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1.  Overexpression of Wild-Type but Not C134W Mutant FOXL2 Enhances GnRH-Induced Cell Apoptosis by Increasing GnRH Receptor Expression in Human Granulosa Cell Tumors 
PLoS ONE  2013;8(1):e55099.
The etiology of granulosa cell tumors (GCTs) is largely unknown. The primary mode of treatment is surgical, however not all women are cured by surgery alone. Thus, it is important to develop improved treatments through a greater understanding of the molecular mechanisms that contribute to this disease. Recently, it has been shown that a FOXL2 402C>G (C134W) mutation is present in 97% of human adult-type GCTs, suggesting an important role for this mutation in the development of GCTs. We have shown previously that gonadotropin-releasing hormone (GnRH)-I and -II induce apoptosis in cultured normal human granulosa cells. Moreover, it has been reported that FOXL2 can bind to the promoter of the mouse GnRH receptor gene and regulate its transcription. Thus, we hypothesized that C134W mutant FOXL2 could modulate the pro-apoptotic effects of GnRH via aberrant regulation of GnRH receptor levels. Using KGN cells, a human GCT-derived cell line which harbors the FOXL2 402C>G mutation, we show that treatment with GnRH-I and -II induces cell apoptosis, and that small interfering RNA-mediated depletion of GnRH receptor abolishes these effects. Overexpression of wild-type FOXL2 increases both mRNA and protein levels of GnRH receptor and consequently enhances GnRH-induced apoptosis. Importantly, neither the expression levels of GnRH receptor nor GnRH-induced apoptosis were affected by overexpression of the C134W mutant FOXL2. Interestingly, knockdown of endogenous FOXL2 down-regulates GnRHR expression in normal human granulosa cells with wild-type FOXL2, but not in KGN cells. These results suggest that the FOXL2 402C>G mutation may contribute to the development of human adult-type GCTs by reducing the expression of GnRH receptor, thus conferring resistance to GnRH-induced cell apoptosis.
PMCID: PMC3553060  PMID: 23372819
2.  HOXA4 protein levels and localization in the aorta and in human abdominal aortic aneurysms 
BMC Physiology  2011;11:18.
This report presents evidence for the specificities of select commercially available HOXA4 antibodies in regards to concerns about the specificity of the HOXA4 antibody used by Lillvis et al. (Regional expression of HOXA4 along the aorta and its potential role in human abdominal aortic aneurysms. BMC Physiol 2011, 11:9). Using an antibody characterized extensively by us, Lillvis et al. report detecting HOXA4 at a size of 33 kDa despite our previous reports that HOXA4 is detected at ~37-39 kDa and that the ~30-33 kDa band is non-specific. Using small interfering RNA targeting HOXA4, forced expression of full-length HOXA4 and HOXA4-positive and -negative ovarian cancer cell lines, we confirm our previous findings that the ~30-33 kDa band is non-specific and that HOXA4 is detected at ~37-39 kDa. Moreover, we demonstrate that HOXA4 small interfering RNA reduces the ~37-39 kDa HOXA4 band, but not the ~30-33 kDa non-specific band, in a human acute monocytic leukemia cell line used by Lillvis et al. Western blot analysis performed with two additional commercially available HOXA4 antibodies also detected HOXA4 at ~37-39 kDa. Lastly, immunofluorescent staining of a HOXA4-negative ovarian cancer cell line with the antibody used by Lillvis et al. yields strong perinuclear staining, similar to that observed by Lillvis et al., which cannot be attributed to HOXA4. Our results highlight and briefly discuss the importance of careful antibody validation and selection for use in various applications.
PMCID: PMC3254126  PMID: 22168796
3.  Kisspeptin and GPR54 immunoreactivity in a cohort of 518 patients defines favourable prognosis and clear cell subtype in ovarian carcinoma 
BMC Medicine  2007;5:33.
Kisspeptins and their G-protein coupled receptor, GPR54 are required for GnRH release and have been associated with anti-metastatic tumour cell behaviour in model systems. The latter might suggest that their overexpression would be associated with a better prognosis in cancer. However, kisspeptin/GPR54 interactions (autocrine, paracrine, and/or endocrine) could also impact tumour behaviour in a negative manner. Here, for the first time, we associate the immunoreactivity of the kisspeptin/GPR54 ligand-receptor pair with favourable prognosis in a large cohort of ovarian carcinomas.
Immunohistochemical analysis for kisspeptin and GPR54 was performed on a tissue microarray (TMA) consisting of 518 early stage ovarian carcinomas, all with linked clinical outcome data. The TMA was scored using a staining intensity scale of 0 (negative), +1 (mild-moderate), and +2 (strong). Strong staining cases were considered either kisspeptin or GPR54 positive and designated as 1, while all other cases were considered negative and designated 0. All statistical analysis was conducted using two-sided tests and a p value equal to or less than 0.05 was considered significant.
Kisspeptin and GPR54 immunoreactive cases show a favourable prognosis in univariable disease specific survival (p = 0.0023, p = 0.0092), as well as in overall survival (p = 0.0006, p = 0.0002). Furthermore, kisspeptin is an independent marker for favourable prognosis as determined by multivariable disease specific (p = 0.0046) and overall survival analysis (p = 0.0170), while GPR54 is an independent marker for overall survival only (p = 0.0303). Both kisspeptin positive and GPR54 positive cases are strongly associated with the ovarian carcinoma clear cell subtype (p < 0.0001, p < 0.0001), and GPR54 is significantly associated with favourable prognosis in overall survival within the clear cell subtype (p = 0.0102).
Kisspeptin and GPR54 immunoreactivity are significantly associated with favourable prognosis in both disease specific and overall survival, as well as being significantly associated with the clear cell ovarian carcinoma subtype, thereby creating the first independent prognostic biomarkers specific for ovarian clear cell carcinomas.
PMCID: PMC2200658  PMID: 18005407

Results 1-3 (3)