Temporal and spatial expression of Sox9, Pax1, TGF-ß1 and type I and II collagen in human intervertebral disc development.

PURPOSE: An accurate understanding of cellular biochemical changes in human intervertebral disc (IVD)s and the corresponding mechanisms during the developmental process still remain unknown and important for investigating the function of critical factors in normal IVD development as well as ascertaining the therapeutic targets for the IVD degeneration. METHODS: Under ethical conditions, human fetal cervical IVDs at 4, 5, and 6 months of pregnancy were collected at abortion surgery. Normal adult human C3-C7 cervical IVDs were taken from cadaveric donors. Sox9, Pax1, TGF-ß1 and type I/II collagen protein and RNA were detected. The number of positive cells was counted to calculate the optical density value for each factor. RESULTS: Sox9, Pax1, and TGF-ß1 expression in the IVD was remarkably reduced with the developmental stage. The location of high expression of Sox9, Pax1, and TGF-ß1 changed with the developmental stage, and migrated from the nucleus pulposus to the annulus fibrosus and endplate. Higher Sox9, Pax1, and TGF-ß1 expression was finally observed around the sclerotome of the vertebral body. The anabolism of type I/II collagens is significantly increased in the IVD in the mid-trimester fetus. CONCLUSIONS: Sox9, Pax1 and TGF-ß1 participate in the developmental process of the human IVD and vertebral body. However, these factors show a separate expression of mRNA and protein, suggesting that they are expressed in the strict time and spatial order.

PAX3-FOXO1A Expression in Rhabdomyosarcoma Is Driven by the Targetable Nuclear Receptor NR4A1.

Alveolar rhabdomyosarcoma (ARMS) is a devastating pediatric disease driven by expression of the oncogenic fusion gene PAX3-FOXO1A. In this study, we report overexpression of the nuclear receptor NR4A1 in rhabdomyosarcomas that is sufficient to drive high expression of PAX3-FOXO1A there. RNAi-mediated silencing of NR4A1 decreased expression of PAX3-FOXO1A and its downstream effector genes. Similarly, cell treatment with the NR4A1 small-molecule antagonists 1,1-bis(3-indolyl)-1-(p-hydroxy or p-carbomethoxyphenyl)methane (C-DIM) decreased PAX3-FOXO1A. Mechanistic investigations revealed a requirement for the NR4A1/Sp4 complex to bind GC-rich promoter regions to elevate transcription of the PAX3-FOXO1A gene. In parallel, NR4A1 also regulated expression of ß1-integrin, which with PAX3-FOXO1A, contributed to tumor cell migration that was blocked by C-DIM/NR4A1 antagonists. Taken together, our results provide a preclinical rationale for the use of NR4A1 small-molecule antagonists to treat ARMS and other rhabdomyosarcomas driven by PAX3-FOXO1A. Cancer Res; 77(3); 732-41. ©2016 AACR.

Assessment of the Utility of PAX8 Immunohistochemical Stain in Diagnosing Endocervical Glandular Lesions.

CONTEXT: PAX8, a member of the paired-box family of genes, is expressed in many tumors of Müllerian origin. However, it is unclear whether PAX8 is a useful marker in diagnosing endocervical glandular lesions because of limited data. OBJECTIVE: To study the expression of PAX8 in endocervical glandular lesions. DESIGN: We first studied a cohort of 29 cervical cone biopsies, followed by a second cohort of 17 cases of endocervical adenocarcinoma and 20 cases of uterine endometrioid adenocarcinoma. RESULTS: In the first cohort, we found that PAX8 was expressed in 23 of 23 (100%) benign endocervical glandular epithelium, 15 of 16 (94%) adenocarcinoma in situ, and 21 of 26 (81%) invasive endocervical adenocarcinoma specimens. In the second cohort, endocervical adenocarcinomas were positive for PAX8 in 14 of 17 (82%), strongly and diffusely positive for p16 in 14 of 17 (82%), positive for carcinoembryonic antigen in 12 of 17 (71%), positive for vimentin in 2 of 17 (12%), and positive for estrogen receptor in 7 of 17 cases (41%). Uterine endometrioid cancer was positive for PAX8 in 20 of 20 (100%), weakly and/or patchy positive for p16 in 17 of 20 (85%), positive for carcinoembryonic antigen in 2 of 20 (10%), positive for vimentin in 19 of 20 (95%), and positive for estrogen receptor in 20 of 20 cases (100%). CONCLUSIONS: PAX8 is expressed in the majority of benign, premalignant, and malignant endocervical glandular lesions. The usefulness of PAX8 in differentiating endocervical from endometrial lesions is limited.

Whole-transcriptome analysis of mouse adipose tissue in response to short-term caloric restriction.

Caloric restriction (CR) has been shown to extend the lifespan of many species by improving cellular function and organismal health. Additionally, fat reduction by CR may play an important role in lengthening lifespan and preventing severe age-related diseases. Interestingly, CR induced the greatest transcriptome change in the epididymal fat of mice in our study. In this transcriptome analysis, we identified and categorized 446 genes that correlated with CR level. We observed down-regulation of several signaling pathways, including insulin/insulin-like growth factor 1 (insulin/IGF-1), epidermal growth factor (EGF), transforming growth factor beta (TGF-ß), and canonical wingless-type mouse mammary tumor virus integration site (Wnt). Many genes related to structural features, including extracellular matrix structure, cell adhesion, and the cytoskeleton, were down-regulated, with a strong correlation to the degree of CR. Furthermore, genes related to the cell cycle and adipogenesis were down-regulated. These biological processes are well-identified targets of insulin/IGF-1, EGF, TGF-ß, and Wnt signaling. In contrast, genes involved in specific metabolic processes, including the tricarboxylic acid cycle and the electron transport chain were up-regulated. We performed in silico analysis of the promoter sequences of CR-responsive genes and identified two associated transcription factors, Paired-like homeodomain 2 (Pitx2) and Paired box gene 6 (Pax6). Our results suggest that strict regulation of signaling pathways is critical for creating the optimal energy homeostasis to extend lifespan.

A GRIA2 and PAX8-positive renal solitary fibrous tumor with NAB2-STAT6 gene fusion.

Solitary fibrous tumor (SFT) is a rare neoplasm composed of mesenchymal-derived spindle cells. Although SFT occurs anywhere in the body, they most frequently affects the thoracic region. Here, we reported an extremely rare case of an extrathoracic SFT occurring primarily in the kidney. To our knowledge, little information has been described on the immunohistochemistry (IHC) and genetics of renal SFT.A 41-year old Japanese female came to our hospital for further examination of a left kidney mass detected incidentally with ultrasound. Extensive investigation of the tumor, including physical, laboratory, and image examinations led to a clinical diagnosis of renal cancer (cT1aN0M0), which were in most parts imbedded in the lower polar parenchyma. The patient underwent laparoscopic radical nephrectomy. The mass was diagnosed pathologically as SFT originating from the kidney, but not as renal carcinoma. Microscopically, the tumor was composed of spindle-shape cells distributed variably in dense collagenous stroma and had a focal hemangiopericytomatous staghorn-like vascular pattern. Mitotic figures, atypical structures, necrosis and hemorrhage were not identified. No adjuvant therapies were given postoperatively. The patient has been free of tumor recurrence for 25 months since the surgery. IHC revealed that the tumor diffusely expressed CD34, CD99, Bcl2, PAX8, NAB2, STAT6, and GRIA2. The tumor stained negatively for desmin, S-100, c-Kit, CK-AE1/AE3, CDK4 and MDM2. A NAB2-SATA6 gene fusion was detected in tumor cells by reverse transcription-polymerase chain reaction, direct sequencing, and an in situ proximity ligation brightfield assay. The gene fusion occurred as an 831 bp truncation of exon 2 in NAB2 connected to the beginning of exon 3 in STAT6. We have reported a case of GRIA2 and PAX8-positive SFT occurring primarily in the kidney with such NAB2-STAT6 gene fusion for the first time. Diffuse expression of PAX8 in the tumor might present with a renal origin. Reportedly, benign histology of SFT cannot necessarily predict favorable clinical prognosis. Genetic alterations recently identified in SFT could possibly refer to risk stratification for tumor recurrence. However, malignant preponderance of extrathoracic SFT over thoracic SFT remains unexplained so far. Long-term follow-up after surgery should be performed in the present case.

Kruppel-Like Factor 4 Regulates Granule Cell Pax6 Expression and Cell Proliferation in Early Cerebellar Development.

Kruppel-like factor 4 (Klf4) is a transcription factor that regulates many important cellular processes in stem cell biology, cancer, and development. We used histological and molecular methods to study the expression of Klf4 in embryonic development of the normal and Klf4 knockout cerebellum. We find that Klf4 is expressed strongly in early granule cell progenitor development but tails-off considerably by the end of embryonic development. Klf4 is also co-expressed with Pax6 in these cells. In the Klf4-null mouse, which is perinatal lethal, Klf4 positively regulates Pax6 expression and regulates the proliferation of neuronal progenitors in the rhombic lip, external granular layer and the neuroepithelium. This paper is the first to describe a role for Klf4 in the cerebellum and provides insight into this gene's function in neuronal development.

In vivo tumor growth of high-grade serous ovarian cancer cell lines.

OBJECTIVE: Genomic studies of ovarian cancer (OC) cell lines frequently used in research revealed that these cells do not fully represent high-grade serous ovarian cancer (HGSOC), the most common OC histologic type. However, OC lines that appear to genomically resemble HGSOC have not been extensively used and their growth characteristics in murine xenografts are essentially unknown. METHODS: To better understand growth patterns and characteristics of HGSOC cell lines in vivo, CAOV3, COV362, KURAMOCHI, NIH-OVCAR3, OVCAR4, OVCAR5, OVCAR8, OVSAHO, OVKATE, SNU119 and UWB1.289 cells were assessed for tumor formation in nude mice. Cells were injected intraperitoneally (i.p.) or subcutaneously (s.c.) in female athymic nude mice and allowed to grow (maximum of 90 days) and tumor formation was analyzed. All tumors were sectioned and assessed using H&E staining and immunohistochemistry for p53, PAX8 and WT1 expression. RESULTS: Six lines (OVCAR3, OVCAR4, OVCAR5, OVCAR8, CAOV3, and OVSAHO) formed i.p xenografts with HGSOC histology. OVKATE and COV362 formed s.c. tumors only. Rapid tumor formation was observed for OVCAR3, OVCAR5 and OVCAR8, but only OVCAR8 reliably formed ascites. Tumors derived from OVCAR3, OVCAR4, and OVKATE displayed papillary features. Of the 11 lines examined, three (Kuramochi, SNU119 and UWB1.289) were non-tumorigenic. CONCLUSIONS: Our findings help further define which HGSOC cell models reliably generate tumors and/or ascites, critical information for preclinical drug development, validating in vitro findings, imaging and prevention studies by the OC research community.

Simultaneous carcinomas of the breast and ovary: utility of Pax-8, WT-1, and GATA3 for distinguishing independent primary tumors from metastases.

Breast carcinomas rarely metastasize to the ovary and are even more rarely present clinically as primary ovarian tumors. However, patients with breast cancer not infrequently develop independent primary ovarian carcinomas. In these cases, distinction between independent primaries and metastatic tumors is crucial. Several comparative immunohistochemical studies have been reported, but few included significant clinicopathologic data and none investigated cases of ovarian and breast carcinomas from the same patients. In this study, we compared 18 cases of patients with bona fide independent breast and ovarian carcinomas (15 high-grade serous and 3 clear cell carcinomas), with 9 cases of patients with known mammary carcinomas (7 lobular and 2 ductal carcinomas) metastatic to the ovary. Immunohistochemical stains for Pax-8, WT-1, and GATA3 were carried out on tissue microarrays (TMA). Most primary ovarian carcinomas were larger than the metastatic tumors (P=0.001) and were diagnosed at an advanced stage. All primary ovarian tumors showed marked nuclear pleomorphism, whereas only 2 metastatic breast carcinomas had Grade 3 nuclei (P=0.000). The vast majority of ovarian metastases (7/9) showed the typical pattern of lobular breast carcinoma. Pax-8 and WT-1 expression were found in 16 of 18 (88%) and 13 of 18 (72%) primary ovarian carcinomas, respectively. In contrast, all primary ovarian carcinomas were negative for GATA3. The 2 Pax-8-negative ovarian carcinomas were also negative for WT-1. With the exception of 3 triple-negative carcinomas, all primary breast carcinomas were positive for GATA3. All metastatic breast carcinomas were positive for GATA3 and negative for Pax-8. WT-1 expression was seen in only 1 of 9 metastatic breast carcinomas (11%). Patients with ovarian metastases had worse prognosis than patients with independent breast and ovarian carcinomas (P=0.000). Pax-8, WT-1, and GATA3 immunoreactions are useful in the distinction between independent primaries and metastatic mammary carcinomas to the ovary in the light of clinicopathologic findings.

Comparison of PAX6 and PAX8 as immunohistochemical markers for pancreatic neuroendocrine tumors.

To compare the utility of PAX6 and PAX8 as immunohistochemical markers for neuroendocrine tumors (NETs) of pancreatic origin, we performed PAX6 and PAX8 immunostains on 178 NETs, including 110 primary NETs (26 pancreatic, 10 gastric, 12 duodenal, 22 jejuno-ileal, 10 rectal, 30 pulmonary) and 68 NETs metastatic to the liver (24 pancreatic, 1 duodenal, 37 jejuno-ileal, 1 rectal, 5 pulmonary). Among primary NETs, PAX6 and PAX8 were positive in 65 % (17/26) and 73 % (19/26) of pancreatic, 0 % (0/10) and 10 % (1/10) of gastric, 92 % (11/12) and 92 % (11/12) of duodenal, 0 % (0/22) and 0 % (0/22) of jejuno-ileal, 90 % (9/10) and 80 % (8/10) of rectal, and 0 % (0/30) and 23 % (7/30) of pulmonary NETs, respectively. PAX6 and PAX8 positivity was seen in 46 % (11/24) and 50 % (12/24) of metastatic pancreatic NETs to the liver, respectively. None of the nonpancreatic NETs metastatic to the liver were immunoreactive for either PAX6 or PAX8. PAX6 showed a slightly but statistically significant higher specificity for pancreatic NETs than did PAX8 (P = 0.039), while the sensitivities were similar (P = 0.51). PAX6 had the additional advantages over PAX8 of not exhibiting nonspecific cytoplasmic staining of tumor cells and only infrequently staining background lymphocytes. Since rectal NETs rarely present with metastatic disease, positive staining of a metastatic NET of unknown primary origin for PAX6 and/or PAX8 favors a pancreatic or duodenal origin. This information may be helpful in directing further diagnostic studies to identify the primary site of the metastatic tumor.

Paired box gene 8 (PAX8) expression is associated with sonic hedgehog (SHH)/wingless int (WNT) subtypes, desmoplastic histology and patient survival in human medulloblastomas.

AIMS: The paired box gene 8 (PAX8) plays crucial roles in organ patterning and cellular differentiation during development and tumorigenesis. Although its function is partly understood in vertebrate development, there is poor data concerning human central nervous system (CNS) development and brain tumours. METHODS: We investigated developing human (n = 19) and mouse (n = 3) brains as well as medulloblastomas (MBs) (n = 113) for PAX8 expression by immunohistochemistry. Human MB cell lines were assessed for PAX8 expression using polymerase chain reaction and immunoblotting and analysed for growth and migration following PAX8 knock-down by small interfering ribonucleic acid (siRNA). RESULTS: PAX8 protein expression was associated with germinal layers in human and murine forebrain and hindbrain development. PAX8 expression significantly decreased over time in the external granule cell layer but increased in the internal granule cell layer. In MB subtypes, we observed an association of PAX8 expression with sonic hedgehog (SHH) and wingless int subtypes but not with group 3 and 4 MBs. Beyond that, we detected high PAX8 levels in desmoplastic MB subtypes. Univariate analyses revealed high PAX8 levels as a prognostic factor associated with a significantly better patient prognosis in human MB (overall survival: Log-Rank P = 0.0404, Wilcoxon P = 0.0280; progression-free survival: Log-Rank P = 0.0225; Wilcoxon P = 0.0136). In vitro assays revealed increased proliferation and migration of MB cell lines after PAX8 siRNA knock-down. CONCLUSION: In summary, high PAX8 expression is linked to better prognosis in MBs potentially by suppressing both proliferative and migratory properties of MB cells. The distinct spatio-temporal expression pattern of PAX8 during brain development might contribute to the understanding of distinct MB subtype histogenesis.

PAX-8 expression in renal tumours and distant sites: a useful marker of primary and metastatic renal cell carcinoma?

AIMS: Immunohistochemical stains have greatly improved the diagnostic accuracy of renal cell carcinoma (RCC) for primary and distant tumours. We evaluate a marker that has recently been incorporated in clinical practice, PAX-8, in primary and metastatic RCCs. METHODS: Two distinct tissue microarrays were used, one consisting of over 334 renal tumours, 294 with adjacent normal kidney and the other with 40 matched nephrectomy and metastatic sites of RCC. PAX-8 expression was assessed by a method of quantitative immunofluorescence. RESULTS: PAX-8 was positive in 96% (146/152) of normal renal tissue and 83% (227/272) of renal tumours. PAX-8 staining was positive in clear cell, papillary and chromophobe tumours in 80% (165/207), 95% (39/41) and 100% (6/6) of samples, respectively. Overall, intensity of PAX-8 expression was significantly higher in RCC metastatic sites than in the primary site (p=0.0047), however, in matched sites there was no statistically significant difference in the proportion of positive versus negative specimens (p=0.274). CONCLUSIONS: As the role of molecular markers expands in the diagnostic algorithm, this study confirms that PAX-8 expression is a useful diagnostic marker for RCC. PAX-8 expression was found in the primary tumour and distant sites. Compared with normal tissue and other histological types, clear cell RCC has lower PAX-8 expression and is less frequently positive, therefore, the lack of expression does not exclude a tumour of renal origin.

Evaluation of PAX2 and PAX8 expression in salivary gland neoplasms.

PAX2 and PAX8 are transcription factors involved in embryogenesis that have been utilized as immunohistochemical indicators of tumor origin. Specifically, PAX2 is a marker of neoplasms of renal and müllerian origin, while PAX8 is expressed by renal, müllerian, and thyroid tumors. While studies examining these transcription factors in a variety of tumors have been published, data regarding their expression in salivary gland neoplasms are limited. The goal of this study was to assess expression of PAX2 and PAX8 in a large cohort of salivary gland tumors. Utilizing tissue microarrays, samples of normal salivary glands (n = 68) and benign and malignant salivary gland neoplasms (n = 442) were evaluated for nuclear immunoreactivity with PAX2 and PAX8. No expression was observed with either marker in the normal salivary glands, and PAX8 was negative in all neoplasms. Focal expression of PAX2 was observed in one example each of oncocytoma and acinic cell carcinoma. These results indicate that evaluation of PAX2 and/or PAX8 expression would be valuable in differentiating primary salivary gland tumors from metastases known to express PAX2 and/or PAX8.

Differential patterns of PAX8, p16, and ER immunostains in mesonephric lesions and adenocarcinomas of the cervix.

Mesonephric remnants, usually located deep in the lateral cervical wall, may become hyperplastic resulting in a florid proliferation. These can be misinterpreted as malignant and confused with endocervical adenocarcinomas. Recent data have shown that PAX2 is diffusely expressed in mesonephric remnants and hyperplasias. PAX8 is a related transcription protein that is expressed in tissues of müllerian and wolffian origin. In this study, we have investigated the utility of an immunohistochemical panel comprising of PAX8, estrogen receptor (ER), and p16 in the differential diagnosis between mesonephric proliferations and cervical adenocarcinomas. A database search was conducted for cases of mesonephric remnants/hyperplasia/carcinoma of cervix and invasive cervical adenocarcinomas. Immunohistochemical stains for PAX8, ER, and p16 were performed using the avidin-biotin peroxidase technique on the most representative tissue. The search yielded 28 cases of mesonephric proliferations of cervix (15 mesonephric remnants, 12 mesonephric hyperplasias, and 1 mesonephric adenocarcinoma) and 16 cases of cervical adenocarcinomas (15 usual type and 1 adenoma malignum). Immunohistochemically, all the mesonephric proliferations, regardless of being benign or malignant, displayed a consistent staining pattern-diffusely and strongly positive for PAX8, negative for ER, and patchy cytoplasmic staining for p16. The usual type cervical adenocarcinomas exhibited a variable staining pattern with PAX8 and ER but all were strongly and diffusely positive for p16. The case of adenoma malignum was PAX8 positive, ER negative, and showed weak and patchy staining with p16. Our study suggests that a panel of immunohistochemical stains composed of PAX8, p16, and ER is useful in the distinction between mesonephric proliferations and cervical adenocarcinomas.

Mesonephric carcinosarcoma involving uterine cervix and vagina: report of 2 cases with immunohistochemical positivity For PAX2, PAX8, and GATA-3.

Mesonephric carcinomas are rare tumors predominantly arising in the uterine cervix from mesonephric remnants. Although the tumor has classic morphologic features, some cases can mimic Müllerian adenocarcinoma and be misdiagnosed, especially those with significant ductal pattern. Moreover, there is an overlap in immunohistochemical results with endometrial and endocervical carcinomas. In this study, we report 2 cases of mesonephric carcinosarcoma, originally diagnosed as Müllerian carcinomas, 1 presenting in the vagina; review immunohistochemical results including positivity for GATA-3, not previously reported and comment on the proposed panel of PAX8, p16, and estrogen receptors as discriminators of Müllerian adenocarcinoma (endocervical or endometrial) versus mesonephric carcinoma.

Use of a murine embryonic stem cell line that is sensitive to high glucose environment to model neural tube development in diabetic pregnancy.

BACKGROUND: Neural tube defects (NTDs) are significantly increased by maternal diabetes. Embryonic stem cells (ESC) that can differentiate into neuroepithelium and can sense supraphysiological glucose concentrations would be very valuable to simulate the effects of maternal diabetes on molecular and cellular processes during neural tube formation. METHODS: LG-ESC, a recently established ESC line that expresses the glucose transporter, Scl2a2, and is sensitive to elevated glucose concentrations, were grown for up to 8 days in a three-dimensional culture to form neural cysts. We tested whether high glucose media inhibits expression of Pax3, a gene that is required for neural tube closure and whose expression is inhibited in embryos of diabetic mice, and inhibits formation of neural cysts. RESULTS: Pax3 expression was detected after 4 days of culture and increased with time. Pax3 expression was inhibited by high glucose media, but not if cells had been cultured in low glucose media for the first 4 days of culture. Pax7, which is also expressed in dorsal neural tube, was not detected. Pax6, which is expressed in the ventral neural tube, was detected only after 8 days of culture, but was not inhibited by high glucose. High glucose media did not inhibit formation of neural cysts. CONCLUSION: LG-ESC can be used as a model of embryonic exposure to a diabetic environment during neural tube development. While high glucose exposure inhibits expression of a gene required for neural tube closure, it may not inhibit all of the processes involved in formation of a neural tube-like structure.

A subset of nondescript axillary lymph node inclusions have the immunophenotype of endosalpingiosis.

We report 2 cases of bland, otherwise nondescript axillary lymph node inclusions that have the immunophenotype of endosalpingiosis in patients with concurrent invasive breast carcinomas. Neither inclusion demonstrated the classic morphology of endosalpingiosis with admixed ciliated and secretory cells. Rather, both cases were composed of nondescript cuboidal to columnar bland epithelial cells situated within the lymph node capsule. Whereas both inclusions labeled diffusely for estrogen receptor and lacked evidence of a myoepithelial component, both labeled diffusely for PAX8 and WT-1, which distinguished them from their corresponding concurrent primary mammary carcinomas. These findings suggest that a subset of otherwise nondescript axillary lymph node inclusions represent endosalpingiosis and highlight the utility of PAX8 and WT-1 immunohistochemistry in distinguishing these from metastatic well-differentiated ductal carcinoma.

Reliability of PAX8 in clinical practice to accurately determine primary site of origin in female pelvic or abdominal lesions.

Female patients with gynecological malignancies can harbor peritoneal pelvic or abdominal lesions; however, other primary tumors can involve the peritoneum as well. Since sampling of the peritoneum now can be easily performed by fine needle aspiration or percutaneous biopsy, we have noticed an increase in such procedures as initial attempts to establish a diagnosis. PAX8 has been used alone or in combination with other tumor markers to accurately classify these lesions and determine primary site of origin; however, prior published studies determined expression of PAX8 within historically diagnosed cases. We reviewed the reliability of PAX8 to determine tumor type or primary site in 135 current clinical pelvic or abdominal lesions and highlight several pitfalls in its routine use, in particular, relying on the presumed expression pattern (positive or negative) within a given primary tumor and that poorly differentiated endometrial endometrioid carcinomas or undifferentiated carcinomas may have patchy PAX8 expression or even lose expression within the primary tumor or the metastasis.

Hyper-activation of Notch3 amplifies the proliferative potential of rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.