: The retina of controls and patients with PVR were collected and their levels of PI3K, phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP-1 were determined by Western blot. The cultured human retinal pigment epithelial cell line D407 was treated with a specific mTOR inhibitor, rapamycin (RAPA) or a PI3K inhibitor, LY294002, of various concentrations and durations. Cell morphology was observed by phase contrast microscopy and the proliferation and apoptosis of treated cells were determined by MTT assay and flow cytometry. RESULTS: Levels of PI3K, phospho-AKT, phospho-mTOR, phospho-P70S6K and phospho-4EBP1 was increased in the retina in PVR (P<0.05). In D407 cells, both RAPA and LY294002 significantly inhibited cell proliferation and cell cycle progression, and promoted apoptosis (P <0.05); morphologically, the cells became smaller. Both RAPA and LY294002 reduced levels of phospho-AKT, phospho-mTOR, phospho-p70S6k and phospho-4EBP1 expression (P <0.05). RAPA, but not LY294002, had no significant effect on PI3K expression. CONCLUSION: PI3K/AKT/mTOR signaling pathway is highly activated in the retinal pigment epithelial cells of PVR. The inhibitors of PI3K/AKT/mTOR signaling pathway, RAPA and LY294002, could inhibited the PI3K/AKT/mTOR signaling pathway by reducing the levels of phosphorylation of mTOR pathway components.

The expression patterns and correlations of chibby, ß-catenin, and DNA methyltransferase-1 and their clinicopathological significance in lung cancers.

Chibby is an inhibitor of the Wnt pathway. The expression and correlation of chibby in lung cancers is unclear. We considered that the expression pattern of chibby might be related to the expression of ß-catenin and DNA methyltransferase-1 (DNMT1). We examined the expressions of chibby, ß-catenin, and DNMT1 in 85 lung cancer tissues and corresponding normal lung tissues using immunohistochemistry. The nuclear expression rate of chibby was reduced in lung cancers (p < 0.001). Increased expression of DNMT1 was correlated with the differentiation (p = 0.034) and TNM stage (p = 0.048). The cytoplasmic expression of ß-catenin was correlated with poor differentiation of lung cancers (p = 0.016). The cytoplasmic and membrane expression of chibby was positively correlated with the cytoplasmic (p = 0.025) and membrane (p = 0.029) expressions of ß-catenin. The membrane expression of chibby was negatively correlated with the expression of DNMT1 (p = 0.006). Moreover, the expression of DNMT1 was correlated with the cytoplasmic expression of ß-catenin (p < 0.001). The nuclear expression of chibby is reduced in lung cancers. Chibby may colocalize with ß-catenin. ß-Catenin and DNMT1 may be concurrently expressed and thereby promote the development of lung cancers.

Overexpression of Frat1 correlates with malignant phenotype and advanced stage in human non-small cell lung cancer.

Frat1 has been reported to be overexpressed in several human malignant tumors, including esophageal squamous, cervical, breast, and ovarian carcinoma, but the role of Frat1 in lung cancer is unknown. Our purpose is to investigate the expression of Frat1 and its correlation with clinicopathologic features and prognosis in lung cancer patients. Immunohistochemistry was performed on 137 cases of non-small cell lung cancer (NSCLC), including 78 cases with clinical follow-up, and Western blot and reverse transcription-polymerase chain reaction (RT-PCR) assays were performed to detect the protein and mRNA expression levels in 30 NSCLC and autologous matched normal tissues. In addition, lung cancer cell line A549 was transfected with Frat1-siRNA plasmids and Matrigel invasive assay was carried out to study the function of Frat1 in cancer cell invasiveness. The results showed that Frat1 was expressed in 85 (62.04%) cases of NSCLC by immunohistochemistry, while all 30 specimens of normal lung tissues were negative. Western blot and RT-PCR results for Frat1 mRNA were in agreement with immunohistochemical findings. Of interest, the expression of Frat1 was strongly correlated with tumor differentiation, TNM stage, and lymph node metastasis (P < 0.05). The Kaplan-Meier survival analysis demonstrated that the cases with Frat1 expression had significantly shorter survival than those without Frat1 (P < 0.001). In addition, down-regulation of Frat1 expression reduced the invasive ability in the A549 cell line, further supporting the idea that Frat1 may play a crucial role in carcinogenesis, tumor invasiveness and dissemination in human lung cancer.