Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity, Density, and Beat

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered

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Basal feet polarity of brain ependymal cells and trachea epithelial cells was lost in the <i>Spag6</i>-deficient mice.

<p>Basal body images were taken with a transmission electron microscope. Notice that the basal feet point to the same orientation in the wild type animals (A: brain; C: trachea). However, they point to different orientation in the <i>Spag6</i>-deficient mice (B: brain; D: trachea). The number of basal body in the <i>Spag6</i>-deficient mice was significantly reduced in both brain and trachea. The arrows point to the basal feet. E. Average basal feet number counted from ten images randomly selected from each group. Horizontal lines represent means and SEMs. Three or four images were counted from each mouse, and three wild-type and three mutant mice were examined. * p<0.05. F. Circular plots of tracheal epithelial cell basal feet orientation in <i>Spag6</i>-deficient (left) and wild-type mice (right). For each mouse, basal foot orientation from five images was analyzed. For each image, the angel for one basal foot orientation was set as 0° (or 360°), angels of the rest basal feet were measured. Each plot represents the combined data from three mice as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0107271#pone.0107271.s002" target="_blank">Figure S2</a> (* p<0.001 between the two groups).</p

Examination of rotational polarity of ciliary axoneme of brain ependymal cells and trachea epithelial cells by transmission electronic miscroscopy.

<p>Axoneme cross-sectional images were taken with a transmission electron microscope. The rotational polarity of each axoneme was evaluated by the angle of the line connecting the central pair. Notice that the orientation of the lines in wild type mice is similar (A: brain; C: trachea). while the orientation of the lines in the <i>Spag6</i>-deficient mice varies among axonemes (B: brain; D: trachea). E. Average axoneme number counted from ten images randomly selected from each group. Three or four images were counted from each mouse, and three wild-type and three mutant mice were examined. Horizontal lines represent the means and SEMs. * p<0.05.</p

Analyses of cilia in the trachea epithelial cells and brain ependymal cells by scanning electronic microscopy and immunofluorescence staining.

<p>Representative images from SEM analyses from wild-type and <i>Spag6</i>-deficient mice. Cilia of the wild type mice were well-organized in both brain ependymal cells (A) and trachea epithelial cells (C). In contrast, there was a dramatic reduction in cilia density in both brain ependymal cells (B) and trachea epithelial cells (D) of the <i>Spag6</i>-deficient mice, and the cilia were disorganized. To calculate the percentage of ciliated cells, cells with cilia and total cells were counted from three (brain) or four (trachea) SEM images from each mouse, and ratio was calculated (E). Three wild-type and three <i>Spag6</i>-deficient mice were analyzed. * Significant differences vs. wild-type (p<0.05). Brain and trachea sections from wild type and <i>Spag6</i>-deficient mice were examined by immunofluorescence staining using an antibody targeting acetylated tubulin. In the wild type mice, the cilia-containing signal was continuously observed along the surface of the epithelial cells. However, in the <i>Spag6</i>-deficient mice, the signal was discontinuous (F).</p

SIAH and EGFR, Two RAS Pathway Biomarkers, are Highly Prognostic in Locally Advanced and Metastatic Breast Cancer

Background: Metastatic breast cancer exhibits diverse and rapidly evolving intra- and inter-tumor heterogeneity. Patients with similar clinical presentations often display distinct tumor responses to standard of care (SOC) therapies. Genome landscape studies indicate that EGFR/HER2/RAS “pathway” activation is highly prevalent in malignant breast cancers. The identification of therapy-responsive and prognostic biomarkers is paramount important to stratify patients and guide therapies in clinical oncology and personalized medicine. Methods: In this study, we analyzed matched pairs of tumor specimens collected from 182 patients who received neoadjuvant systemic therapies (NST). Statistical analyses were conducted to determine whether EGFR/HER2/RAS pathway biomarkers and clinicopathological predictors, alone and in combination, are prognostic in breast cancer. Findings: SIAH and EGFR outperform ER, PR, HER2 and Ki67 as two logical, sensitive and prognostic biomarkers in metastatic breast cancer. We found that increased SIAH and EGFR expression correlated with advanced pathological stage and aggressive molecular subtypes. Both SIAH expression post-NST and NST-induced changes in EGFR expression in invasive mammary tumors are associated with tumor regression and increased survival, whereas ER, PR, and HER2 were not. These results suggest that SIAH and EGFR are two prognostic biomarkers in breast cancer with lymph node metastases. Interpretation: The discovery of incorporating tumor heterogeneity-independent and growth-sensitive RAS pathway biomarkers, SIAH and EGFR, whose altered expression can be used to estimate therapeutic efficacy, detect emergence of resistant clones, forecast tumor regression, differentiate among partial responders, and predict patient survival in the neoadjuvant setting, has a clear clinical implication in personalizing breast cancer therapy. Funding: This work was supported by the Dorothy G. Hoefer Foundation for Breast Cancer Research (A.H. Tang); Center for Innovative Technology (CIT)-Commonwealth Research Commercialization Fund (CRCF) (MF14S-009-LS to A.H. Tang), and National Cancer Institute (CA140550 to A.H. Tang)