High-grade Tumor Cells Research Articles

Adrenocortical Carcinoma Arising in an Adrenal Rest: a Case Report and Review of the Literature

Carcinomas arising from embryonic adrenal rests are rare with only a handful of reported cases. We report a case of an adrenocortical carcinoma arising from an adrenal rest located between the bladder and prostate in a 51 year-old man. The patient presented following a year of rectal pain and constipation. Computed tomography (CT) scan revealed a 9 cm pelvic mass that appeared to arise from the soft tissue between the bladder and prostate, with displacement of the organs and narrowing of the rectal lumen, suspected to be a sarcoma. The surgically resected specimen showed a well-circumscribed, partially encapsulated tumor measuring 10.0 cm in greatest dimension. Both adrenal glands were identified intraoperatively. Grossly, the lesion was heterogeneous tan-brown to yellow, hemorrhagic and necrotic. Histology revealed sheets and nests of high-grade pleomorphic tumor cells with abundant clear to vacuolated cytoplasm with areas of necrosis, a high mitotic index (>10 mitoses/10 HPF) and foci suspicious for lymphovascular invasion. Adjacent adrenal cortical-type tissue was identified. Immunohistochemical stains revealed the tumor cells were weakly and focally positive for MiTF, Melan-A, inhibin and synaptophysin, and negative for CKAE1/AE3, HMB-45, calretinin, EMA, SMA, chromogranin, PAX8, MDM2 and CDK4. Based upon the morphologic and immunohistochemical profile, this was diagnosed as an adrenocortical carcinoma, arising in an adrenal rest. To our knowledge, no such tumor has been previously described in this location.

ISY-12-5 - Current status and perspective of systemic chemotherapies for GEP-NEC

According to the World Health Organization grading system for neuroendocrine neoplasms, neuroendocrine carcinoma (NEC) is a poorly differentiated, high-grade malignant tumor, including small and large cell carcinoma. The primary sites of NEC vary, with gastroenteropancreatic NEC (GEP-NEC) accounting for 20%–68% of extra-pulmonary NEC. Treatment guidelines for advanced extra-pulmonary NEC recommend platinum-based chemotherapy regimens, which are suitable for small cell lung carcinoma. There are two large-scale, multicenter retrospective analyses of advanced GEP-NEC: the NORDIC NEC study [Sorbye H et al. Ann Oncol. 2013;24(1)] and the Japanese study [Yamaguchi et al. Cancer Sci. 2014;105(9)]. In both studies, GEP-NEC prognoses varied according to primary organs. Although only etoposide plus cisplatin regimen (EP) was the mainstream first line chemotherapy in NORDIC, EP and irinotecan plus cisplatin regimen (IP) were commonly used in Japan. Both regimens (EP, IP) have been demonstrated to show favorable efficacy and have been acknowledged as de facto standard regimens for advanced NEC, although it remains unclear which of the two regimens might yield more favorable outcomes. Therefore, a phase III study comparing EP and IP in terms of overall survival for patients with recurrent or unresectable GEP-NEC (TOPIC-NEC, UMIN000014795) has been commenced by the Japan Clinical Oncology Group (JCOG). The sample size was set as 140 (70 patients per arm). For second line regimens, amrubicin, irinotecan, and S-1 were commonly utilized in Japan, but no standard regimens have been established. A multicenter phase II study to evaluate the efficacy of everolimus in pancreatic NEC patients who are unresponsive to/intolerant of platinum-based regimens (NECTOR: UMIN000012752) is currently in progress.

Impact of tumor histology and grade on treatment success of percutaneous renal cryoablation.

We analyzed oncological outcomes in patients who underwent percutaneous renal cryoablation (PRC) with documented renal cell carcinoma (RCC) by perioperative biopsy. Multicenter retrospective analysis of 153 patients [median follow-up 48months] who underwent PRC from 09/2005 to 08/2014 was performed. We divided the cohort into patients who developed recurrence versus no recurrence. Kaplan-Meier analyses examined recurrence-free survival (RFS) according to grade and histology. Multivariable analysis (MVA) was performed to identify factors associated with tumor recurrence. One hundred and fifty-three patients were analyzed [18 patients (11.8%) with recurrence and 135 (88.2%) patients without recurrence]. There were no differences between the groups with respect to demographics, RENAL score, and number of probes utilized. Recurrence group had larger tumor size (3.1 vs. 2.4cm; p=0.011), upper pole tumor location (p=0.016), and greater proportions of high-grade tumor (33 vs. 0.7%; p<0.001) and clear cell histology (77.8 vs. 45.9%; p=0.011). Four-year RFS was 100 versus 80% for grade 1 versus grade 2/3 tumors (p=0.0002), and 97 versus 88% for other RCC versus clear cell RCC (p=0.07). MVA demonstrated tumor size>3cm (OR 2.46; p=0.019), clear cell histology (OR 2.12; p=0.027), and high tumor grade (OR 2.33, p<0.001) as independent risk factors associated with tumor recurrence. Association of higher grade and clear cell histology with recurrence and progression suggests need for increased emphasis on preoperative risk stratification by biopsy, with grade 1 and non-clear cell RCC being associated with improved treatment success than higher grade and clear cell RCC.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Tubular epithelial cells in renal clear cell carcinoma express high RIPK1/3 and show increased susceptibility to TNF receptor 1-induced necroptosis.

We previously reported that renal clear cell carcinoma cells (RCC) express both tumor necrosis factor receptor (TNFR)-1 and -2, but that, in organ culture, a TNF mutein that only engages TNFR1, but not TNFR2, causes extensive cell death. Some RCC died by apoptosis based on detection of cleaved caspase 3 in a minority TUNEL-positive cells but the mechanism of death in the remaining cells was unexplained. Here, we underpin the mechanism of TNFR1-induced cell death in the majority of TUNEL-positive RCC cells, and show that they die by necroptosis. Malignant cells in high-grade tumors displayed threefold to four fold higher expression of both receptor-interacting protein kinase (RIPK)1 and RIPK3 compared with non-tumor kidney tubular epithelium and low-grade tumors, but expression of both enzymes was induced in lower grade tumors in organ culture in response to TNFR1 stimulation. Furthermore, TNFR1 activation induced significant MLKLSer358 and Drp1Ser616 phosphorylation, physical interactions in RCC between RIPK1-RIPK3 and RIPK3-phospho-MLKLSer358, and coincidence of phospho-MLKLser358 and phospho-Drp1Ser616 at mitochondria in TUNEL-positive RCC. A caspase inhibitor only partially reduced the extent of cell death following TNFR1 engagement in RCC cells, whereas three inhibitors, each targeting a different step in the necroptotic pathway, were much more protective. Combined inhibition of caspases and necroptosis provided additive protection, implying that different subsets of cells respond differently to TNF-α, the majority dying by necroptosis. We conclude that most high-grade RCC cells express increased amounts of RIPK1 and RIPK3 and are poised to undergo necroptosis in response to TNFR1 signaling.

Decreased Expression of Bioinformatically Predicted piwil2-targetting microRNAs, miR-1267 and miR-2276 in Breast Cancer.

Human Piwil2, a member of Piwi subfamily of Argonaute proteins, is primarily expressed in testis, where it regulates self-renewal of germ cells. However, its ectopic expression has been reported with several tumors, including breast cancer. The upregulation of piwil2 in various stages of breast cancer suggested its suitability as a novel tumor biomarker. Considering the vital role of microRNAs (miRNAs) in regulating the expression of most human genes, we hypothesized a concomitant downregulation of the bioinformatically-predicted piwil2-targetting microRNAs in breast cancer. We employed different bioinformatic tools to predict piwil2-targeting miRNAs. Then, from the list of predicted miRNAs, we chose two less studied miRNAs (miR-1267 and miR-2276) for experimental validation. Using a real-time RT-PCR approach, we quantified the relative expression of the miRNAs in 31 pairs of formalin-fixed paraffin-embedded tumor/non-tumor tissue samples. Our data revealed a noticeable but not statistically significant (P = 0.133) downregulation of miR-1267 in tumor samples, compared to non-tumor samples obtained from the same patients. Downregulation of miR-1267 was more significant in higher grades of malignancies (fold change = 2.39, P = 0.033) and also in lymph nodes containing high-grade tumor cells (fold change = 6.66, P = 0.02). Interestingly, a significant upregulation of miR-1267 was observed in tumors at high stages (stage 3a, 3b), compared to low stages (stage 2a, 2b) (fold change = 8.05, P = 0.048). Similar patterns of expression alteration were also observed for miR-2276. Altogether, our findings suggest a probable tumor suppressor role for miR-1267 and miR-2276 in breast tumor initiation and progression, but a probable promoting role for them in invasion and metastasis.

Abstract A59: Prognostic relevance of PTEN and TP53 deletion in breast cancer

Abstract Inactivation of PTEN and TP53 have been suggested to promote unfavorable phenotypes in various cancers, including breast cancer. Chromosomal deletion is one well established mechanism for inactivation of these genes. In breast cancer, deletions of TP53 and PTEN are known to occur, but systematic analyses of its clinical relevance are lacking. We thus analyzed a tissue microarray (TMA) with 2,197 breast cancers by fluorescence in-situ hybridization (FISH) using specific probes against TP53 and PTEN in combination with respective centromere probes. We found, that TP53 deletion (29%/1026 cancers) occur more frequent in breast cancers than deletions of PTEN (19%/1,239 cancers). The frequency of PTEN deletion varied between histological subtypes with highest deletion rate in medullary cancers (18 of 39, 46%) and lowest deletion rate in mucinous cancers (1 of 28; 3%; p&amp;lt;0.0001) for mucinous vs. medullary). PTEN deletions were significantly linked to various unfavorable tumor features, including advanced tumor stage (p=0.0054), high-grade (p&amp;lt;0.0001), high tumor cell proliferation (Ki67 Labeling Index; p&amp;lt;0.0001), and shortened overall survival (p=0.0090). Deletions of TP53 were significantly linked to high-grade (p&amp;lt;0.0001) and high tumor cell proliferation (p=0,0193) but unrelated to histological subtypes, tumor stage, nodal status, and patient survival. Deletion of PTEN and TP53 were unrelated to each other (p=0.6469). Combined analysis of PTEN and TP53 deletions showed that TP53 deletion had no additional effect on patient prognosis in cancers with or without PTEN deletion. In conclusion, our data support a role of PTEN and TP53 deletions in breast cancer. However, only PTEN deletion is inked to aggressive tumor behavior, while TP53 deletion has no prognostic relevance in breast cancer. Citation Format: Martina Kluth, Anette Lebeau, Isabell Witzel, Sven Mahner, Linn Woelber, Stefan Geist, Peter Paluchowski, Christian Wilke, Uwe Heilenkoetter, Volkmar Mueller, Ronald Simon, Guido Sauter, Luigi Terracciano, Rainer Krech, Albert von der Assen, Eike Burandt, Patrick Lebok. Prognostic relevance of PTEN and TP53 deletion in breast cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A59.

IMPS-26HARNESSING THE MYELOID CHECKPOINT CD47-SIRPa AXIS AGAINST ADULT AND PEDIATRIC MALIGNANT BRAIN TUMORS: A NOVEL IMMUNOTHERAPEUTIC MODALITY

INTRODUCTION: Adult and pediatric brain tumors are among the most fatal cancers known with limited treatment options. One of the key tumor features facilitating its growth and progression is evading the immune system. Recent data suggests that the interaction between the cell surface antigen CD47 and its binding partner SIRPα is a mechanism by which non-solid and solid tumors can evade the innate immune system. Here, we show that disrupting the interaction of this anti-phagocytic surface protein, CD47 with its interacting partner on macrophages (SIRPa) induces the macrophages to 'eat and kill' brain cancer cells through modulating the innate immune system. METHODS: CD47 expression was evaluated on surgical patient and post-mortem samples from different brain tumor: Adult and pediatric Glioblastoma, Oligodendroglioma, DIPG, Medulloblastoma, Ependymoma, ATRT, PNET tissue samples were analyzed for CD47 expression. Using a novel humanized anti-CD47 monocloncal antibody we tested whether blocking CD47- SIRPα interaction induces phagocytosis. Orthotopic patient derived xenograft models were treated them with the humanized anti-CD47 mAb. Additionally, we tested the effect of huCD47-Ab antibody on normal neural cells. The ability of blocking the CD47-SIRPa axis to enhance the therapeutic response of available clinical monoclonal antibodies was also tested. RESULTS: CD47 expression was upregulated in all tumor types and was present in >90% of the cells in high-grade tumors. Blocking the CD47- SIRPα interaction increased phagocytosis by macrophages in tumor but not on normal neural cells. Systemic treatment with anti-CD47 antibody significantly reduced tumor burden and enhanced survival. NHP studies show that the huCD47 antibody can be administered safely at potentially therapeutic serum levels. CONCLUSION: Anti-CD47 therapy is a viable and effective treatment modality for human high-grade brain tumors. Currently a Phase I study is ongoing to evaluate its safety in patients with non-CNS tumors to be followed by safety in patients with CNS malignancies

Cytology in uropathological diagnostics

Cytology in uropathological diagnostics is mainly performed for oncological purposes. The assessment of malignancy by urothelial cell morphology is therefore decisive; however, cytology is only sensitive enough to detect high-grade tumor cells and the different low-grade tumors cannot be reliably diagnosed. Thus, the four-tier classification system of cytological findings (i.e. negative, atypical cells but significance uncertain, suspicious and positive) refers to high-grade tumor cells only. Furthermore, for valid cytological diagnostics not only the cytological specimen but also clinical information on cystoscopy findings and, if applicable, a biopsy should be evaluated together. In difficult differential diagnostic settings, e.g. differentiation between reactive versus neoplastic atypia or difficult to access lesions in the upper urinary tract, additional fluorescence in situ hybridization of cytological preparations might be helpful. At the moment there are no indications for further immunocytology or additional biomarker tests.

Abstract 5408: Liposomal doxorubicin with modified EGCG increased antitumor activity by topical targeting efficiency into tumor

Abstract Purpose: The aim of this study is increase of antitumor activity using novel liposome with (-)-epigallocatechin-3-gallate (EGCG) as one of the green tea components. Liposome has been known as a one of useful drug carriers for delivering drug into targeting site. Moreover, some ligand is able to modify around liposomal membrane by a simple method. On the other hand, it was reported that surface of high grade tumor cell expresses 67kDa laminin receptor (67LR) which intermediates of cytostatic effect of EGCG, and EGCG is able to induce apoptosis selectively. Our strategy was to prepare liposomal doxorubicin with EGCG as carrier ligand for getting antitumor activity by both apoptosis and doxorubicin at tumor site. In this study, we tried to prepare EGCG-modified liposome and evaluated the antitumor activity in vitro. Methods: The liposomal doxorubicin was prepared by L-α-distearoylphosphatidylcholine, cholesterol, L-α-distearoylphosphatidyl-DL-glycerol and doxorubicin (100:100:60:18 μmol). Doxorubicin included EGCG-modified liposome (EL-DOX) was prepared to add EGCG-dipalmitate. Amount of EGCG modification on liposomal membrane was determined using DPPH assay. On P388 leukemia cells and M5076 ovarian sarcoma cells, cytotoxicity was evaluated by WST-8 assay and shown IC50. DOX uptake into tumor cells was evaluated in vitro. The concentration of doxorubicin into tumor cells was determined with a fluorescence spectrophotometer at an excitation wavelength of 500 nm and an emission wavelength of 550 nm. Results and Discussion: Particle sizes of EL-DOX were 147 nm on average, zeta potential was -33.1 mV and encapsulated ratio of doxorubicin was 96.5 %. These data of sizes and zeta potentials were better for intravenous administration of liposomes. EGCG ratio on liposomal membrane (71.0 %) was enough to show its ability. In the study of doxorubicin uptake into tumor cells, EL-DOX group was increased amount of doxorubicin for 30 min. The doxorubicin level of EL-DOX was significantly higher compared with that of unmodified liposomal doxorubicin (p&amp;lt;0.05). At cytotoxicity, EL-DOX was stronger than unmodified liposomal doxorubicin. It was provided that IC50 of EL-DOX was also 125 times higher than EGCG solution which was reported to have been antitumor activity. Furthermore, co-incubation of EGCG solution and liposomal doxorubicin was not shown the enhancement effect both doxorubicin uptake into cells and the cytotoxicity. It was suggested that superior effect were shown in modification of EGCG around liposomal membrane, not mixture of EGCG and liposome. In conclusion, EL-DOX exhibited strong antitumor activity efficiently. Citation Format: Ikumi Sugiyama, Kunihiro Kaihatsu, Nobuo Kato, Yasuyuki Sadzuka. Liposomal doxorubicin with modified EGCG increased antitumor activity by topical targeting efficiency into tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5408. doi:10.1158/1538-7445.AM2014-5408

Morphometry in the cytological diagnosis of cervical smears.

Morphometry of cells found in normal and abnormal smears taken from the vagina and the uterine cervix is the assessment of the size and diameter of their nuclei. The values of these quantities provide information on the origin of these cells and the degree of possible anomalies. Determining the morphometric traits of different types of cells found in the cervix and the uterus is a very important element in the diagnosis of disorders that often lead to cervical tumors. The aim of this research is to determine the morphometric characteristics of cells found in cervical smears by measuring the cell circumference, the diameter of the nucleus and the cell surface areain order to identify which clinical group the cells belong to, which facilitates diagnosis. The study material consisted of cervical smears that demonstrated the presence of cells in various phases of the clinical Bethesda classification. For each clinical classification, the values of the cell circumference, the cell surface area and the diameter of the nucleus were measured for 100 cells. The largest cells are normal cells in the surface layer. In relation to these cells, the atrophic cells from the groups containing atypical squamous cells of undetermined significance (ASC-US), low-grade squamous intraepithelial lesions (LSIL), high-grade squamous intraepithelial lesion (HSIL) and tumor cells tend to decrease in size, with small variations. Considering the mean values of the parameters analyzed, the cells of the LSIL group are larger than those from the ASC-US group. According to the mean values, normal cells have the smallest nucleus and the HSIL cells and tumor cells have the largest. The statistical analysis shows significant differences between the morphometric traits in the different clinical groups, which indicates that morphometry can be used in cytological diagnosis.

High expression of spindle assembly checkpoint proteins CDC20 and MAD2 is associated with poor prognosis in urothelial bladder cancer

Aneuploidy is a result of the abnormal expression of spindle assembly checkpoint (SAC) proteins and resulting abnormal spindle function during mitosis. High expression of cell division cycle 20 homolog (CDC20) and mitotic arrest defective protein 2 (MAD2), key components of the SAC, has been reported in various carcinomas. However, the clinicopathological significance of CDC20 and MAD2 expressions in urothelial carcinoma of the human bladder (UCB) is unknown. We therefore studied the expression of CDC20 and MAD2 in UCB specimens by immunohistochemistry. High expression of CDC20 and MAD2 was observed in 59.0 % (200/339) and 51.0 % (173/339) of UCB cases, respectively. Most high-grade tumor cells exhibited diffuse nuclear and/or cytoplasmic staining for CDC20 and MAD2, whereas most low-grade tumor cells and normal urothelial cells were not stained. CDC20 overexpression was associated with advanced age (p = 0.010), high grade (p < 0.001), advanced stage (p < 0.001), non-papillary growth pattern (p < 0.001), and distant metastasis (p = 0.042). Similarly, high MAD2 expression correlated with high grade (p < 0.001), advanced stage (p < 0.001), and non-papillary growth pattern (p < 0.001). In univariate survival analyses, high CDC20 expression correlated with shorter recurrence-free survival (RFS) (p = 0.032) and poorer overall survival (OS) (p = 0.007) in patients with UCB, whereas high MAD2 expression was associated with poorer OS (p = 0.008). In multivariate analyses, high CDC20 expression correlated with shorter RFS of patients with Ta stage UCB (hazard ratio, 1.91; p = 0.01). In conclusion, increased expression of CDC20 and MAD2 is related to poor prognosis of UCB.

Primary resistance phenomena to oncolytic measles vaccine viruses

Measles vaccine virus (MeV) has been shown to possess profound oncolytic capabilities. However, tumor cell resistance to MeV may endanger broad clinical success. Here, this hypothesis is underlined by our analysis of the NCI-60 tumor cell panel infected with a suicide gene-armed MeV vector (MeV-SCD). Quantification of the MeV-SCD-induced oncolytic effect exhibited a 50% rate of NCI-60 solid tumor cell lines being susceptible to MeV-SCD induced oncolysis. In contrast, nearly 40% of the NCI-60 tumor cell lines had to be categorized as partially resistant (exhibiting 50-75% remnant tumor cells) and six tumor cell lines even showed high resistance to MeV-SCD-induced oncolysis with remnant tumor cell masses >75%. According to our further analysis, these high-grade resistant tumor cell lines i) exhibited a high variation in primary infectability rates and also different patterns of alterations ii) in virus replication and iii) in interferon response. This diversity of virotherapy resistance phenomena seems to go along with the diversity of genetic and epigenetic changes accompanying malignant transformation. Of paramount clinical importance, this plethora of resistance phenomena was shown to be overcome in vitro by employment of an increased MOI together with addition of the prodrug 5-FC, thus exploiting the highly efficient suicide gene function of vector MeV-SCD used in this study.

Abstract 5218: Development of Anti-CD47 therapy for pediatric brain tumors.

Abstract Introduction: Extensive molecular characterization of Brain tumors done in recent years has proven valuable for tumor classification, risk stratification and outcome prediction for current treatment modalities. However the current standard of care has not improved the prognosis and carries an increased risk of cognitive impairments for children with brain tumors. A characteristic feature of tumor progression and recurrence is its ability to evade the immune system. Our hypothesis is that by modulating the innate immune system we can enhance the ability of macrophages to ‘eat and kill’ brain cancer cells. Recent data suggests that the interaction between the cell surface antigen CD47 and its binding partner SIRPα is a mechanism by which non-solid and solid tumors can evade the innate immune system. To see if anti-CD47 therapy is a potential therapy in malignant pediatric brain tumors we first looked at CD47 expression in freshly isolated patient and postmortem samples from 4 different tumor types; Diffused Intrinsic Pontine Glioma, Medulloblastoma, Ependymoma and ATRTs. We tested the hypothesis that blocking CD47- SIRPα interaction induced phagocytosis in an in-vitro phagocytosis assay. We next established orthotopic xenografts models in immune compromised mice and treated them with anti-CD47 humanized antibody, which is currently being developed for clinical trials in hematopoietic and non-CNS malignancies. Result: CD47 expression was upregulated in all tumor types and was present in &amp;gt;90% of the cells in high grade tumors. Increased CD47 expression was observed in CD15+ and CD133+ putative cancer stem cell population. Blocking the CD47- SIRPα interaction increases tumor phagocytosis by macrophages in-vitro. Systemic treatment with anti-CD47 antibody significantly reduced tumor burden in an orthotopic xenograft setting. Conclusion: Anti-CD47 therapy is a viable and effective treatment modality for pediatric high grade brain tumors. Citation Format: Sharareh Gholamin, Siddhartha S. Mitra, Chase Elliott Richard, Achal Achrol, Doosik Kong, Jun Jae Shin, Michelle Monje-Deisseroth, Yoon-Jae Cho, Irving Weissman, Samuel H. Cheshier. Development of Anti-CD47 therapy for pediatric brain tumors. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5218. doi:10.1158/1538-7445.AM2013-5218

Abstract 4942: Deletion of the asymmetry regulator Lgl1 contributes to tumor stemness and invasiveness.

Abstract In the developing and adult brain asymmetric cell division (ACD) maintains a balance between self-renewing and differentiated progeny of neural stem and progenitor cells. The WD protein lethal giant larvae 1 (Lgl1) regulates the distribution of polarity proteins in dividing cells and thereby establishes an axis of polarity, which is crucial for proper asymmetric divisions to occur (Baek et al. Mutat Res. 1999; 436; 131-6). Lgl, along with other asymmetry regulators, have been identified as a putative tumor suppressor gene in Drosophila. There remains debate as to whether disrupting ACD by itself is sufficient to cause tumor initiation by mammalian stem and progenitor cells. We have previously shown that expression of multiple asymmetry regulators is disrupted in human glioma (Sugiarto et al. Cancer Cell; 2011; 20; 320-340). Here we address our hypothesis that ACD defects are crucial for tumorigenesis, by delineating the role of Lgl1 in tumor initiation and progression. Using mouse neural stem cells (NSCs) isolated from genetically modified Lgl1lox/lox mice (Klezovitch et al. Genes Dev. 2004; 18; 559-71) we investigated the contribution of Lgl1 to various hallmarks of cancer in vitro and in vivo. We demonstrate that loss of Lgl1 in mouse NSC and oligodendrocyte precursor cells disrupts ACD and increases the expression of early-stage precursor cell markers. Deletion of Lgl1 does not increase proliferation in vitro and deletion of Lgl1 alone does not transform cells transplanted into immunocompromised mice. However properties associated with a high-grade tumor, including invasiveness and stem cell marker expression, are increased when Lgl1 is deleted in the context of oncogenic EgfrVIII expression. Increase in tumor invasiveness has previously been demonstrated to be associated with an increase in stem cell marker expression (Molina et al. Neoplasia. 2010; 12; 453-63). Our data suggest that loss of asymmetry in tumor precursor cells blocks differentiation and contributes to tumor progression by increasing stemness and hence invasiveness. Citation Format: Robin G. Lerner, Loan Nguyen, Claudia Petritsch. Deletion of the asymmetry regulator Lgl1 contributes to tumor stemness and invasiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4942. doi:10.1158/1538-7445.AM2013-4942

Aquaporin3 Is Required for FGF-2-Induced Migration of Human Breast Cancers

PurposeThe aquaporin (AQP) family consists of a number of small integral membrane proteins that transport water and glycerol. AQPs are critical for trans-epithelial fluid transport. Recent reports demonstrated that AQPs, particularly AQP1 and AQP5, are expressed in high grade tumor cells of a variety of tissue origins, and that AQPs are involved in cell migration and metastasis. Based on this background, we examined whether AQP3, another important member of the AQP family, could facilitate cell migration in human breast cancers.MethodsPotential role of AQP3 was examined using two representative breast cancer cell lines (MDA-MB-231 and Bcap-37). Briefly, AQP3 expression was inhibited with a lentivirus construct that stably expressed shRNA against the AQP3 mRNA. AQP3 expression inhibition was verified with Western blot. Cell migration was examined using a wound scratch assay in the presence of fibroblast growth factor-2 (FGF-2). In additional experiments, AQP3 was inhibited by CuSO4. Fibroblast growth factor receptor (FGFR) kinase inhibitor PD173074, PI3K inhibitor LY294002, and MEK1/2 inhibitor PD98059 were used to dissect the molecular mechanism of FGF-2 induced AQP3 expression.ResultsFGF-2 treatment increased AQP3 expression and induced cell migration in a dose dependent manner. Silencing AQP3 expression by a lentiviral shRNA inhibited FGF-2 induced cell migration. CuSO4, a water transport inhibitor selective for AQP3, also suppressed FGF-2-induced cell migration. The FGFR kinase inhibitor PD173074, significantly inhibited FGF-2-induced AQP3 expression and cell migration. The PI3K inhibitor LY294002 and MEK1/2 inhibitor PD98059 inhibited, but not fully blocked, FGF-2-induced AQP3 expression and cell migration.ConclusionsAQP3 is required for FGF-2-induced cell migration in cultured human breast cancer cells. Our findings also suggest the importance of FGFR-PI3K and FGFR-ERK signaling in FGF-2-induced AQP3 expression. In summary, our findings suggest a novel function of AQP3 in cell migration and metastasis of breast cancers.

BMP and TGFbeta pathways in human central chondrosarcoma: enhanced endoglin and Smad 1 signaling in high grade tumors

BackgroundAs major regulators of normal chondrogenesis, the bone morphogenic protein (BMP) and transforming growth factor β (TGFB) signaling pathways may be involved in the development and progression of central chondrosarcoma. In order to uncover their possible implication, the aim of this study was to perform a systematic quantitative study of the expression of BMPs, TGFBs and their receptors and to assess activity of the corresponding pathways in central chondrosarcoma.MethodsGene expression analysis was performed by quantitative RT-PCR in 26 central chondrosarcoma and 6 healthy articular cartilage samples. Expression of endoglin and nuclear localization of phosphorylated Smad1/5/8 and Smad2 was assessed by immunohistochemical analysis.ResultsThe expression of TGFB3 and of the activin receptor-like kinase ALK2 was found to be significantly higher in grade III compared to grade I chondrosarcoma. Nuclear phosphorylated Smad1/5/8 and Smad2 were found in all tumors analyzed and the activity of both signaling pathways was confirmed by functional reporter assays in 2 chondrosarcoma cell lines. Immunohistochemical analysis furthermore revealed that phosphorylated Smad1/5/8 and endoglin expression were significantly higher in high-grade compared to low-grade chondrosarcoma and correlated to each other.ConclusionsThe BMP and TGFβ signaling pathways were found to be active in central chondrosarcoma cells. The correlation of Smad1/5/8 activity to endoglin expression suggests that, as described in other cell types, endoglin could enhance Smad1/5/8 signaling in high-grade chondrosarcoma cells. Endoglin expression coupled to Smad1/5/8 activation could thus represent a functionally important signaling axis for the progression of chondrosarcoma and a regulator of the undifferentiated phenotype of high-grade tumor cells.

Expression of the PTTG1 Oncogene Is Associated with Aggressive Clear Cell Renal Cell Carcinoma

The pituitary tumor transforming gene (PTTG1) is a recently discovered oncogene implicated in malignant progression of both endocrine and nonendocrine malignancies. Clear cell renal cell carcinoma (ccRCC) is cytogenetically characterized by chromosome 3p deletions that harbor the ccRCC-related von Hippel-Lindau, PBRM1, BAP1, and SETD2 tumor suppressor genes, along with chromosome 5q amplifications where the significance has been unclear. PTTG1 localizes to the chromosome 5q region where amplifications occur in ccRCC. In this study, we report a functional role for PTTG1 in ccRCC tumorigenesis. PTTG1 was amplified in ccRCC, overexpressed in tumor tissue, and associated with high-grade tumor cells and poor patient prognosis. In preclinical models, PTTG1 ablation reduced tumorigenesis and invasion. An analysis of gene expression affected by PTTG1 indicated an association with invasive and metastatic disease. PTTG1-dependent expression of the RhoGEF proto-oncogene ECT2 was observed in a number of ccRCC cell lines. Moreover, ECT2 expression correlated with PTTG1 expression and poor clinical features. Together, our findings reveal features of PTTG1 that are consistent with its identification of an oncogene amplified on chromsome 5q in ccRCC, where it may offer a novel therapeutic target of pathologic significance in this disease.

Epidermal growth factor enhances MPC-83 pancreatic cancer cell migration through the upregulation of aquaporin 3

Aquaporin (AQP) water channels are expressed in high-grade tumor cells of different tissue origins. In this study, we investigated whether AQP3 is expressed in cultured MPC-83 pancreatic cancer cells, whether AQP3 enhances cell migration and the signal pathway mechanism involved. MPC-83 pancreatic cancer cells were pre-treated and treated with EGF at different time points and then analyzed using western blotting. Results showed that epidermal growth factor (EGF) induced the phosphorylation of the EGF receptor (EGFR) and extracellular signal-regulated kinase (ERK), which peaked at 5min after EGF treatment. EGFR and ERK phosphorylation induced by EGF were inhibited by PD153035 (EGFR tyrosine kinase inhibitor) and U0126 (ERK inhibitor), respectively. EGF increased the activity of AQP3 in a dose- and time-dependent manner in MPC-83 pancreatic cancer cells, which peaked at 24h after treatment. The activity of AQP3 and cell migration were inhibited by PD153035, U0126 and CuSO4 (AQP3 water transport inhibitor). EGFR/ERK pathway-mediated AQP3 activation and cell migration were stimulated by EGF in cultured MPC-83 pancreatic cancer cells invitro and this cell signaling pathway is inhibited by the EGFR and ERK inhibitors, which may be used as potential therapeutic targets in the treatment of pancreatic cancer.

CD25 Is Expressed by Canine Cutaneous Mast Cell Tumors but not by Cutaneous Connective Tissue Mast Cells

Canine cutaneous mast cell tumors (MCT) of different histological grades have distinct biological behaviors. However, little is known about underlying molecular mechanisms that lead to tumor development and increasing malignancy with higher tumor grade. Recent studies have identified the interleukin-2 receptor (IL-2R) subunits CD25 and CD2 as markers that distinguish nonneoplastic from neoplastic mast cells in human systemic mastocytosis. In this study, their potential as a marker for canine MCT and their possible impact on MCT carcinogenesis were evaluated. mRNA expression levels of both genes were compared between grade 1 (n = 12) and grade 3 (n = 8) MCT, and protein expression levels of CD25 were compared in 90 MCT of different tumor grades. mRNA expression levels of both CD25 and CD2 were upregulated in grade 3 MCT. In contrast, CD25 protein was expressed by fewer tumor cells and at decreased levels in grade 3 tumors, while most grade 1 MCT had strong CD25 protein expression. Moreover, CD25 was not expressed by nonneoplastic, resting cutaneous mast cells, while few presumably activated mast cells in tissue samples from dogs with allergic dermatitis had weak CD25 expression. Taken together, these findings suggest that CD25 may play a critical role in early MCT development and may be a stimulatory factor in grade 1 MCT, while grade 3 MCT seem to be less dependent on CD25. Because of the low number of CD25-positive tumor cells in high-grade tumors, the usefulness of CD25 as a tumor marker is, however, questionable.