KCNJ3 is a new independent prognostic marker for estrogen receptor positive breast cancer patients

Numerous studies showed abnormal expression of ion channels in different cancer types. Amongst these, the potassium channel gene KCNJ3 (encoding for GIRK1 proteins) has been reported to be upregulated in tumors of patients with breast cancer and to correlate with positive lymph node status. We aimed to study KCNJ3 levels in different breast cancer subtypes using gene expression data from the TCGA, to validate our findings using RNA in situ hybridization in a validation cohort (GEO ID GSE17705), and to study the prognostic value of KCNJ3 using survival analysis. In a total of > 1000 breast cancer patients of two independent data sets we showed a) that KCNJ3 expression is upregulated in tumor tissue compared to corresponding normal tissue (p < 0.001), b) that KCNJ3 expression is associated with estrogen receptor (ER) positive tumors (p < 0.001), but that KCNJ3 expression is variable within this group, and c) that ER positive patients with high KCNJ3 levels have worse overall (p < 0.05) and disease free survival probabilities (p < 0.01), whereby KCNJ3 is an independent prognostic factor (p <0.05). In conclusion, our data suggest that patients with ER positive breast cancer might be stratified into high risk and low risk groups based on the KCNJ3 levels in the tumor

Critical evaluation of KCNJ3 gene product detection in human breast cancer: mRNA in situ hybridisation is superior to immunohistochemistry

Increased expression levels of KCNJ3 have been correlated with lymph node metastases and poor prognosis in patients with breast cancer, suggesting a prognostic role of KCNJ3. We aimed to establish protocols for the detection of KCNJ3 in formalin-fixed, paraffin-embedded (FFPE) breast cancer tissue. Several antibodies were tested for sensitivity and specificity by western blot, followed by optimisation of the immunohistochemistry (IHC) procedure and establishment of KCNJ3 mRNA in situ hybridisation (ISH). Methods were validated by processing 15 FFPE breast cancer samples for which microarray data were available. Spearman's rank correlation analysis resulted in borderline significant correlation for IHC versus ISH (r(S): 0.625; p<0.05) and IHC versus microarray (r(S): 0.668; p<0.01), but in significant correlation for ISH versus microarray (r(S): 0.861; p<0.001). The ISH method was superior to IHC, regarding robustness, sensitivity and specificity and will aid to further study expression levels of KCNJ3 in both malignant and physiological conditions

Lipopolysaccharide- And lipoteichoic acid-mediated pro-inflammatory cytokine production and modulation of TLR2, TLR4 and MyD88 expression in human endometrial cells

Background: Toll-like receptor (TLR)-mediated inflammatory processes are supposed to be involved in pathophysiology of spontaneous abortion and preterm labor. Here, we investigated functional responses of human endometrial stromal cells (ESCs) and whole endometrial cells (WECs) to lipopolysaccharide (LPS) and lipoteichoic acid (LTA). Methods: Endometrial tissues were obtained from 15 cycling women who underwent laparoscopic tubal ligation. Modulation of TLR2, TLR4 and MyD88 expression and production of pro-inflammatory cytokines by WECs and ESCs in response to LPS and LTA were assessed. Results: WECs and ESCs expressed significant levels of TLR4 and MyD88 transcripts but, unlike WECs, ESCs failed to express TLR2 gene. Regardless of positive results of Western blotting, ESCs did not express TLR4 at their surface as judged by flow cytometry. Immunofluorescent staining revealed intracellular localization of TLR4 with predominant perinuclear pattern. LPS stimulation marginally increased TLR4 gene expression in both cell types, whereas such treatment significantly upregulated MyD88 gene expression after 8 hr (p<0.05). At the protein level, however, LPS activation significantly increased TLR4 expression by ESCs (p<0.05). LTA stimulation of WECs was accompanied with non-significant increase of TLR2 and MyD88 transcripts. LPS and LTA stimulation of WECs caused significant production of IL-6 and IL-8 in a dosedependent manner (p<0.05). Similarly, ESCs produced significant amounts of IL-6, IL-8 and also TNF-α in response to LPS activation (p<0.05). Conclusion: Our results provided further evidence of initiation of inflammatory processes following endometrial TLR activation by bacterial components which could potentially be harmful to developing fetus

Placental Kisspeptins Differentially Modulate Vital Parameters of Estrogen Receptor-Positive and -Negative Breast Cancer Cells.

Kisspeptins (KPs) are major regulators of trophoblast and cancer invasion. Thus far, limited and conflicting data are available on KP-mediated modulation of breast cancer (BC) metastasis; mostly based on synthetic KP-10, the most active fragment of KP. Here, we report for the first time comprehensive functional effects of term placental KPs on proliferation, adhesion, Matrigel invasion, motility, MMP activity and pro-inflammatory cytokine production in MDA-MB-231 (estrogen receptor-negative) and MCF-7 (estrogen receptor-positive). KPs were expressed at high level by term placental syncytiotrophoblasts and released in soluble form. Placental explant conditioned medium containing KPs (CM) significantly reduced proliferation of both cell types compared to CM without (w/o) KP (CM-w/o KP) in a dose- and time-dependent manner. In MDA-MB-231 cells, placental KPs significantly reduced adhesive properties, while increased MMP9 and MMP2 activity and stimulated invasion. Increased invasiveness of MDA-MB-231 cells after CM treatment was inhibited by KP receptor antagonist, P-234. CM significantly reduced motility of MCF-7 cells at all time points (2-30 hr), while it stimulated motility of MDA-MB-231 cells. These effects were reversed by P-234. Co-treatment with selective ER modulators, Tamoxifen and Raloxifene, inhibited the effect of CM on motility of MCF-7 cells. The level of IL-6 in supernatant of MCF-7 cells treated with CM was higher compared to those treated with CM-w/o KP. Both cell types produced more IL-8 after treatment with CM compared to those treated with CM-w/o KP. Taken together, our observations suggest that placental KPs differentially modulate vital parameters of estrogen receptor-positive and -negative BC cells possibly through modulation of pro-inflammatory cytokine production

Effect of term placental kisspeptins on proliferation of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF7 (B) cells were treated with different media as indicated in the figure for 24, 48 and 72 hr. All treatments were performed in six replicas. The extent of proliferation was measured by Propidium Iodide (PI) fluorometric assay. Significant differences were analyzed by Kruskal-Wallis test with a Dunnett’s posthoc test. Different dilutions from 1:2 to 1:8 were tested. CM: Term placenta conditioned medium containing KP, CM-w/o KP: KP-free CM, anti-KP: anti-Kp-54/Kp-145 antibody, Rb Ig: Rabbit immunoglobulin, Medium: Culture medium alone, KP: Kisspeptin. Results are representative of 7 term placentas. * CM <i>vs</i>. CM-w/o KP(p<0.05–0.01 for MDA-MB-231 and p<0.05–0.001 for MCF-7), φ CM <i>vs</i>. medium (p<0.01–0.001 for MDA-MB-231 and p<0.05–0.01 for MCF-7), β CM <i>vs</i>. CM+anti-KP-10 (p<0.05–0.01 for MDA-MB-231 and p<0.05 for MCF-7), Φ CM-w/o KP <i>vs</i>. medium (p<0.05–0.001 for MDA-MB-231 and p<0.05 for MCF-7), ψ KP-10 <i>vs</i>. medium (p<0.05–0.01 for MDA-MB-231 and p<0.05–0.01 for MCF-7).</p

Effect of term placental kisspeptins on adhesion of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF-7 (B) cells were treated with different media as indicated in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153684#pone.0153684.g003" target="_blank">Fig 3</a> for 150 min and their adhesion to fibronectin-coated plates was assessed by a colorimetric assay. Different dilutions from 1:2 to 1:8 were tested. CM: Term placenta conditioned medium containing KP, CM-w/o KP: KP-free CM, anti-KP: anti-Kp-54/Kp-145 antibody, Rb Ig: Rabbit immunoglobulin, Medium: Culture medium alone, KP: Kisspeptin. Results are representative of 11 term placentas. * CM <i>vs</i>. CM-w/o KP(p<0.05 for MDA-MB-231).</p

Effect of term placental kisspeptins on invasiveness of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF-7 (B) cells were treated with supernatants of cultured term placental explants containing KP (CM), CM plus 1 μM KISS1R antagonist; P-234, KP-free CM (CM-w/o KP), KP-10 or culture medium alone and their invasion toward chemo attractant (FCS) through Matrigel-coated filters was investigated. Invading cells was enumerated in 50 random fields (Ab and Bb) and expressed as percentage of corresponding controls (Aa and Ba). C) Gelatin zymographic analysis for matrix metalloproteinase (MMP) expression. MDA-MB-231 cells treated with CM released higher amounts of MMP2 and MMP9 compared to those treated with either CM-w/o KP. In parallel, cells treated with KP-10 showed slightly higher MMP9 activity in comparison to cultured in medium alone. Cells treated with Phorbol 12-myristate 13-acetate (PMA) served as positive control. CM failed to express detectable levels of MMP2 and MMP9. Results of two (denoted by numbers) out of four samples are shown. KP: Kisspeptin, MW: Molecular weight.</p

Effect of term placental kisspeptins on motility of MDA-MB-231 and MCF-7 breast cancer cells.

<p>MDA-MB-231 (A) and MCF-7 (B) cells were treated with supernatants of cultured term placental explants containing KP (CM), KP-free CM (CM-w/o KP), KP-10 or culture medium alone and their migration toward scratch was monitored during a period of 30 hr. Representative photographs are shown in Aa (MDA-MB-231) and Ba (MCF-7). Percent of reduction in scratched areas at each time point compared to initial time point was measured and analyzed using Image J software (Ab for MDA-MB-231 and Bb for MCF-7). Treatment with P-234 reversed the effect of CM on motility of MDA-MB-231 (Ac) and MCF-7 (Bc). Co-treatment with selective ER modulators inhibited the effect of CM on motility of MCF-7 cells (Bc). * CM <i>vs</i>. CM-w/o KP (p<0.05–0.001 for MDA-MB-231 and p<0.05–0.001), φ CM <i>vs</i>. medium (p<0.01–0.001 for MDA-MB-231 and p<0.001–0.0001 for MCF-7), ψ KP-10 <i>vs</i>. medium (p<0.05–0.01 for MDA-MB-231 and p<0.01–0.0001 for MCF-7),? CM <i>vs</i>. CM-w/o KP and CM+P-234 (p<0.05),? CM-w/o KP, CM+P-234, CM+100 nm Tam and CM+1 nm Ral <i>vs</i>. CM (p<0.05). Results are representative of 4 term placentas. Tam: Tamoxifen, Ral: Raloxifene.</p