Search

To treat facial asymmetry with severe inclination of the frontal occlusal plane, unilateral maxillary impaction in the molar region must be performed to reposition the maxilla. We herein report the...

Highlights • Neurofibromatosis is a disease that causes various abnormalities such as neurofibroma, mainly in the skin and nerves. • The common sites of occurrence in the oral cavity are the palate, gingiva, etc., occurrence in the mandible is rare. • The patient had already been prenatally diagnosed with neurofibromatosis type 1. • The lesion was clinically diagnosed with a radicular cyst, but histopathological diagnosis showed a neurofibroma. • Tumor extirpation was performed under general anesthesia., Introduction Neurofibromatosis is a disease that causes various abnormalities such as neurofibroma, mainly in the skin and nerves. The common sites in the oral cavity are the palate, gingiva, tongue, buccal mucosa, and lips but, occurrence in the mandible is rare. Presentation of case A 26-year-old woman was referred to our clinic because of percussion pain. Radiographic findings showed a radiolucent area. The patient was clinically diagnosed with a radicular cyst by a previous doctor. Multiple café-au-lait spots were found disseminated on her body, and she had already been prenatally diagnosed with neurofibromatosis type 1 (NF1). We performed a biopsy and suggested a neurofibroma. Tumor extirpation was performed under general anesthesia. The histopathological diagnosis showed a neurofibroma. Clinical discussion and conclusion NF1 is a systemic nevus that causes abnormalities in melanocytes and Schwann cells, and various lesions appear, but intramandibular lesions are extremely rare. Diagnosis of NF1 and radicular cysts in the mandible is difficult due to their image resemblance. However, it should be kept in mind if the underlying disease is NF1. In our case, it was easy to detach and may have originated from small peripheral nerve endings in the mandible.

Many anatomical variations of the superficial veins of the head and neck have been reported throughout the literature. Accordingly, anatomists and surgeons must have a comprehensive understanding of these variations to avoid confusion. Duplication of the external jugular vein (EJV) is occasionally observed during routine cadaveric dissections; however, this variation seems to be reported less often than actual experience suggests. Therefore, to gain a better understanding of its anatomical and clinical implications, an analysis of the available data should be available. Thus, in this article, we reviewed the current available literature for studies reporting duplication of the EJV. We conducted a search using PubMed and Google Scholar with the following keywords: “duplication of the external jugular vein,” “division of the external jugular vein,” and “fenestration of the external jugular vein,” “double external jugular vein,” and “doubled external jugular vein.” As a case illustration, we also describe a case of a duplicated EJV found during a right neck dissection of a female cadaver. Twenty sides across sixteen different studies were analyzed including the present case. All studies were published between 2009 and 2020. EJV division patterns were classified as either duplication, fenestration, fenestration followed by duplication, or double fenestrations. We have reviewed the literature regarding cases documenting duplication/fenestration of the EJV. As it is often difficult to find recent studies that report on classic anatomical variations, therefore, revisiting older articles and textbooks is necessary for achieving a “comprehensive” review, especially across different languages.

Head and neck squamous cell carcinomas (HNSCCs) frequently invade the bones of the facial skeleton. Semaphorin 4D (Sema4D) is an axon guidance molecule produced by oligodendrocytes. Sema4D was also identified in the bone microenvironment and many cancer tissues including HNSCC. To date, however, the role of Sema4D in cancer-associated bone disease is still unknown. This is the first study to demonstrate the role of Sema4D in bone invasion of cancer. In the clinical tissue samples of bone lesion of HNSCC, Sema4D was detected at high levels, and its expression was correlated with insulin-like growth factor-I (IGF-I) expression. In vitro experiments showed that IGF-I regulates Sema4D expression and Sema4D increased proliferation, migration and invasion in HNSCC cells. Sema4D also regulated the expression of receptor activator of nuclear factor κβ ligand (RANKL) in osteoblasts, and this stimulated osteoclastgenesis. Furthermore, knockdown of Sema4D in HNSCC cells inhibited tumor growth and decreased the number of osteoclasts in a mouse xenograft model. Taken together, IGF-I-driven production of Sema4D in HNSCCs promotes osteoclastogenesis and bone invasion.

Background/aim Retinoid signaling is important for the maturation of growth-plate chondrocytes. The effect of retinoid receptor gamma (RARγ) signaling on the expression of genes in hypertrophic chondrocytes is unclear. This study investigated the role of RARγ signaling in regulation of hypertrophic chondrocyte-specific genes. Materials and methods The gene expression in mouse E17.5 tibial cartilage was examined by in situ hybridization analysis. Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunoblotting were used for analysis of mRNA and phosphorylated mitogen-activated protein kinase (MAPK). Results mRNA expression of Rarg and connective tissue growth factor (Ccn2) was detected in maturing chondrocytes throughout the cartilaginous skeletal elements. In chondrogenic ATDC5 cells, an RARγ agonist induced the gene expression of type-X collagen (Col10A1), transglutaminase-2 (Tg2), matrix metalloproteinase-13 (Mmp13), and Ccn2 mRNA, whereas a retinoic acid pan-agonist suppressed RARγ agonist-stimulated gene expression. Phosphorylated extracellular signal regulated-kinases (pERK1/2), p-p38, and phosphorylated c-Jun N-terminal kinase (pJNK) MAPK were time-dependently increased by RARγ agonist treatment. Experimental p38 inhibition led to a severe drop in the RARγ agonist-stimulated expressions of Col10A1, Tg2, Mmp13, and Ccn2 mRNA. Conclusion RARγ signaling is required for the differentiation of hypertrophic chondrocytes, with differential cooperation with p38 MAPK.

The sublingual space is superomedial to mylohyoid muscle and lateral to genioglossus and geniohyoid muscles in the oral cavity. Its major contents are the sublingual salivary gland and its ducts (the major sublingual duct, Bartholin’s duct; the smaller sublingual ducts, Rivinus’s ducts), the submandibular duct (Wharton’s duct), the superior portion of the submandibular salivary gland, the lingual nerve, artery and vein, and the glossopharyngeal (IX) and hypoglossal (XII) nerves. The sublingual space communicates with the submandibular space at the posterior margin of mylohyoid muscle where there is a gap between this muscle and hyoglossus muscle. Many lesions such as ranula and submandibular duct obstruction, various malignancies, inflammation, and vascular abnormalities arise uniquely in the sublingual space. Better knowledge of the complex muscular, vascular, glandular, ductal, and neural anatomy of this region is important for accurate diagnosis and treatment planning by both general dentists and oral surgeons in order to avoid unnecessary complications. In this chapter, the normal anatomy and variations of the sublingual space are reviewed and its clinical relevance is discussed.

Tumors of the sublingual salivary gland are extremely rare. Most of the sublingual tumors are malignant, adenoid cystic carcinoma (ACC) and mucoepidermoid carcinoma being the most common histological types. Here we report the first case of a familial occurrence of ACC in a family in which the father and daughter were treated for ACC of the sublingual salivary gland. A 75-year-old man was referred with a swelling of the floor of the mouth on his right side and diagnosed as ACC of sublingual salivary gland synchronous with adenocarcinoma of the lung. One year later, his daughter presented, at the age of 46 years, with a swelling of the floor of the mouth on her right side, which was also diagnosed as ACC. This is the first case of familial recurring ACC of sublingual salivary gland worldwide.

BACKGROUND/AIM This study evaluated the associations between bone invasion of gingival squamous cell carcinoma (SCC) and clinicopathological manifestations, and aimed to determine whether bone invasion is an independent prognostic factor in gingival SCC. PATIENTS AND METHODS The study was a retrospective review of 78 patients with gingival SCC who underwent surgery with curative intent. The level of bone invasion was pathologically categorized as medullary, cortical or no bone invasion. RESULTS Cortical and medullary bone invasion was present in 29 and 22 patients, respectively. There was a significant association between medullary bone invasion and tumor size (p=0.017), pathological N classification (p

Highlights • The patient was a 59-year-old man presenting with a swollen right submandibular gland. Laboratory tests revealed IgG4 levels of 176 mg/dl (reference range: 4.8–105). • Histological examination of the submandibular gland showed an adenoid cystic carcinoma with lymphocytic infiltration containing many IgG4-positive plasma cells in the tumor stroma., Introduction Immunoglobulin G4-related disease (IgG4-RD) is an inflammatory condition associated with elevated serum IgG4 levels and tissue infiltration by IgG4-expressing plasma cells. We present a case of adenoid cystic carcinoma (ACC) of the submandibular gland with possible involvement of IgG4-RD. Presentation of case The patient was a 59-year-old man presenting with a swollen right submandibular gland. Laboratory tests revealed IgG4 levels of 176 mg/dl (reference range: 4.8–105). An initial open biopsy for histological diagnosis showed chronic sialadenitis. The region was monitored on an outpatient basis, and finally the right submandibular was totally resected because malignant tumor could not be excluded. Histological examination of the submandibular gland showed an ACC with lymphocytic infiltration containing many IgG4-positive plasma cells in the tumor stroma. Discussion We have described a case that indicated a possible involvement of ACC with IgG4-RD. This allows us to speculate that longstanding IgG4-RD may progress to malignancy or infiltration of IgG4-positive plasma cells through the signals of tumor stimuli. Further investigations are required to determine the potential pathogenic mechanism underlying this unique tumor. Conclusion This case underscores that caution is needed in the diagnosis of masses with high serum IgG4 levels, as the differential diagnosis includes malignancy.

BACKGROUND Sonic hedgehog (SHH) signaling is related to the pathogenesis of oral squamous cell carcinoma (OSCC), but its role in OSCC is not yet well understood. In this study, we analyzed the role of SHH signaling in OSCC. MATERIALS AND METHODS We examined the expression pattern of SHH and its signal proteins in clinically resected OSCC samples by immunohistochemistry. We also evaluated the function of SHH signaling using the hedgehog signaling inhibitor cyclopamine in vivo and in vitro by proliferation, migration and angiogenesis analyses. RESULTS We found that SHH was highly expressed in human tongue OSCC, whereas patched (PTCH1), glioma-associated oncogene 1 (GLI1) and GLI2 proteins were expressed in the microvascular cells in the tumor invasive front. Administration of cyclopamine to mice suppressed the growth and angiogenesis of OSCC xenografts in vivo. Moreover, cyclopamine inhibited endothelial cell proliferation and migration, and reduced aorta vascular length in the rat. CONCLUSION These findings suggest that OSCC-derived SHH stimulates angiogenesis at the tumor invasive front.

Background: According to the report of Japan’s Ministry of Health, Labor and Welfare, looking at the main reasons requiring nursing care by age group, elderly people aged 75 and over, falls and fractures increase. The purpose of this study was to investigate the trends and characteristic features of maxillofacial fractures in over 75 years old, late stage of elderly patients. Patients and Methods: Records of patients who were treated for maxillofacial fractures at the Oral and Maxillofacial Surgery department (Biopathology) of Okayama University hospital from January 2008 to December 2013 were retrospectively analyzed. Clinical records were analyzed in terms of age, gender, etiology, relevant medical history, and anatomical site of fracture. Patients (n=103) were divided into two groups by age

Background/aim The neurokinin 3 receptor (NK-3R) is differentially expressed in the central nervous system including cases of human oral squamous cell carcinoma. However, the role of NK-3R signaling in oral squamous cell carcinoma is not well known. Materials and methods NK-3R expression in surgically resected oral squamous cell carcinoma was examined immunohistochemically and the strength of the expression was quantified. We evaluated the function of NK-3R signaling using NK-3R antagonist in human oral squamous cell carcinoma bone invasion mouse model. Results NK-3R was significantly expressed in tumor cells that had invaded the bone matrix compared to the oral side tumor cells. SB222200, a selective antagonist of NK-3R, significantly suppressed the radiographic osteolytic lesion and tumorigenesis. Conclusion NK-3R signaling is a potential target for the treatment of oral squamous cell carcinoma in cases of bone destruction.

Objective: In recent years, the need for dental treatment and dental hygiene management within the perioperative period and supportive advanced medical treatment has been increasing. However, perioperative intervention in diseases requiring oral surgical treatment is not well understood at present. Here we report our investigation of diseases requiring oral surgical treatment in which Department of Oral and Maxillofacial Surgery (Biopathology) at Okayama University Hospital conducted medicaldental cooperation via Division of Hospital Dentistry. Methods: In the five years from April 2011 to March 2016, we have identified 310 cases that reached the Okayama University Hospital Department of Oral and Maxillofacial Surgery (Biopathology) via the Division of Hospital Dentistry. Results: The patients were treated in 22 clinical departments, the most prevalent treatment was chemotherapy, followed by surgery and then transplantation. The surgical diseases for which our department was consulted were most commonly diseases of the teeth and periodontal disease (269 cases), followed by inflammatory conditions such as implantitis and osteomyelitis, and mucosal diseases such as lichen planus and leukoplakia. The surgical procedure performed was most often tooth extraction (243 cases); there are few cases of cystectomy and sequestrum. In the majority of cases, the surgical treatment was performed on the initial examination day; in about 84% of cases, surgery was performed within 2 weeks. Conclusion: Further investigation is necessary into the practice of preventing adverse events non-dental medical departments by increasing the frequency of oral surgical intervention in appropriate cases during the perioperative period.

Key Clinical Message We report a case of Behcet's disease which was aggravated by psychological stress and oral infection. The control of oral infection under medical and dental collaboration is important for providing Behcet's disease patients with the optimal medical care and for facilitating the relief of the primary disease.

Highlights • The management of medication-related osteonecrosis of the jaw (MRONJ) is controversial. • To date, there is no established treatment for cases of advanced stage 3 MRONJ osteoporosis in elderly patients. • We describe a case in which a pathological mandible fracture induced by MRONJ was healed and functional recovery of the occlusion was obtained by complete dentures after treatment with teriparatide., Introduction The management of medication-related osteonecrosis of the jaw (MRONJ) is controversial. To date, there is no established treatment for cases of advanced stage 3 MRONJ osteoporosis in elderly patients. Presentation An 87-year-old osteoporotic woman with osteonecrosis-induced left mandible fracture related to minodronate therapy was referred to us for treatment. She had a vertebral compression fracture concurrently and had started subcutaneous injection of teriparatide. After 18 months of treatment with teriparatide the pathological mandible fracture was healed and functional recovery of the occlusion was obtained by complete dentures. Discussion Teriparatide may have a powerful anabolic effect on bone, and promote bone regeneration against pathologic mandible fracture induced by MRONJ. Conclusion Based upon these findings, teriparatide might be beneficial for advanced stage 3 MRONJ osteoporosis in elderly patients.

Purpose of the research Tooth germ development involves multiple events, including cell proliferation and cell differentiation. Connective tissue growth factor (CTGF/CCN2) is a signaling protein involved in tooth germ development, and we investigated how it is expressed and what roles it may have in primary cultures of mesenchymal cells derived from the developing tooth germ. We also examined the expression of CCN2 in a human odontogenic myxofibroma, a benign tumor of odontogenic mesenchymal origin, and considered the possible roles of CCN2 in the development of myxofibromas. Materials and methods Mesenchymal cells of early bell-stage tooth germs were isolated from Day-90 bovine embryos and placed in primary culture. A resected specimen from a patient with odontogenic myxofibroma was prepared for immunohistochemical studies. Principal results The CCN2 expression level in proliferating odontogenic mesenchymal cells freshly isolated from the early bell stage of developing bovine tooth germs and placed in primary culture was 3 times higher than that in the confluent non-proliferating cells. Recombinant CCN2 significantly increased the proliferation and type I collagen expression in odontogenic mesenchymal cells in primary culture. Immunohistochemical analysis on myxofibroma case revealed that CCN2 was detectable in MIB-1, a cellular marker of proliferation-positive odontogenic mesenchymal cells adjacent to capillary blood vessels and in the endothelial cells of the vessels in the tumor. Major conclusion CCN2 signaling would influence the proliferation of and extracellular matrix production by dental mesenchymal cells. Our results suggest that the same mechanisms of CCN2 action toward dental mesenchymal cells would also be operative in the odontogenic myxofibroma microenvironment.

Bone metastasis is a common occurrence in human malignancies, including breast, prostate, and lung cancer, and is associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the heart. This assay can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Bone metastasis is a common occurrence in human malignancies, including breast, prostate, and lung cancer, and is associated with a high morbidity rate because of intractable bone pain, pathological fractures, hypercalcemia, and nerve compression. Animal models of bone metastasis are important tools to investigate the pathogenesis and develop treatment strategies. However, there are few models of spontaneous bone metastasis despite the fact that animals often spontaneously develop cancer. Here, we describe methods for developing a mouse model of breast cancer bone metastasis achieved by injection of MDA-MB-231 breast cancer cells into the heart. This assay can be applied to studies on roles of CCN proteins in tumor metastasis and development of treatment strategies targeting CCN proteins.

Summary Objectives Angiogenin (ANG) is a prominent angiogenic factor that has been shown to have a dual effect on tumor progression by inducing both angiogenesis and cancer cell proliferation through stimulating ribosomal RNA transcription in both endothelial cells and cancer cells. In the present study, we investigated the expression profiles of ANG and vascular endothelial growth factor (VEGF) in oral cancer and their correlation with hypoxia and evaluated the possible value of ANG as a therapeutic target for oral cancer. Materials and Methods Immunohistochemistry (IHC), ELISA, real-time RT-PCR and Western blotting were used to examine the expression of ANG, VEGF, and hypoxia-inducible factor 1α (HIF-1α) in oral squamous cell carcinoma (OSCC) specimens and human OSCC cell lines. In order to examine the role of ANG, we knocked down ANG expression in HSC-2 cells by means of plasmid-mediated RNA interference. Results IHC showed that the expression of ANG was significantly correlated with that of HIF-1α in 50 OSCC specimens ( P = 0.031). However, no significant correlation between VEGF and HIF-1α expression was found ( P = 0.243). Consistently, ANG secretion increased under hypoxia in all of the 10 OSCC cell lines tested; and a significant increase was observed in 6 of them. In contrast, there was no noticeable increase in VEGF secretion under hypoxia in any of these cell lines. In HSC-2 and SAS OSCC cells, the increase in ANG mRNA expression correlated very well with that of HIF-1α protein expression after hypoxia onset. However, no noticeable increase in VEGF mRNA expression was observed even after 12 h of hypoxia. Down-regulation of ANG expression in HSC-2 cells highly expressing and secreting VEGF inhibited ribosome biogenesis, cell proliferation, tumor angiogenesis, and xenograft growth in athymic mice. Conclusion These results suggest that ANG is up-regulated in the hypoxic environment of oral cancers and that its inhibition can have a therapeutic implication.

Highlights • Lengthening of the mandible by distraction osteogenesis using an internal device is the preferred method for the treatment of hemifacial microsomia. Despite its advantages, this technique can lead to various complications after the surgery. • We describe a case in which rod unaccessible pain developed after the initial mandibular distraction activation, and the pain was resolved by installing an original sleeve., Introduction Lengthening of the mandible by distraction osteogenesis using an internal device is the preferred method for the treatment of hemifacial microsomia. Despite its advantages, this technique can lead to various complications after the surgery. Presentation of case We report the case of an 8-yr-old Japanese girl whose case presented practical difficulties in device activation because of rod unaccessible pain after the initial mandibular distraction with an internal device, and this complication was addressed with the installation of an original sleeve. Discussion In the present patient, the region of the bend rod was located at the inferior border of the right mandible, causing rod unaccessible pain by contacting the surrounding tissue including a sensory nerve. Careful vertical ramus distractor position planning and tools to resolve complications are the key factors for accomplishing the scheduled elongation. Conclusion Alternative techniques using a sleeve for safer and gentle distraction for rod unaccessible pain on activation should be considered.

Angiogenin (ANG), originally identified as an angiogenic ribonuclease, has recently been shown to play a direct role in prostate cancer cell proliferation by mediating rRNA transcription. ANG is up-regulated in human prostate cancer and is the most significantly up-regulated gene in AKT-driven prostate intraepithelial neoplasia (PIN) in mice. Enhanced cell proliferation in the PIN lesions requires increased ribosome biogenesis, a multistep process involving an orchestrated production of ribosomal proteins and rRNA. AKT is known to enhance ribosomal protein production through the mammalian target of rapamycin pathway. However, it was unknown how rRNA is proportionally increased. Here, we report that ANG is essential for AKT-driven PIN formation and survival. We showed that up-regulation of ANG in the AKT-overexpressing mouse prostates is an early and lasting event. It occurs before PIN initiation and lasts beyond PIN is fully developed. Knocking down ANG expression by intraprostate injection of lentivirus-mediated ANG-specific small interfering RNA prevents AKT-induced PIN formation without affecting AKT expression and its signaling through the mammalian target of rapamycin pathway. Neomycin, an aminoglycoside that blocks nuclear translocation of ANG, and N65828, a small-molecule enzymatic inhibitor of the ribonucleolytic activity of ANG, both prevent AKT-induced PIN formation and reverse established PIN. They also decrease nucleolar organizer region, restore cell size, and normalize luminal architectures of the prostate despite continuous activation of AKT. All three types of the ANG inhibitor suppress rRNA transcription of the prostate luminal epithelial cells and inhibit AKT-induced PIN, indicating an essential role of ANG in AKT-mediated cell proliferation and survival. (Mol Cancer Res 2009;7(3):415–24)

Connective tissue growth factor (CCN2) regulates diverse cellular functions, including tooth development. In order to delineate the precise role of CCN2 in the epithelium during odontogenesis, we investigated how it is expressed and what roles it may have in primary cultures of epithelial cells derived from developing tooth germ of the bovine fetus. Ccn2 mRNA and protein were strongly expressed in the inner dental epithelium, which is consistent with the expression of transforming growth factor-β2 mRNA and proliferating cell nuclear antigen. Bone morphogenetic protein 4 (BMP4) and fibroblast growth factor 2 (FGF2) were also expressed in the inner dental epithelium, indicating that CCN2 functionally interacts with these factors in the epithelium. The stimulatory effects of FGF2 on cell proliferation and BMP4 on cell differentiation were additively up-regulated by CCN2 in a newly-established dental epithelium cell culture. Taken together, our data provide clear evidence that CCN2 is synthesized by inner dental epithelial cells, and appears to act as an autocrine factor, which regulates dental epithelial cell proliferation and differentiation in concert with growth factors.

Angiogenin undergoes nuclear translocation and stimulates ribosomal RNA transcription in both endothelial and cancer cells. Consequently, angiogenin has a dual effect on cancer progression by inducing both angiogenesis and cancer cell proliferation. The aim of this study was to assess whether neamine, a blocker of nuclear translocation of angiogenin, possesses antitumor activity toward oral cancer.The antitumor effect of neamine on oral cancer cells was examined both in vitro and in vivo.Neamine inhibited the proliferation of HSC-2, but not that of SAS oral cancer cells in vitro. Treatment with neamine effectively inhibited growth of HSC-2 and SAS cell xenografts in athymic mice. Neamine treatment resulted in a significant decrease in tumor angiogenesis, accompanied by a decrease in angiogenin- and proliferating cell nuclear antigen-positive cancer cells, especially of HSC-2 tumors.Neamine effectively inhibits oral cancer progression through inhibition of tumor angiogenesis. Neamine also directly inhibits proliferation of certain types of oral cancer cells. Therefore, neamine has potential as a lead compound for oral cancer therapy.

The androgen receptor (AR) is a critical effector of prostate cancer development and progression. Androgen-dependent prostate cancer is reliant on the function of AR for growth and progression. Most castration-resistant prostate cancer (CRPC) remains dependent on AR signaling for survival and growth. Ribosomal RNA (rRNA) is essential for both androgen-dependent and castration-resistant growth of prostate cancer cells. During androgen-dependent growth of prostate cells, androgen-AR signaling leads to the accumulation of rRNA. However, the mechanism by which AR regulates rRNA transcription is unknown. Here, investigation revealed that angiogenin (ANG), a member of the secreted ribonuclease superfamily, is upregulated in prostate cancer and mediates androgen-stimulated rRNA transcription in prostate cancer cells. Upon androgen stimulation, ANG undergoes nuclear translocation in androgen-dependent prostate cancer cells, where it binds to the rDNA promoter and stimulates rRNA transcription. ANG antagonists inhibit androgen-induced rRNA transcription and cell proliferation in androgen-dependent prostate cancer cells. Interestingly, ANG also mediates androgen-independent rRNA transcription through a mechanism that involves its constitutive nuclear translocation in androgen-insensitive prostate cancer cells, resulting in a constant rRNA overproduction and thereby stimulating cell proliferation. Critically, ANG overexpression in androgen-dependent prostate cancer cells enables castration-resistant growth of otherwise androgen-dependent cells. Thus, ANG-stimulated rRNA transcription is not only an essential component for androgen-dependent growth of prostate cancer but also contributes to the transition of prostate cancer from androgen-dependent to castration-resistant growth status. Implications: The ability of angiogenin to regulate rRNA transcription and prostate cancer growth makes it a viable target for therapy. Mol Cancer Res; 11(10); 1203–14. ©2013 AACR.

Purpose: Angiogenin (ANG) undergoes nuclear translocation and stimulates rRNA transcription in both prostate cancer cells and endothelial cells. The purpose of this study is to assess the antitumor activity of neamine, a nontoxic degradation product of neomycin that blocks nuclear translocation of ANG. Experimental Design: The anti-prostate cancer activity of neamine was first evaluated in a xenograft animal model. It was then examined in the murine prostate-restricted AKT transgenic mice that develop prostate intraepithelial neoplasia (PIN) owing to AKT transgene overexpression. Results: Neamine inhibits xenograft growth of PC-3 human prostate cancer cells in athymic mice. It blocks nuclear translocation of ANG and inhibits rRNA transcription, cell proliferation, and angiogenesis. Neamine also prevents AKT-induced PIN formation as well as reverses fully developed PIN in murine prostate-restricted AKT mice, accompanied by a decrease in rRNA synthesis, cell proliferation, and angiogenesis and an increase in prostate epithelial cell apoptosis. Conclusion: We confirmed that ANG is a molecular target for cancer drug development and that blocking nuclear translocation of ANG is an effective means to inhibit its activity. Our results also suggested that neamine is a lead compound for further preclinical evaluation.

Angiogenin is progressively upregulated in prostate cancer, in particular in androgen-independent diseases. The objective of this project is to explore the role angiogenin plays in the development of androgen-independent disease. In this reporting period, we have demonstrated that nuclear translocation of angiogenin is specific for prostate cancer cells and does not occur in normal prostate epithelial cells. Angiogenin is translocated to the nucleus of androgen-dependent cells (LNCaP) only when the cells are stimulated with androgen (DHT). But angiogenin is constitutively translocated to the nucleus of androgen-independent cells (PC-3, PC-3M, DU145). Angiogenin is required for DHT to stimulate rRNA transcription and cell proliferation of androgen-dependent cells. Overexpression of angiogenin enables androgen-independent growth of otherwise androgen-dependent prostate cancer cells in vitro and in vivo. Finally, we have shown that angiogenin binds to the promoter region of rDNA in vivo and stimulates rRNA transcription.

Human angiogenin is progressively up-regulated in the prostate epithelial cells during the development of prostate cancer from prostate intraepithelial neoplasia (PIN) to invasive adenocarcinoma. Mouse angiogenin is the most up-regulated gene in AKT-induced PIN in prostate-restricted AKT transgenic mice. These results prompted us to study the role that angiogenin plays in prostate cancer. Here, we report that, in addition to its well established role in mediating angiogenesis, angiogenin also directly stimulates prostate cancer cell proliferation. Angiogenin undergoes nuclear translocation in PC-3 human prostate cancer cells grown both in vitro and in mice. Thus, knocking down angiogenin expression in PC-3 human prostate adenocarcinoma cells inhibits ribosomal RNA transcription, in vitro cell proliferation, colony formation in soft agar, and xenograft growth in athymic mice. Blockade of nuclear translocation of angiogenin by the aminoglycoside antibiotic neomycin inhibited PC-3 cell tumor growth in athymic mice and was accompanied by a decrease in both cancer cell proliferation and angiogenesis. These results suggest that angiogenin has a dual effect, angiogenesis and cancer cell proliferation, in prostate cancer and may serve as a molecular target for drug development. Blocking nuclear translocation of angiogenin could have a combined benefit of antiangiogenesis and chemotherapy in treating prostate cancer.

The role of CTGF/CCN2 in osteolytic metastasis by breast cancer cells and its mechanism of action were studied. Osteolytic metastasis accompanied by CCN2 and PTHrP overproduction was efficiently inhibited by an anti-CCN2 antibody. Furthermore, we found that CCN2 was induced by PTHrP through PKA-, PKC-, and ERK-mediated pathways therein. Introduction: Connective tissue growth factor (CTGF/CCN2) is a mediator of local angiogenesis induced by breast cancer, but its role in osteolytic metastasis has not been evaluated. PTH-related peptide (PTHrP) is another critical factor in the development of the osteolytic metastasis. Using both in vivo and in vitro approaches, we studied whether/how neutralization of CCN2 prevented bone metastasis and how PTHrP signaling is related. Materials and Methods: A mouse model of bone metastasis by human breast cancer cell line MDA231 was treated with a CCN2-neutralizing antibody, and osteolytic bone metastases were assessed on radiographs and immunohistochemistry. Ccn2 gene expression and transcription were examined by Northern blot and luciferase analysis. Immunoblot analysis and kinase inhibitors were used to identify the signaling pathways implicated. Anti-angiogenic/osteoclastogenic effects of ccn2 downregulation were also evaluated. Results: Treatment of mice with a CCN2-neutralizing antibody greatly decreased osteolytic bone metastasis, microvasculature, and osteoclasts involved. The antibody also suppressed the growth of subcutaneous tumor in vivo and proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro. Downregulation of ccn2 also repressed osteoclastogenesis. CCN2 expression was specifically observed in cancer cells producing PTHrP and type I PTH/PTHrP receptor (PTH1R) invaded the bone marrow, and PTHrP strongly upregulated ccn2 in MDA231 cells in vitro. Activation of protein kinase C (PKC) and protein kinase A (PKA) was necessary and sufficient for the stimulation of ccn2 by PTHrP. Indeed, inhibition of the extracellular signal-regulated kinase (ERK1/2), PKC, or PKA by specific inhibitors counteracted the stimulation of ccn2 expression. Incubation of MDA231 cells with PTHrP induced the activation of ERK1/2. Consistent with these findings, inhibition of PKC prevented PTHrP-induced ERK1/2 activation, whereas 12-O-tetradecanoylphorbol13-acetate (TPA), a stimulator of PKC, upregulated it. Conclusions: CCN2 was critically involved in osteolytic metastasis and was induced by PKA- and PKC-dependent activation of ERK1/2 signaling by PTHrP. Thus, CCN2 may be a new molecular target for anti-osteolytic therapy to shut off the PTHrP–CCN2 signaling pathway.