Daohu Wang

Vasculogenic Mimicry in Prostate Cancer: The Roles of EphA2 and PI3K.

BACKGROUND. Aggressive tumor cells can form perfusable networks that mimic normal vasculature and enhance tumor growth and metastasis. A number of molecular players have been implicated in such vasculogenic mimicry, among them the receptor tyrosine kinase EphA2, which is aberrantly expressed in aggressive tumors. Here we study the role and regulation of EphA2 in vasculogenic mimicry in prostate cancer where this phenomenon is still poorly understood. METHODS. Vasculogenic mimicry was characterized by tubules whose cellular lining was negative for the endothelial cell marker CD34 but positive for periodic acid-Schiff staining, and/or contained red blood cells. Vasculogenic mimicry was assessed in 92 clinical samples of prostate cancer and analyzed in more detail in three prostate cancer cell lines kept in three-dimensional culture. Tissue samples and cell lines were also assessed for total and phosphorylated levels of EphA2 and its potential regulator, Phosphoinositide 3-Kinase (PI3K). In addition, the role of EphA2 in vasculogenic mimicry and in cell migration and invasion were investigated by manipulating the levels of EphA2 through specific siRNAs. Furthermore, the role of PI3K in vasculogenic mimicry and in regulating EphA2 was tested by application of an inhibitor, LY294002. RESULTS. Immunohistochemistry of prostate cancers showed a significant correlation between vasculogenic mimicry and high expression levels of EphA2, high Gleason scores, advanced TNM stage, and the presence of lymph node and distant metastases. Likewise, two prostate cancer cell lines (PC3 and DU-145) formed vasculogenic networks on Matrigel and expressed high EphA2 levels, while one line (LNCaP) showed no vasculogenic networks and lower EphA2 levels. Specific silencing of EphA2 in PC3 and DU-145 cells decreased vasculogenic mimicry as well as cell migration and invasion. Furthermore, high expression levels of PI3K and EphA2 phosphorylation at Ser897 significantly correlated with the presence of vasculogenic mimicry and in vitro inhibition of PI3K by LY294002 disrupted vasculogenic mimicry, potentially through a reduction of EphA2 phosphorylation at Ser897. CONCLUSIONS. The expression levels of PI3K and EphA2 are positively correlated with vasculogenic mimicry both in vivo and in vitro. Moreover, phosphorylation levels of EphA2 regulated by PI3K are also significantly associated with vasculogenic mimicry in vivo. Based on its functional implication in vasculogenic mimicry in vitro, EphA2 signaling may be a potential therapeutic target in advanced prostate cancer.

Association of DSC3 mRNA down-regulation in prostate cancer with promoter hypermethylation and poor prognosis.

Background Desmocollin 3 (DSC3), a member of the cadherin gene superfamily, is associated with pathogenesis of some cancers, but its role in prostate cancer (PCa) remains largely unknown. Methods DSC3 gene expression level in available PCa microarray dataset was examined using the Oncomine database. DSC3 transcript expression in prostate cell line panel and an independent tissue cohort (n = 52) was estimated by quantitative PCR (Q-PCR). Epigenetic status of DSC3 gene promoter in PCa was investigated by uploading three dataset (ENCODE Infinium 450K array data and two methylation sequencing) in UCSC genome browser. While pyrosequencing analysis measured promoter DNA methylation, Q-PCR estimates were obtained for DSC3 transcript re-expression after 5-Aza-deoxycytidine (5-Aza) treatment. Clinical relevance of DSC3 expression was studied by Kaplan-Meier survival analysis. Finally, functional studies monitoring cell proliferation, migration and invasion were performed in prostate cell lines after siRNA mediated DSC3 knockdown or following 5-Aza induced re-expression. EMT markers Vimentin and E-cadherin expression was measured by Western Blot. Results Microarray data analyses revealed a significant decrease in DSC3 transcript expression in PCa, compared to benign samples. Q-PCR analysis of an independent cohort revealed DSC3 transcript down-regulation, both in PCa cell lines and tumor tissues but not in their benign counterpart. Examination of available NGS and Infinium data identified a role for epigenetic regulation DSC3 mRNA reduction in PCa. Pyrosequencing confirmed the increased DSC3 promoter methylation in cancer cell lines and restoration of transcript expression upon 5-Aza treatment further corroborated this epigenetic silencing mechanism. Importantly Kaplan-Meier analysis of an outcome cohort showed an association between loss of DSC3 expression and significantly increased risk of biochemical recurrence. Functional studies indicate a role for epithelial–mesenchymal transition in DSC3 regulated cell migration/invasion. Conclusion Taken together, our data suggests that DNA methylation contributes to down-regulation of DSC3 in prostate cancer, and loss of DSC3 predicts poor clinical outcome.

Matrix metalloproteinase-9 is required for vasculogenic mimicry by clear cell renal carcinoma cells☆

BACKGROUND Vasculogenic mimicry (VM), a new pattern of tumor microcirculation system, has been proved to be important for tumor growth and progression and may be one of the causes of antiangiogenesis resistance. Matrix metalloproteinase-9 (MMP9) was shown to correlate with VM formation in some other cancers. However, the relationship between VM formation and MMP9 in renal cell carcinoma (RCC) has not been determined. METHODS The VM formation and MMP9 expressions were analyzed by CD34/periodic acid-Schiff dual staining and immunohistochemistry in 119 RCC specimens. We used a well-established 3-dimention culture model to compare VM formation in 786-O, 769-P, and HK-2 cell lines in vitro. MMP9 expressions on either messenger RNA or protein levels were compared among the cell lines by quantitative polymerase chain reaction or Western blot. To determine further the relationship between MMP9 and VM in RCC, 786-O and 769-P were treated with specific MMP9 inhibitor or small interfering RNA. VM formation, cell migration, and invasion were subsequently assessed by 3-dimention culture, wound-healing, and transwell assays. RESULTS Immunohistochemistry demonstrated both VM formation and MMP9 overexpression were positively associated with clinical staging, pathological grade, and metastasis (P<0.01). VM formation was closely correlated with MMP9 overexpression in RCC (r = 0.602, P<0.01). Lower MMP9 expression level was observed in normal kidney cell line HK-2, which was unable to form VM on Matrigel, whereas higher expression of MMP9 was found in VM-forming cancer cell lines 786-O and 769-P. Inhibition of MMP9 not only disrupted VM formation in 786-O and 769-P but also reduced cell migration and invasion. CONCLUSIONS These results indicate an intimate relationship between MMP9 overexpression and VM formation in RCC. Treatments targeting VM formation by inhibiting the activity of MMP9 could be beneficial in RCC therapy.

Sunitinib monotherapy instead of mitotane combination therapy for the treatment of refractory adrenocortical carcinoma.

DOI: 10.1111/iju.12903 ACC is a rare malignancy with a poor prognosis. Only complete surgical resection can offer the potential for cure; however, even after successful excision, local recurrences and distant metastases frequently occur. Thus, adjuvant therapy options need to be considered for advanced ACC, but the effectiveness remains controversial. As is well known, mitotane is the only recognized adrenal cytotoxic agent currently available, and might prolong progressionfree survival. However, the objective response rate was just 23%, and long-term disease control was achieved in <15% of patients. Therefore, several studies have investigated targeted therapies, such as imatinib, erlotinib, sorafenib and sunitinib, in patients with ACC, but the responses have been disappointing. Here, we report a case of refractory ACC that showed a good response to sunitinib treatment. A 33-year-old woman presented to First Affiliated Hospital of Sun Yat-sen University, Guangzhou, China, with a palpable mass in her right upper quadrant of the abdomen. CT scan showed a 13.8 9 10.4-cm mass in the right adrenal gland and liver involvement; a tumor thrombus in the inferior vena cava and lung metastases were also found (Fig. S1a,b). Endocrine examination was almost normal. She was diagnosed with advanced ACC and received surgical resection in March 2011. The pathological evaluation confirmed an ACC with liver invasion, and immunohistochemical staining for VEGF was positive in the tumor cells. The Weiss score was nine (Fig. S1c-f). Then she received adjuvant chemotherapy. Mitotane plus etoposide, doxorubicin and cisplatin chemotherapies were initiated, but failed after two cycles of chemotherapy. After the failure of mitotane-based cytotoxic chemotherapy, targeted therapy with sunitinib was chosen after careful discussion with the patient’s family. She received sunitinib plus mitotane treatment from August 2011. However, after 8 months of sunitinib treatment, the patient developed a severe cough, and a CT scan showed progressive disease in April 2012. Her entire lungs were bestrewn with increasing pulmonary nodules (Fig. 1a,b). Because of the disease progression and adverse events, mitotane was withdrawn in May 2012 and sunitinib monotherapy was continued. In May 2013, a CT scan showed a significant remission (Fig. 1c,d) and the patient’s condition improved. In May 2014, a further CT scan showed that there were fewer metastatic pulmonary nodules and their sizes were smaller (Fig. 1e,f). The patient’s condition stabilized and she survived postoperatively for 4 years. Sunitinib is an oral multitargeted tyrosine kinase inhibitor that targets VEGFR1, VEGFR2, c-KIT and Fms-like tyrosine kinase 3, and thus combines direct antitumor effects with antiangiogenic activity. The results of a phase II study in patients progressing after mitotane and one to three cytotoxic chemotherapies showed that sunitinib had modest activity in advanced refractory ACC. Furthermore, the results were highly suggestive of a negative impact of concomitant mitotane therapy on the outcome. Fortunately, we have gained great encouragement from our successful experience with that refractory ACC patient. After the failure of mitotane-based chemotherapy, we continued her treatment with a combination of sunitinib and mitotane, but the tumor still progressed. This result was in good agreement with published data. Mitotane was then withdrawn and sunitinib monotherapy was continued. A year later, unexpected results were observed. A CT scan showed that the patient’s metastatic pulmonary nodules had nearly disappeared and her condition stabilized. The clinical benefit of sunitinib has persisted until the present time. However, the present results again suggest that a drug interaction between sunitinib and mitotane might have a major influence on their effects. The pharmacokinetic properties of the two drugs could be the reason for this interaction. It is well established that sunitinib is metabolized in the liver and intestine by CYP3A4 monooxygenase to its active metabolite SU12662, which is then inactivated in a second step by CYP3A4 monooxygenase. However, mitotane is an extraordinarily strong inducer of CYP3A4, which will markedly increase the inactivation of sunitinib, and therefore reduce

A New Modified Renal Vein Anastomosis in Unilateral Dual Renal Transplantation

Transplantation of renal allografts inadequate to meet the recipient’s metabolic needs has been proposed as a cause of chronic allograft failure. Dual renal transplant is a novel idea to transplant enough nephron mass to solve this problem. According to the current literature, graft renal veins are anastomosed to the external iliac vein or inferior vena cava as venous drainage in the setting of dual renal transplantation. In the presented case, renal blood was returned through one of the grafts back to the systematic circulation through the other renal vein graft. This provides a feasible option for unilateral dual renal transplantation to accomplish renal vein anastomosis in some difficult situations.

Vasculogenic mimicry plays an important role in adrenocortical carcinoma

To determine the prognostic role of vasculogenic mimicry in adrenocortical carcinoma, and to explore its relationship with vascular endothelial growth factor receptor 2 expression.

AB241. Cancer stem cell-like side population cells in clear cell renal cell carcinoma cell line 769P

Background Although cancers are widely considered to be maintained by stem cells, the existence of stem cells in renal cell carcinoma (RCC) has seldom been reported, in part due to the lack of unique surface markers. We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Methods We here identified cancer stem cell-like cells with side population (SP) phenotype in five human RCC cell lines. Results Flow cytometry analysis revealed that 769P, a human clear cell RCC cell line, contained the largest amount of SP cells among five cell lines. These 769P SP cells possessed characteristics of proliferation, self-renewal, and differentiation, as well as strong resistance to chemotherapy and radiotherapy that were possibly related to the ABCB1 transporter. In vivo experiments with serial tumor transplantation in mice also showed that 769P SP cells formed tumors in NOD/SCID mice. Conclusions Taken together, these results indicate that 769P SP cells have the properties of cancer stem cells, which may play important roles in tumorigenesis and therapy-resistance of RCC.

Primary Paraganglioma of Seminal Vesicle

To the Editor: Extra-adrenal paragangliomas are tumors that arise from neural crest-derived endocrine cells. They are most frequently found in the organ of Zuckerkandl but can be found anywhere along the sympathetic chain.[1] Seminal vesicle paragangliomas have been reported to involve bladder and prostate. However, only two cases of primary seminal vesicle paragangliomas were reported in medical literature to the best of our knowledge.[1,2] Here, we reported on one case. A 44-year-old male patient with the family history of hypertension was admitted to the local hospital with a chief complaint of the lower abdominal discomfort and chest distress for more than 2 years. Subsequent examination revealed hypertension and coronary artery computed tomography (CT) did not show any abnormality. As a result, the patient received antihypertensive therapy. Symptoms happened more frequently, about one to two times per day since 6 months ago. Blood pressure was 200–260/120–150 mmHg at onset and partially responded to therapy as 120–130/90–100 mmHg. Therefore, the patient came to our hospital and was admitted in the endocrinology department. The elevated urine vanillylmandelic acid (VMA) and blood catecholamine were confirmed, and abdominal CT was taken. An occupying lesion in the left seminal vesicle was found, considering ectopic pheochromocytoma [Figure 1], while the adrenal glands and other organs are normal. After oral intake of phenoxybenzamine for 14 days, the patient was admitted to the urology department and underwent laparoscopic resection of the mass. The surgery went well and the mass was found to be paraganglioma of seminal vesicle on pathology. Postoperatively, the symptoms disappeared and the blood pressure returned to normal. Antihypertensive therapy was discontinued and no symptoms occurred during the short-term follow-up of 3 months. Written informed consent was obtained from the patient for publication of this case report and accompanying images. Figure 1 Abdominal computed tomography showing an occupying lesion in the left seminal vesicle. Extra-adrenal paraganglioma (pheochromocytoma) is a rare tumor which can be found anywhere along the sympathetic chain from the base of the skull and neck to the bladder and prostate gland. About 90% of sympathetic paragangliomas are intra-adrenal (pheochromocytomas). Furthermore, approximately 10% of all extra-adrenal paraganglioma are malignant.[3] In the genitourinary tract, the urinary bladder is the most common site for paraganglioma (79.2%), followed by the urethra (12.7%), pelvis (4.9%), and ureter (3.2%).[3,4] The histogenesis of seminal vesicle paraganglioma is unknown. Histologically, all paragangliomas show similar morphologic characteristics. Immunohistochemistry shows positive staining for the synaptophysin, chromogranin A, and CD56 neuroendocrine markers. Protein S100 highlights the sustentacular and tumor cells, which is in accordance with our case. Mitotic activity and necrosis have been regarded as some of the unfavorable prognostic factors in histopathology. The only consistent criterion for malignancy in paraganglioma at any site is metastasis. The clinical manifestation of seminal vesicle paraganglioma may largely depends on the size and the functional status of the tumor.[1] The symptoms and signs of catecholamine overproduction such as hypertension, headaches, palpitations, sweating, and tachycardia may be seen. In general, an elevation of the 24 h urinary catecholamine metabolites and VMA levels can be detected as in our case. The standard treatment for localized or locally advanced seminal vesicle paraganglioma is surgery. Laparoscopic resection has been an effective management because it can magnify the anatomic structure for easier removal and decrease the trauma. Primary paraganglioma of the seminal vesicle is rare but important to diagnosis in the differential diagnosis of extra-adrenal paragangliomas. Laparoscopic resection appears to be a safe and effective management for localized seminal vesicle paraganglioma. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

The performance of transrectal ultrasound in the diagnosis of seminal vesicle defects: a comparison with magnetic resonance imaging

Obstructive azoospermia (OA) is one of the most common causes of male infertility. Transrectal ultrasound (TRUS) has been used to diagnose OA for many years. From 2009 to 2013, we evaluated a prospective cohort of 1249 patients with suspected OA using TRUS. It was found that dilation of the ejaculatory duct (ED) (29.9%, 374/1249) was the most common cause of OA, followed by seminal vesicle (SV) abnormalities (28.5%, 356/1249). A total of 237 patients were diagnosed with congenital defects (agenesis and/or hypoplasia) of the SV, constituting more than half of the cases of SV disease in OA (19.0%, 237/1249). In contrast to ED, congenital defects of the SV could not be corrected with surgical treatment. Therefore, it is meaningful to compare TRUS and magnetic resonance imaging (MRI) for accurate diagnosis of SV defects. Among our patients, 30 with agenesis or/and hypoplasia of the SV on TRUS were further evaluated using pelvic MRI within 2 years, with the objective of verifying the TRUS results. The concordance rate for diagnosing congenital defects of the SV was 73.3% (22/30). We concluded that TRUS is a reliable and convenient method for diagnosing agenesis or hypoplasia of the SV in OA patients with a high concordance with MRI while MRI is useful in patients with inconclusive TRUS findings.

Ureteral Triplication Combined with Right Renal Ectopia and Ureteral Cyst

Ureteral triplication is one of the rarest malformations of the upper urinary tract. We report the case of a 12-year-old girl with right ureteral triplication combined with renal ectopia and ureteral cyst with stenosis at the junction of the ureteral cyst and distal ureter. The ureteral cyst was tailored and tubularized, and the tight junction was removed, as in Hynes-Anderson ureteropyeloplasty; on reevaluation almost 4 years later, kidney function was normal and computed tomography showed a normal kidney and ureter.