Quantification of testicular fat content: the value of evaluating testicular function after cryptorchidism surgery.

BACKGROUND: To investigate the correlation between testicular fat content (TFC) and sex hormone levels in patients with cryptorchidism and its value in assessing postsurgical testicular function. METHODS: Pelvic MRI with the mDIXON Quant sequence was performed on 23 cryptorchidism patients and 15 normal controls. The TFC before and after surgery was measured and compared. The correlations between cryptorchid TFC and testosterone (TSTO), follicle-stimulating hormone (FSH), and estradiol (E2) levels were analyzed, as was the specificity of TFC and each hormone for assessing testicular function after surgery. RESULTS: The preoperative cryptorchid TFC (3.06% ± 0.74) was higher than that of the normal controls (1.36% ± 0.49). TSTO was negatively correlated with the cryptorchid TFC (r = -0.698), while FSH and E2 were positively associated with the cryptorchid TFC (r = 0.658, 0.676). Cryptorchid TFC after surgery (2.01% ± 0.55) was lower than the preoperative TFC, but hormone levels were not significantly different. The TFC after surgery (0.864) had a larger AUC value than did TSTO (0.639), FSH (0.597), and E2 (0.586). CONCLUSION: Noninvasive quantification of cryptorchid TFC using the mDIXON Quant sequence is more specific than hormone levels for assessing postsurgical changes in testicular function. IMPACT: The cryptorchid testicular fat content is significantly higher than the normal testicular fat content. Cryptorchid testicular fat content is negatively correlated with presurgical serum TSTO levels and positively correlated with presurgical FSH and E2 levels. Pre- and postoperative changes in cryptorchid testicular fat content change are more sensitive than changes in TSTO, FSH, or E2 levels. Noninvasive cryptorchid testicular fat content quantified by the mDIXON Quant sequence is more specific than serum TSTO, FSH, and E2 levels for assessing changes in testicular function after cryptorchidism surgery.

Biomarkers of preschool children with autism spectrum disorder: quantitative analysis of whole-brain tissue component volumes, intelligence scores, ADOS-CSS, and ages of first-word production and walking onset.

BACKGROUND: Preschooling is a critical time for intervention in children with autism spectrum disorder (ASD); thus, we analyzed brain tissue component volumes (BTCVs) and clinical indicators in preschool children with ASD to identify new biomarkers for early screening. METHODS: Eighty preschool children (3-6 years) with ASD were retrospectively included. The whole-brain myelin content (MyC), white matter (WM), gray matter (GM), cerebrospinal fluid (CSF), and non-WM/GM/MyC/CSF brain component volumes were obtained using synthetic magnetic resonance imaging (SyMRI). Clinical data, such as intelligence scores, autism diagnostic observation schedule-calibrated severity scores, age at first production of single words (AFSW), age at first production of phrases (AFP), and age at walking onset (AWO), were also collected. The correlation between the BTCV and clinical data was evaluated, and the effect of BTCVs on clinical data was assessed by a regression model. RESULTS: WM and GM volumes were positively correlated with intelligence scores (both P < 0.001), but WM and GM did not affect intelligence scores (P = 0.116, P = 0.290). AWO was positively correlated with AFSW and AFP (both P < 0.001). The multivariate linear regression analysis revealed that MyC, AFSW, AFP, and AWO were significantly different (P = 0.005, P < 0.001, P < 0.001). CONCLUSIONS: This study revealed positive correlations between WM and GM volumes and intelligence scores. Whole-brain MyC affected AFSW, AFP, and AWO in preschool children with ASD. Noninvasive quantification of BTCVs via SyMRI revealed a new visualizable and quantifiable biomarker (abnormal MyC) for early ASD screening in preschool children.

NF-κB represses retinoic acid receptor-mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia.

Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulation plays a critical role. Lung tumor suppressor G protein-coupled receptor, family C, member 5A (GPRC5A), is repressed in most non-small cell lung cancer (NSCLC); however, the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induced GPRC5A repression both in vitro and in vivo. Intriguingly, transactivation of NF-κB downstream targets was not required, but the transactivation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in the GPRC5A promoter. Instead, p65 was complexed with retinoic acid receptor α/ß (RARα/ß) and recruited to the RA response element site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complexing and suppressed transcription. Notably, phosphorylation on serine 276 of p65 was required for interaction with RARα/ß and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression was through suppression of acetylated histone H3K9 (H3K9ac), but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a histone deacetylase inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via a complex with RARα/ß and mediates epigenetic repression via suppression of H3K9ac.

Comparison of the modified laparoscopic Vecchietti and Davydov colpoplasty techniques in Mayer-Rokitansky-Küster-Hauser syndrome: A long-term follow-up analysis.

AIMS: To introduce and compare the modified laparoscopic Vecchietti and Davydov techniques for vaginoplasty in patients with Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome. Moreover, the long-term treatment of vaginal agenesis was followed-up. METHODS: This comparative retrospective cohort study enrolled a total of 53 women with MRKH syndrome. The patients underwent surgical creation of a neovagina including 32 patients who underwent the modified laparoscopic Vecchietti technique, and 21 patients who underwent the modified laparoscopic Davydov technique from January 2009 to February 2019. The perioperative parameters, complications, anatomical, and functional outcomes of the two groups were compared. Patients' sexual functions were evaluated over a long-term follow-up using the female sexual function index (FSFI) and the revised female sexual distress scale (FSDS-R). RESULTS: The medians (25th-75th) of the surgery duration for modified Vecchietti procedures was 50.0 (40.0-59.0) minutes, comparing to 135.0 (117.5-162.5) min for Davydov procedures (p < 0.001). The intraoperative blood loss was 20 (7.5-20.0) mL versus 50.0 (50.0-100.0) mL using the modified Vecchietti and Davydov approaches (p < 0.001), respectively. In the 39 follow-up cases, the lengths of the neovagina of the patients for Vecchietti group versus Davydov group were 7.9 ± 1.0 cm versus 8.6 ± 1.2 cm at 6 months after the vaginoplasty and 8.3 ± 0.7 cm versus 8.5 ± 0.9 cm after 2 years. There was no statistical difference in the FSFI and FSDS-R scores between the two groups. CONCLUSIONS: Both the modified Davydov and Vecchietti laparoscopic procedures successfully achieved optimal anatomic and functional outcomes in treatments of vaginal agenesis. The modified Vecchietti technique is relatively simpler than the modified Davydov technique.

Epithelial neoplasia coincides with exacerbated injury and fibrotic response in the lungs of Gprc5a-knockout mice following silica exposure.

Exposure to crystalline silica is suggested to increase the risk for a variety of lung diseases, including fibrosis and lung cancer. However, epidemiological evidences for the exposure-risk relationship are ambiguous and conflicting, and experimental study from a reliable animal model to explore the relationship is lacking. We reasoned that a mouse model that is sensitive to both lung injury and tumorigenesis would be appropriate to evaluate the exposure-risk relationship. Previously, we showed that, Gprc5a-/- mice are susceptible to both lung tumorigenesis and endotoxin-induced acute lung injury. In this study, we investigated the biological consequences in Gprc5a-/- mouse model following silica exposure. Intra-tracheal administration of fine silica particles in Gprc5a-/- mice resulted in more severe lung injury and pulmonary inflammation than in wild-type mice. Moreover, an enhanced fibrogenic response, including EMT-like characteristics, was induced in the lungs of Gprc5a-/- mice compared to those from wild-type ones. Importantly, increased hyperplasia or neoplasia coincided with silica-induced tissue injury and fibrogenic response in lungs from Gprc5a-/- mice. Consistently, expression of MMP9, TGFß1 and EGFR was significantly increased in lungs from silica-treated Gprc5a-/- mice compared to those untreated or wild-type ones. These results suggest that, the process of tissue repair coincides with tissue damages; whereas persistent tissue damages leads to abnormal repair or neoplasia. Thus, silica-induced pulmonary inflammation and injury contribute to increased neoplasia development in lungs from Gprc5a-/- mouse model.

Lung Tumor Suppressor GPRC5A Binds EGFR and Restrains Its Effector Signaling.

GPRC5A is a G-protein-coupled receptor expressed in lung tissue but repressed in most human lung cancers. Studies in Gprc5a(-/-) mice have established its role as a tumor-suppressor function in this setting, but the basis for its role has been obscure. Here, we report that GPRC5A functions as a negative modulator of EGFR signaling. Mouse tracheal epithelial cells (MTEC) from Gprc5a(-/-) mice exhibited a relative increase in EGFR and downstream STAT3 signaling, whereas GPRC5A expression inhibited EGFR and STAT3 signaling. GPRC5A physically interacted with EGFR through its transmembrane domain, which was required for its EGFR inhibitory activity. Gprc5a(-/-) MTEC were much more susceptible to EGFR inhibitors than wild-type MTEC, suggesting their dependence on EGFR signaling for proliferation and survival. Dysregulated EGFR and STAT3 were identified in the normal epithelia of small and terminal bronchioles as well as tumors of Gprc5a(-/-) mouse lungs. Moreover, in these lungs EGFR inhibitor treatment inhibited EGFR and STAT3 activation along with cell proliferation. Finally, overexpression of ectopic GPRC5A in human non-small cell lung carcinoma cells inhibited both EGF-induced and constitutively activated EGFR signaling. Taken together, our results show how GPRC5A deficiency leads to dysregulated EGFR and STAT3 signaling and lung tumorigenesis. Cancer Res; 75(9); 1801-14. ©2015 AACR.

G9a is essential for EMT-mediated metastasis and maintenance of cancer stem cell-like characters in head and neck squamous cell carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a particularly aggressive cancer with poor prognosis, largely due to lymph node metastasis and local recurrence. Emerging evidence suggests that epithelial-to-mesenchymal transition (EMT) is important for cancer metastasis, and correlated with increased cancer stem cells (CSCs) characteristics. However, the mechanisms underlying metastasis to lymph nodes in HNSCC is poorly defined. In this study, we show that E-cadherin repression correlates with cancer metastasis and poor prognosis in HNSCC. We found that G9a, a histone methyltransferase, interacts with Snail and mediates Snail-induced transcriptional repression of E-cadherin and EMT, through methylation of histone H3 lysine-9 (H3K9). Moreover, G9a is required for both lymph node-related metastasis and TGF-ß-induced EMT in HNSCC cells since knockdown of G9a reversed EMT, inhibited cell migration and tumorsphere formation, and suppressed the expression of CSC markers. Our study demonstrates that the G9a protein is essential for the induction of EMT and CSC-like properties in HNSCC. Thus, targeting the G9a-Snail axis may represent a novel strategy for treatment of metastatic HNSCC.

Gprc5a-deficiency confers susceptibility to endotoxin-induced acute lung injury via NF-κB pathway.

Susceptibility to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) varies greatly among patients in sepsis/septic shock. The genetic and biochemical reasons for the difference are not fully understood. G protein coupled receptor family C group 5 member A (GPRC5A), a retinoic acid target gene, is predominately expressed in the bronchioalveolar epithelium of lung. We hypothesized that Gprc5a is important in controlling the susceptibility to ALI or ARDS. In this study, we examined the susceptibility of wild-type and Gprc5a-knockout (ko) mice to induced ALI. Administration of endotoxin LPS induced an increased pulmonary edema and injury in Gprc5a-ko mice, compared to wild-type counterparts. Consistently, LPS administration induced higher levels of inflammatory cytokines (IL-1ß and TNFα) and chemokine (KC) in Gprc5a-ko mouse lungs than in wild-type. The enhanced pulmonary inflammatory responses were associated with dysregulated NF-κB signaling in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs. Importantly, selective inhibition of NF-κB through expression of the super-repressor IκBα in the bronchioalveolar epithelium of Gprc5a-ko mouse lungs alleviated the LPS-induced pulmonary injury, and inflammatory response. Thus, Gprc5a is critical for lung homeostasis, and Gprc5a deficiency confers the susceptibility to endotoxin-induced pulmonary edema and injury, mainly through NF-κB signaling in bronchioalveolar epithelium of lung.

EGFR phosphorylates and inhibits lung tumor suppressor GPRC5A in lung cancer.

BACKGROUND: GPRC5A is a retinoic acid inducible gene that is preferentially expressed in lung tissue. Gprc5a- knockout mice develop spontaneous lung cancer, indicating Gprc5a is a lung tumor suppressor gene. GPRC5A expression is frequently suppressed in majority of non-small cell lung cancers (NSCLCs), however, elevated GPRC5A is still observed in a small portion of NSCLC cell lines and tumors, suggesting that the tumor suppressive function of GPRC5A is inhibited in these tumors by an unknown mechanism. METHODS: In this study, we examined EGF receptor (EGFR)-mediated interaction and tyrosine phosphorylation of GPRC5A by immunoprecipitation (IP)-Westernblot. Tyrosine phosphorylation of GPRC5A by EGFR was systematically identified by site-directed mutagenesis. Cell proliferation, migration, and anchorage-independent growth of NSCLC cell lines stably transfected with wild-type GPRC5A and mutants defective in tyrosine phosphorylation were assayed. Immunohistochemical (IHC) staining analysis with specific antibodies was performed to measure the total and phosphorylated GPRC5A in both normal lung and lung tumor tissues. RESULT: We found that EGFR interacted with GPRC5A and phosphorylated it in two conserved double-tyrosine motifs, Y317/Y320 and Y347/ Y350, at the C-terminal tail of GPRC5A. EGF induced phosphorylation of GPRC5A, which disrupted GPRC5A-mediated suppression on anchorage-independent growth of NSCLC cells. On contrary, GPRC5A-4 F, in which the four tyrosine residues have been replaced with phenylalanine, was resistant to EGF-induced phosphorylation and maintained tumor suppressive activities. Importantly, IHC analysis with anti-Y317/Y320-P sites showed that GPRC5A was non-phosphorylated in normal lung tissue whereas it was highly tyrosine-phosphorylated in NSCLC tissues. CONCLUSION: GPRC5A can be inactivated by receptor tyrosine kinase via tyrosine phosphorylation. Thus, targeting EGFR can restore the tumor suppressive functions of GPRC5A in lung cancer.

Repression of G protein-coupled receptor family C group 5 member A is associated with pathologic differentiation grade of oral squamous cell carcinoma.

BACKGROUND: G protein-coupled receptor family C group 5 member A (GPRC5A), a member of G protein-coupled receptor family, has been shown to function as a tumor suppressor in lung tissue. The biological functions of GPRC5A have therefore been linked to lung tissue. However, the biological significance of this gene product remains obscure. In this study, we investigated the expression of GPRC5A proteins in normal oral tissue and oral squamous cell carcinoma (OSCC), and we characterized its biological activity in OSCC cell lines. METHODS: Western blot analysis and immunohistochemical staining were used to investigate the expression of GPRC5A in both OSCC cell lines and clinical samples. GPRC5A stable transfectants and their parental OSCC cells were characterized for their biological activities in anchorage-independent growth. RESULTS: High levels of immunohistochemical GPRC5A expression were detected in normal oral tissue, especially differentiated area. In contrast, GPRC5A expression was dramatically repressed in OSCCs (P < 0.01). The immunohistochemical GPRC5A expression was moderately well differentiated, but greatly repressed in moderately differentiated OSCCs and completely repressed in poorly differentiated OSCCs. Overexpression of GPRC5A in OSCC CAL27 cells resulted in a suppressed anchorage-independent growth activity, a transforming phenotype. CONCLUSIONS: GPRC5A is expressed in normal oral epithelium. Repression of GPRC5A is associated with poorly differential grade of OSCCs. Overexpression of GPRC5A in OSCC cell line reversed the malignant phenotype. Thus, GPRC5A is important for homeostasis in oral tissue, and deletion or repression of this gene may involve in tumorigenesis of OSCCs and may serve as a prognostic marker for malignant type of OSCCs.