In Vitro and In Vivo Study on the Effect of Lysosome-associated Protein Transmembrane 4 Beta on the Progression of Breast Cancer.

PURPOSE: Although the effect of lysosome-associated protein transmembrane 4 beta (LAPTM4B) on the proliferation, migration, and invasion of breast cancer (BC) cells has already been studied, its specific role in BC progression is still elusive. Here, we evaluated the effect of different levels of LAPTM4B expression on the proliferation, invasion, adhesion, and tumor formation abilities of BC cells in vitro, as well as on breast tumor progression in vivo. METHODS: We investigated the influence of LAPTM4B expression on MCF-7 cell proliferation, invasion, adhesion, and tube formation abilities in vitro through its overexpression or knockdown and on breast tumor progression in vivo. RESULTS: Cell growth curves and colony formation assays showed that LAPTM4B promoted the proliferation of breast tumor cells. Cell cycle analysis results revealed that LAPTM4B promoted the entry of cells from the G1 into the S phase. Transwell invasion and cell extracellular matrix adhesion assays showed that LAPTM4B overexpression increased the invasion and adhesion capabilities of MCF-7 cells. More branches were observed in MCF-7 cells overexpressing LAPTM4B under an electron microscope. In comparison with LAPTM4B overexpression, LAPTM4B knockdown decreased the expression of vascular endothelial growth factor-A and significantly inhibited the vasculogenic tube formation ability of tumors. These results were also verified with western blot analysis. CONCLUSION: LAPTM4B promoted the proliferation of MCF-7 cells through the downregulation of p21 (WAF1/CIP1) and caspase-3, and induced cell invasion, adhesion, and angiogenesis through the upregulation of hypoxia-inducible factor 1 alpha, matrix metalloproteinase 2 (MMP2), and MMP9 expression. This specific role deems LAPTM4B as a potential therapeutic target for BC treatment.

Quantitative assessment of human uterine fibroid xenograft using sequential in vivo bioluminescence imaging.

Uterine fibroid is one of the most common solid tumors occurring in reproductive age women. Lack of accurate methods for In vivo quantitative assessment of uterine fibroid progression severely impedes the basic research and drug screen of this disease. To solve this problem, the correlation between bioluminescence imaging (BLI) and initial cell number used to form xenograft was investigated in this study. The results showed that both subcutaneous (SC) and intraperitoneal (IP) D-luciferin administration led to fast increase of bioluminescence signal (BLS) intensity and caused large variation of peak signal intensity of xenografts through the analysis of BLI kinetic curves. We found that a distinct linear stage appeared in xenograft BLI curve for each mouse subjected to IP-injection of D-luciferin. Moreover, a high positive correlation was found between linear slope and the initial number of human uterine fibroid smooth muscle cells (fSMCs) used for xenograft formation. Our research indicates that the slope of linear stage in BLI curve is more appropriate for in vivo quantitative assessment of human uterine fibroid xenograft.

Circular RNA circZFR contributes to papillary thyroid cancer cell proliferation and invasion by sponging miR-1261 and facilitating C8orf4 expression.

In recent years, more and more circular RNAs (circRNAs) have been identified in multiple tissues and cells. Increasing evidences show circRNAs play important roles in human cancers. However, the role of circRNAs in papillary thyroid carcinoma (PTC) remains largely unknown. In this study, we identified a new circRNA circZFR that was significantly upregulated in PTC tissues compared to adjacent normal tissues. Furthermore, circZFR expression level was negatively correlated with clinical severity. We found that circZFR knockdown dramatically inhibited the proliferation, migration and invasion of PTC cells in vitro. Mechanistically, we found circZFR could promote C8orf4 expression via serving as a competing endogenous RNA (ceRNA) of miR-1261 in PTC cells. Rescue assays indicated that restoration of C8orf4 significantly attenuated the inhibitory effects of circZFR knockdown on PTC cell proliferation, migration and invasion. In summary, our findings demonstrated that circRNA circZFR exerted oncogenic roles via regulating miR-1261/C8orf4 axis in PTC, which suggested circZFR might be a potential therapeutic target.

KIT and BRAF heterogeneous mutations in gastrointestinal stromal tumors after secondary imatinib resistance.

BACKGROUND AND AIMS: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the digestive tract and characterized by expression of KIT protein. Imatinib is the frontline therapy for metastatic and unresectable GIST patients showing clinical responses in 80 % of cases. Despite the often long-lasting clinical benefit seen in most patients treated with imatinib, many will eventually suffer disease progression. The most frequent mechanism of imatinib resistance in GIST is the acquisition of secondary mutations in either KIT or PDGFRA. There are also some imatinib-resistant GIST patients lacking an identifiable mechanism of treatment failure. Recently, activating BRAF mutation was detected in a small percentage of GISTs. In this study, we report a case of GIST with acquired resistance to imatinib during therapy. METHODS: Histological, immunohistochemical, Western blot and mutational analyses were performed on GIST tissues before and after imatinib resistance. RESULTS: The imatinib-resistant tumor showed not only heterogeneous mutations of KIT and BRAF besides the primary mutation, but also transdifferentiation into a rhabdomyosarcoma phenotype. According to Western blot analysis, in imatinib-resistant GIST with both KIT V559D and BRAF V600E mutations, the inhibition of KIT V559D by imatinib caused a strong decrease of AKT phosphorylation, while ERK1/2 phosphorylation was not affected. CONCLUSIONS: This finding, in combination with the loss of KIT expression, suggests the possibility of activation of RAS-RAF-MEK-ERK pathways driven by a KIT-independent oncogenic mechanism. Understanding the genetic aberrations beyond KIT and PDGFRA may lead to the identification of additional therapeutic targets for GISTs.

B-Myb regulates snail expression to promote epithelial-to-mesenchymal transition and invasion of breast cancer cell.

Breast cancer is the leading cause of cancer death in women worldwide, which is closely related to metastasis. Recent studies argue that breast cancer cells that have undergone epithelial-to-mesenchymal transition (EMT) acquire aggressive malignant properties, but the molecular mechanisms underlying this transition are poorly understood. In this study, we found that siRNA-mediated attenuation of B-Myb expression restored E-cadherin expression and cell-cell junction formation in breast cancer cells, suppressing cell invasion, anchorage-independent growth, and tumor formation. In contrast, the forced B-Myb expression decreased the expression of the epithelial marker E-cadherin, but increased the mesenchymal markers in breast cancer cells. We found that B-Myb upregulated expression of the key EMT regulator snail and that it mediated EMT activation and cell invasion by B-Myb.

B-myb is a gene implicated in cell cycle and proliferation of breast cancer.

B-myb belongs to the myb family of transcription factors that include A-myb and c-myb. While A-myb and c-myb are tissue-specific, B-myb is broadly expressed in rapidly dividing cells of developing adult mammals. Results of our study showed that increased B-myb expression of was associated with the progression of breast cancer and that B-myb protein levels were significantly elevated in matched metastases. High B-myb levels also predict shorter overall survival of breast cancer patients. Moreover, B-myb stimulated transcription of target genes that promoted entry into the S and M-phases of the cell cycle, cell proliferation, migration and invasion in breast cancer. Taken together, our results strongly demonstrated that B-myb had a critical role in both cell cycle progression and tumorigenesis, and might serve as a novel potential target in the diagnosis and/or treatment of human breast cancer.

Rhabdomyosarcomatous differentiation in gastrointestinal stromal tumors after imatinib resistance: a potential diagnostic pitfall.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumor of the digestive tract and characterized by expression of protein-tyrosine kinase (KIT) protein. Treatment of advanced GISTs has been improved dramatically following the development of imatinib. Despite the often long-lasting clinical benefit seen in most patients treated with imatinib, many will eventually suffer disease progression. In general, progressing GISTs retain their typical morphology. In this study, we present a patient with metastatic GISTs, who received more than 16 months of treatment with imatinib and whose tumors changed their morphological and immunohistochemical characteristics after imatinib-resistance. Histological, immunohistochemical and mutational analysis was performed on the prior and post-imatinib treatment GIST samples. The imatinib-resistant tumor cells in the progressing metastases showed marked pleomorphism which proved to be rhabdomyoblastic differentiation with Desmin and Myogenin immunopositivity. However, there was no secondary mutation of KIT, PDGFRA, KRAS and BRAF genes found in the imatinib-resistant lesion, except primary KIT V559D mutation. To our knowledge, this case represents the few reports on this unusual type of transdifferentiation in GISTs under imatinib therapy. Awareness of this phenomenon would help to avoid diagnostic confusion when evaluating post-imatinib samples from GISTs.

[Establishment and pathologic analysis of imatinib-resistant gastrointestinal stromal tumor xenografts].

OBJECTIVE: To establish and characterize imatinib-resistant gastrointestinal stromal tumor (GIST) xenografts. Further provided an ideal experimental platform through the imatinib-resistant GIST xenografts to investigate the mechanism of resistance to imatinib. METHODS: Imatinib-resistant GIST cells were injected under the skin of athymic nude mice to establish animal models of human imatinib-resistant GIST. The molecular and histopathologic features of GIST xenografts were also analysed and compared with their counterpart of cell lines. RESULTS: The xenograft tumor models had been established by subcutaneously injection of GIST cells into nude mice. Immunohistochemistry results showed CD117 expression was positive in GIST-PR2 xenograft tumor, but negative in GIST-R. In GIST-PR1, tumor areas showing rhabdomyoblastic differentiation were presented next to areas with classic GIST morphology. The rhabdomyoblastic component demonstrated consistently positivity for desmin and myogenin, whereas CD117 was completely negative. The mutation profiles of these xenograft tumors were the same as their counterpart of cell lines. CONCLUSIONS: Human GIST xenografts with mutation in c-kit have been established from imatinib-resistant GIST lines. Those models will enable further studies on mechanisms of resistance, combination therapies and allow testing of novel targeted therapies.

Gene mutations and prognostic factors analysis in extragastrointestinal stromal tumor of a Chinese three-center study.

BACKGROUND: Most gastrointestinal stromal tumors (GISTs) have gain-of-function mutation of the c-kit gene, and some have mutation of the platelet-derived growth factor receptor-α (PDGFR-α) gene. Extragastrointestinal stromal tumors (EGISTs) are mesenchymal tumors that occur outside the digestive tract. But the clinicopathologic characteristics of EGISTs are still poorly understood. METHODS: Paraffin-embedded tissues from 25 cases of EGIST were analyzed for CD117, CD34, Ki-67, S-100, smooth muscle actin, and desmin expression by immunohistochemical method. These cases of EGISTs were also evaluated for the presence of c-kit exons 9, 11, 13, and 17 mutations and PDGFR-α exons 12 and 18 mutations. Survival analysis was used to evaluate the prognostic factors. RESULTS: c-kit mutations were detected in 44% of EGIST patients and all were exon 11 mutations. PDGFR-α mutations were found in 12% of the 25 cases and all were exon 18 mutations. Survival analysis indicated that mitotic count and Ki-67 labeling index (Ki-67 LI) were significant predictors of survival. CONCLUSION: The pattern of c-kit and PDGFR-α mutation in EGISTs was essentially similar to that in GISTs. From the molecular genetics aspect, EGISTs may be a special subtype of GISTs. The results also show that the combination of mitotic counts and Ki-67 LI may be useful for predicting the prognosis of EGISTs.

Survival and prognostic factors analysis in surgically resected gastrointestinal stromal tumor patients.

BACKGROUND/AIMS: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor of the digestive tract and predicting the clinical behavior and prognosis of GISTs has still been problem for both pathologists and clinicians. The aim of this study was to investigate the survival and prognostic factors of gastrointestinal stromal tumors after surgery. METHODOLOGY: Hematoxylin and eosin (H&E) stained histopathological slides of tumors from patients with GISTs were reviewed. Immunohistochemical staining was performed to demonstrate CD117, CD34, platelet -derived growth factor receptor (PDGFR-alpha) and Ki-67 protein expression. Clinicopathologic features (age, sex, tumor location and size, cell type, mitotic count, risk category, necrosis, surgical method, expression of CD117, CD34, PDGFR-alpha and Ki-67 protein) were evaluated by univariate and multivariate analyses in 135 patients with resected primary GISTs to identify independent prognostic factors. RESULTS: The overall disease-specific survival of 135 patients was 94.1% at 1 year, 76.3% at 3 years and 65.9% at 5 years. Multivariate analyses indicated that the tumor size, primary location, mitotic count, risk category, necrosis and Ki-67 index were independent significant predictors of survival (p < 0.05). Ki-67 index was strong poor predictors of survival as tumor size and mitotic count. CONCLUSIONS: Fletcher's biological behavior ranking method was a good approach to predict prognosis of GIST patients and had significant clinical value. It's better to combine other factors such as Ki-67 index and tumor primary location et al to predict prognosis accurately. Accurate prognostic prediction could provide evidence for postoperative adjuvant targeted therapy.

Secondary C-kit mutation is a cause of acquired resistance to imatinib in gastrointestinal stromal tumor.

C-kit gene gain of function mutations are important in the pathogenesis of gastrointestinal stromal tumors (GISTs). Imatinib is a selective tyrosine kinase inhibitor of KIT and achieves a partial response or stable disease in most patients with metastatic GIST, but there is increasing evidence of acquired resistance. We report a case of GIST with acquired resistance to imatinib during therapy and secondary c-kit mutation besides the primary mutation.