Precise pathologic diagnosis and individualized treatment improve the outcomes of invasive micropapillary carcinoma of the breast: a 12-year prospective clinical study.

Invasive micropapillary carcinoma of the breast is a histologic subtype of breast cancer and associated with high incidence of lymphovascular invasion, lymph node metastasis and poor prognosis. The aim of this prospective study was to investigate the impact of precise pathologic diagnosis and individualized treatment on the outcomes of invasive micropapillary carcinoma of the breast. The study group included 2299 women with invasive micropapillary carcinoma diagnosed at Tianjin Medical University Cancer Institute and Hospital between January 2004 and December 2015. In the study group, specimens were examined with the method of whole-specimen orientation and serial sectioning, and patients received precise pathological diagnosis and individualized treatment. The control group of invasive micropapillary carcinoma consisted of 163 cases, identified through a retrospectively review of 9056 invasive carcinomas diagnosed at our institution between January 1989 and December 2003 using the standard pathology-evaluation method (i.e., not using the whole-specimen orientation and serial-sectioning method). The clinicopathological features, treatments and outcomes were compared between the two groups. The incidence of invasive micropapillary carcinoma in the study group was 6% (2299/39,714 cases), significantly higher than that of the control group (2%; 163/9056 cases). The 5-year disease-free survival in the study group was significantly higher than that in the control group (83.8 vs.45.4%; p < 0.05). The 5-year overall survival was significantly increased from 57.4% in the control group to 90.9% in the study group (p < 0.05). In the multivariate analysis, lymphovascular invasion, estrogen receptor status and lymph node metastasis were independent prognostic factors. Although invasive micropapillary carcinoma of the breast is associated with poor prognosis, precise pathologic diagnosis and individualized treatment improved the disease-free survival and overall survival of invasive micropapillary carcinoma patients. Precise pathological diagnosis is the premises for individualized treatments and for improving the outcomes of patients with invasive micropapillary carcinoma of the breast.

CD44 targets Wnt/ß-catenin pathway to mediate the proliferation of K562 cells.

BACKGROUND: Chronic myeloid leukemia is a clonal myeloproliferative disorder disease in which BCR/ABL plays an important role as an oncoprotein and molecular target. Despite the success of targeted therapy using tyrosine kinase inhibitors, CML remains largely incurable, most likely due to the treatment resistance after firstly chemical therapy. So know well the unique molecular pathway of CML is very important. METHODS: The expressions of CD44 in different leukemia patients and cell lines were detected by real-time PCR and western blotting. The effects of CD44 on proliferation of K562 cells were determined using the MTT and colony formation assays, and even in a nude mouse transplantation model. Then, the cell cycle changes were detected by flow cytometric analysis and the early apoptosis of cells was detected by the annexin V/propidium iodide double-staining assay. The expressions of the cycles and apoptosis-related proteins p21, Cyclin D1 and Bcl-2 were analyzed by western blot and real-time PCR assay. Finally, the decreased nuclear accumulation of ß-catenin was detected by western blotting and immunefluorescence. RESULTS: Firstly, we showed that CD44 expression was increased in several kinds of leukemia patients and K562 cells. By contrast, the down-regulation of CD44 resulted in decreased proliferation with a G0/G1 arrest of cell cycle in K562 cells according to the MTT assay and the flow cytometric analysis. And no significant induction of both the early and late phases of apoptosis was shown by the annexin V-FITC and PI staining. During this process, p21 and cyclin D1 are the major causes for cell cycle arrest. In addition, we found CD44 down-regulation decreased the expression of ß-catenin and increased the expression of phosphorylated ß-catenin. The instability of Wnt/ß-catenin pathway induced by increased expression of p-ß-catenin resulted in a decreased nuclear accumulation in CD44 silenced K562 cells. In the nude mouse transplantation model, we also found the same results. CONCLUSIONS: These results show that K562 cells depend to a greater extent on CD44 for proliferation, and CD44 down-regulation may induce a cell cycle arrest through Wnt/ß-catenin pathway. CD44 blockade may be beneficial in therapy of CML.

sLex expression in invasive micropapillary breast carcinoma is associated with poor prognosis and can be combined with MUC1/EMA as a supplementary diagnostic indicator.

OBJECTIVE: Mucin 1 (MUC1/EMA) and sialyl Lewis X (sLex) indicate polarity reversal in invasive micropapillary carcinoma (IMPC). The purpose of this study was to evaluate the expression of MUC1/EMA and sLex and to assess their diagnostic and prognostic value in patients with IMPC. METHODS: The expression of sLex and MUC1/EMA in 100 patients with IMPC and a control group of 89 patients with invasive ductal carcinoma not otherwise specified (IDC-NOS) were analyzed with IHC. Fresh tumor tissues were collected from patients with IMPC or IDC-NOS for primary culture and immunofluorescence analysis. RESULTS: The rate of nodal metastasis was higher in patients with IMPC than those with IDC-NOS, and IMPC cells tended to express more sLex and MUC1/EMA in the cytomembranes (the stroma-facing surfaces of the micropapillary clusters) than IDC-NOS cells. In IMPC, high cytomembrane expression of sLex, but not MUC1/EMA, indicated poor prognosis. In addition, among the 100 patients with IMPC, 10 patients had sLex+/EMA- expression patterns, and 8 patients had sLex-/EMA+ expression patterns. The primary IMPC cells were suspended, non-adherent tumor cell clusters, whereas the primary IDC cells were adherent tumor cells. Immunofluorescence analysis showed that MUC1/EMA and sLex were co-expressed on the cytomembranes in IMPC cell clusters and in the cytoplasm in IDC-NOS cells. CONCLUSIONS: sLex can be used as a prognostic indicator and can be combined with MUC1/EMA as a complementary diagnostic indicator to avoid missed IMPC diagnosis.

Expression of Polo-Like Kinase 4(PLK4) in Breast Cancer and Its Response to Taxane-Based Neoadjuvant Chemotherapy.

PURPOSE: Polo-like kinase 4(PLK4) is an important evolutionarily regulator involved in centrosome duplication. We here investigated the expression of PLK4 mRNA and PLK4 in breast cancer, and evaluated its predictive value for response to taxane-based neoadjuvant chemotherapy. METHOD: The PLK4 mRNA expression was measured in breast cancer tissues and corresponding normal breast tissues from 30 breast cancer patients by quantitative real-time polymerase chain reaction (PCR).The association of the expression of PLK4 with clinicopathological parameters and prognostic significance was evaluated in 154 cases of invasive breast cancer. In addition, we immunohistochemically examined the changes of PLK4 expression in biopsy and postoperative tumor specimens of another 64 breast cancer patients who received taxane-based neoadjuvant chemotherapy. RESULTS: The level of PLK4 mRNA expression in cancerous tissues had a significant difference compared to the corresponding normal breast tissues (P=0.021). There is a correlation of PLK4 expression with higher incidence of lymph node metastasis and distant metastasis or surrounding recurrence (P=0.043; P=0.006). High PLK4 expression was found to be a detrimental prognostic factor measured by overall survival (OS) (P=0.003) and progress-free survival (PFS) (P=0.003). Moreover, the results demonstrated that PLK4 expression was a negative predictor of response to taxane-based neoadjuvant chemotherapy (rs= - 0.253, P=0.044). CONCLUSION: The findings of this current study indicated that PLK4 expression in breast cancer could be a potential prognostic factor and a negative predictor of response to taxane-based neoadjuvant chemotherapy.

SIRT2 mediates multidrug resistance in acute myelogenous leukemia cells via ERK1/2 signaling pathway.

SIRT2, one of nicotinamide adenine dinucleotide (NAD+)-dependent class Ⅲ histone deacetylase family proteins, has been found to be involved in the proliferation and survival of acute myeloid leukemia (AML) cells. However, its effect on drug resistance on chemoresistant AML cells is unclear. In the present study, we first found that SIRT2 was expressed at higher level in the relapsed AML patients than the newly diagnosed patients. Consistent with this observation, the expression level of SIRT2 was higher in HL60/A cells than that in HL60 cells. Depletion of SIRT2 by shRNAs in HL60/A cells resulted in decreased MRP1 level, enhanced drug accumulation and triggered more apoptosis. By contrast, overexpression of SIRT2 in HL60 cells led to increased MRP1 level, drug efflux and attenuated drug sensitivity. Moreover, the decreased expression of phosphorylated ERK1/2 was detected in SIRT2-depleted HL60/A cells and increased expression of phosphorylated ERK1/2 was observed in SIRT2 overexpressed HL60 cells. Furthermore, blockage of ERK1/2 signaling pathway with the chemical inhibitor PD98059, further induced apoptosis of HL60/A cells conferred by SIRT2 depletion. Importantly, ERK1/2 inhibition was able to reverse the drug resistance of HL60 conferred by SIRT2 overexpression. Thus, our findings collectively suggested that the expression level of SIRT2 has a positive relationship with DNR/Ara-C resistance and activity of ERK1/2 signaling pathway. SIRT2 might regulate DNR/Ara-C sensitivity in AML cells at least partially through the ERK1/2 pathway.

Knock-down of CIAPIN1 sensitizes K562 chronic myeloid leukemia cells to Imatinib by regulation of cell cycle and apoptosis-associated members via NF-κB and ERK5 signaling pathway.

CIAPIN1 (cytokine-induced apoptosis inhibitor 1) was recently identified as an essential downstream effector of the Ras signaling pathway. However, its potential role in regulating myeloid leukemia cells sensitivity to Imatinib remains unclear. In this study, we found depletion of CIAPIN1 inhibited proliferation and triggered more apoptosis of K562CML (chronic myeloid leukemia) cells with or without Imatinib treatment. Meanwhile, CIAPIN1 depletion decreased ERK5 phosphorylation and NF-κB activity. Importantly, treating CIAPIN1-depleted K562 cells with ERK5 signaling pathway specific inhibitor, XMD8-92, further inhibited proliferation and promoted apoptosis with or without Imatinib treatment. Treatment with the NF-κB specific inhibitor, Bay 11-7082, induced nearly the same inhibition of proliferation and promotion of apoptosis conferred by CIAPIN1 depletion as was observed with XMD8-92 treatment. Further, XMD8-92 and Bay 11-7082 synergistically inhibited proliferation and promoted apoptosis of CIAPIN1-depleted K562 cells with or without Imatinib treatment. The nude mice transplantation model was also performed to confirm the enhanced sensitivity of CIAPIN1-depleted K562 cells to Imatinib. Thus, our results provided a potential management by which CIAPIN1 knock-down might have a crucial impact on enhancing sensitivity of K562 cells to Imatinib in the therapeutic approaches, indicating that CIAPIN1 knock-down might serve as a combination with chemotherapeutical agents in leukemia diseases therapy.

Therapeutic effects of micheliolide on a murine model of rheumatoid arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease and collagen-induced arthritis (CIA) is an animal model for RA. Micheliolide (MCL) is a novel compound with strong anti-inflammatory effects. The present study was conducted to evaluate the therapeutic effects of MCL on RA. Mice were randomly divided into four groups and the CIA model mice were treated with methotrexate (MTX), MCL and dimethyl sulfoxide. A score associated with the severity of arthritis was assigned on alternate days from the 22nd day for 60 days. Histopathological changes and the serum levels of cytokines were measured on day 85. The results demonstrated that the MCL treatment group had arthritis scores lower than the CIA group and higher than the MTX group; compared with the CIA group, MCL and MTX significantly reduced the swelling of the paws and suppressed the degeneration of articular cartilage. Expression levels of macrophage colony-stimulating factor (M-CSF), tissue inhibitors of metalloproteinases-1 (TIMP-1) and complement component 5a (C5/C5a) were lower in the mice with arthritis compared with normal mice, however, following treatment with MCL and MTX, all the mice exhibited significant recovery to differing degrees. Unlike the MTX group, the MCL group failed to recover the level of soluble intercellular adhesion molecule-1. In addition, the cytokine of B-lymphocyte chemoattractant (BLC) solely presented in the MCL group. These results suggest that MCL may be considered for use as a novel therapeutic treatment against RA and that changes in the expression of cytokines C5/C5a, TIMP-1, M-CSF and BLC may underlie the mechanism by which MCL effects changes in this disease.

CIAPIN1 targets Na+/H+ exchanger 1 to mediate K562 chronic myeloid leukemia cells' differentiation via ERK1/2 signaling pathway.

CIAPIN1 (cytokine-induced antiapoptotic inhibitor 1) was recently identified as an essential downstream effector of the Ras signaling pathway. However, its potential role in regulating myeloid differentiation remains unclear. In this study, we found depletion of CIAPIN1 by shRNAs led to granulocytic differentiation of K562 cells. Meanwhile, the decrease of NHE1 and up-regulation of phosphorylated ERK1/2 were observed after CIAPIN1 depletion. Interestingly, targeted inhibition of NHE1 further promoted the differentiation of K562 cells with CIAPIN1 silencing. Accordingly, ectopic expression of NHE1 reversed this phenotype. Furthermore, ERK1/2 inhibition with the chemical inhibitor, PD98059, abolished CIAPIN1 silencing-induced differentiation of K562 cells after NHE1 inhibition. Thus, our results revealed important mechanism that CIAPIN1 targeted NHE1 to mediate differentiation of K562 cells via ERK1/2 pathway. Our findings implied CIAPIN1 and NHE1 could be new targets in developing therapeutic strategies against leukemia.

[Effects of Sam68 gene silence on proliferation of acute T lymphoblastic leukemia cell line Jurkat].

This study was purpose to investigate the effect of Sam68 gene silence on proliferation of human acute T lymphoblastic leukemia cell line Jurkat. The sequence of shRNA targeting the site 531-552 of Sam68 mRNA was designed and chemically synthesized, then a single-vector lentiviral, Tet-inducible shRNA-Sam68 system (pLKO-Tet-On) was constructed; next the Jurkat cells were infected with lentivirus to create stable cell clones with regulatable Sam68 gene expression. The inhibitory efficiency of Sam68 gene was assayed by Real-time PCR and Western blot; the cell activity of Jurkat cells was detected with MTT assay; the change of colony forming potential of Jurkat cells was analyzed by colony forming test; the cell cycle distribution was tested by flow cytometry. The results indicated that the expression of Sam68 in experimental cells was statistically decreased as compared with that of the control cells; the cells activity and colony forming capacity of the Jurkat cells with Sam68 gene silence were significantly inhibited; with Sam68 gene silencing, the percentage of S phase cells was significantly increased, while the percentage of G2 phase cells was significantly decreased. It is concluded that the silencing Sam68 gene using shRNA interference can effectively inhibit the proliferation of human acute T lymphoblastic leukemia cell line Jurkat.