Frequent Lung Metastases Research Articles

Abstract A023: RNF185 modulates prostate cancer metastatic potential through regulation of epithelial-mesenchymal transition

Abstract RNF185 is a RING finger domain-containing ubiquitin ligase which is anchored to the ER and resemble RNF5, a ubiquitin ligase implicated in ER associated degradation. In prostate cancer, deregulation of the expression or activity of several ubiquitin ligases has been shown to contribute to disease progression (e.g., Siah2, SPOP, RNF6). Analysis of prostate tumor patient data revealed a negative correlation between RNF185 expression and prostate adenocarcinoma progression and metastasis. Supporting these observations, several prostate cancer cell lines exhibited greater migration and invasion capabilities upon RNF185 depletion, in vitro. Subcutaneous inoculation of mouse prostate cancer MPC3 cells, stably expressing shRNA against RNF185, resulted in larger tumors and more frequent lung metastases in NSG mice. These results suggest that the increased metastatic potential associated with lower RNF185 levels may depend on molecular changes intrinsic to the tumors cells. RNA-sequencing and Ingenuity Pathway Analysis of RNF185-depleted compared to control MPC3 cells identified wound healing and cellular movement among the most significantly upregulated pathways in RNF185-depleted cells. Transcription regulators of Epithelial-Mesenchymal Transition (EMT) Snai2 and Zeb1 were among the most upregulated genes that were predicted to be activated based on the expression of their target genes. Comparison between differentially expressed genes in patients with low RNF185 expression, and RNF185-depleted cell lines (from mouse and human prostate tumors) confirmed deregulation of genes implicated in EMT. Taken together, our results identify RNF185 as gatekeeper of prostate cancer metastasis, via its control of EMT genes implicated in cell motility and metastasis. Citation Format: Benjamin Van Espen, Ladan Fazli, Htoo Z. Oo, Rabi Murad, Martin Gleave, Ze'ev A. Ronai. RNF185 modulates prostate cancer metastatic potential through regulation of epithelial-mesenchymal transition [abstract]. In: Proceedings of the AACR Special Conference: Advances in Prostate Cancer Research; 2023 Mar 15-18; Denver, Colorado. Philadelphia (PA): AACR; Cancer Res 2023;83(11 Suppl):Abstract nr A023.

Cathelicidin LL-37 promotes EMT, migration and metastasis of hepatocellular carcinoma cells in vitro and mouse model

ABSTRACT The effect of cathelicidin hCAP18/LL-37 in hepatocellular carcinoma (HCC) metastasis remains unclear. Here, we confirmed that LL-37 expression enhanced endothelial-mesenchymal transition (EMT), migration and invasion in HCC cells. And the HER2/EGFR-MAPK/ERK signal participated in the process above. More frequent lung metastases were observed in an LL-37-overexpressing hematogenous metastasis model. Interestingly, 1,25(OH)2D3 together with si-LL-37 significantly enhanced 1,25(OH)2D3-induced inhibition of migration and invasion in PLC/PRF-5 cells, and also enhanced reversion of the EMT process. Therefore, LL-37 is involved in HCC metastases, and may act as an important factor to attenuate the inhibitory activity of 1,25(OH)2D3 on HCC metastasis. Targeting hCAP18/LL-37 may offer a potential strategy to improve the anticancer activity of 1,25(OH)2D3 in HCC therapy.

MULTIPLE LUNG NODULES AND AN UNCOMMON FINDING: TWO DISTINCT PULMONARY METASTASIS

TOPIC: Lung Cancer TYPE: Global Case Reports INTRODUCTION: The solitary lung nodule is a classical entity usually described under solid guidelines and strong scientific evidence, with well-known management and follow-up. However, dual or multiple pulmonary nodules might be a propedeutic challenge, although they are usually linked to a single nosological agent and when related to a tumoral origin are habitually provenient from breast, colorectal, renal cell, head and neck cancer. More uncommon primary tumors with frequent lung metastasis are pancreatic, testicular and thyroid cancer followed by osteosarcoma. This case report demonstrates the management of a patient with double main pulmonary nodules with distinct causes. CASE PRESENTATION: A 69 year old, non-smoker male patient from Rio de Janeiro, Brazil presented on a scheduled follow-up for a nephrectomy in 2009 caused by a renal clear cell carcinoma with a chest tomography with two lung nodules in lingula, both measuring about 1,5 cm and clinically asymptomatic. Further investigation revealed nodule enlargement but low avidity on PET-CT 2 months later. Patient underwent exploratory thoracotomy and lobectomy which revealed both nodules to be a clear renal cell carcinoma metastasis, but surprisingly, a third nodule of 1,5 mm was found on the procedure and consisted of a papillary thyroid carcinoma. On sequence, in 2021 his thyroid was surgically removed, revealing a malignant nodule, corroborating the previous diagnosis. DISCUSSION: Thyroid cancer is the most common subsequent tumor related to a previous renal cell carcinoma and some scientific research proposes a BRAF mutation being linked to this finding. That being said, renal cell carcinoma has as risk factor a history of thyroid carcinoma. Being both tumors with slow onset and poor clinical presentation and low metabolic profile, they are usually difficult to diagnose at early stages. CONCLUSIONS: This case demonstrates that a high suspicion level and thorough management can achieve reasonable levels of diagnosis and high survival rates even when two rare entities are bound in the same case. REFERENCE #1: Immunotargets Ther . 2013; 2: 73 90. Jul 22 REFERENCE #2: Thyroid cancer : Coexisting papillary thyroid carcinoma and renal cell carcinoma: 4 cases ( Mehmet Celik1, Semra Ayturk1, Buket Yilmaz Bulbul1, Nuray Can2, Ebru Tastekin2, Atakan Sezer3, Funda Ustun4 & Sibel Guldiken1) : Endocrine Abstracts (2016) 41 EP1146 DISCLOSURES: no disclosure on file for Barbara Bracarense; No relevant relationships by Michelle Cailleaux-Cezar, source=Web Response No relevant relationships by Fábio Kunita, source=Web Response

Aggressive Subsets of Metastatic Triple Negative Breast Cancer

BackgroundRelative to other metastatic breast cancer subtypes, metastatic triple-negative breast cancer (mTNBC) has a shorter duration of response to therapy and worse overall survival. Among patients with mTNBC, it is hypothesized that inflammatory breast cancer (IBC) and young women have particularly aggressive phenotypes. We investigated clinical and cell-free DNA (cfDNA) characteristics of inflammatory-mTNBC and young-mTNBC. Patients and MethodsWe evaluated 158 patients with mTNBC who were stratified into 3 groups: (1) IBC; (2) patients aged 45 years or younger at primary diagnosis without IBC (non-IBC young); and (3) patients over age 45 at diagnosis without IBC. We evaluated clinicopathologic characteristics, sites of metastasis, survival outcomes, and the fraction of DNA in circulation derived from tumor (TFx). ResultsAnalysis of metastatic sites revealed that young patients without IBC had the most frequent lung metastases (P = .002). cfDNA analyses of first sample showed that TFx was highest in the non-IBC young group but not elevated in the IBC group (analysis of variance P = .056 for first TFx). Individually, median overall survival from metastatic diagnosis for the IBC group was 15.2 months; for the non-IBC young group, 21.2 months, and for the non-IBC over 45 group, 31.2 months. Patients with IBC and young patients without IBC had worse prognosis relative to patients over 45 without IBC (log-rank P = .023). ConclusionsAmong patients with mTNBC in this single-institution cohort, patients with IBC and young patients without IBC had significantly worse overall survival compared with patients over 45 without IBC. Young patients without IBC had significantly higher cfDNA TFx, whereas patients with IBC did not have elevated TFx despite a poor prognosis. These findings demonstrate that further analyses of mTNBC subsets are warranted.

Abstract P4-01-17: Aggressive subgroups of metastatic triple-negative breast cancer: Inflammatory breast cancer and young patients in the Dana-Farber cell-free DNA cohort

Abstract Background: Relative to other metastatic breast cancer subtypes, metastatic triple-negative breast cancer (mTNBC) has a shorter duration of response to therapy and worse overall survival. Within mTNBCs, there is a prevailing belief that inflammatory breast cancer and young women tend to have among the most aggressive phenotypes. We investigated clinical and cell-free DNA (cfDNA) characteristics of inflammatory-mTNBC and young-mTNBC. We hypothesized that inflammatory-mTNBC may have distinct clinical and cfDNA characteristics, offering potential novel biomarker and therapeutic strategies. Methods: 164 patients from the Dana-Farber metastatic triple-negative cell-free DNA cohort (Stover DG, et al J Clin Oncol 2018) were included in this secondary analysis. Patients were stratified into three groups: 1) inflammatory breast cancer ('IBC'); 2) non-IBC patients aged 45 years (yr) or younger at primary diagnosis ('non-IBC young'); and 3) non-IBC patients over age 45 yr at diagnosis. For each subset population, we evaluated clinicopathologic characteristics, sites of metastasis, survival outcomes, and cfDNA 'tumor fraction' – the fraction of DNA in circulation derived from tumor. Those patients with adequate cfDNA tumor content for high confidence copy number calls (n=101) were included in an analysis of copy number alterations. Results: Among 164 patients with metastatic TNBC, 13.4% (22/164) had IBC, 37.8% (62/164) were non-IBC young, and 48.8% (80/164) were non-IBC and over 45 yr. Race and primary receptor status were similar. IBC patients were diagnosed at a higher stage (Chi-square p=0.0009) while non-IBC young patients were significantly more likely to harbor a BRCA mutation (Chi-square p=0.03). Analysis of metastatic sites revealed that IBC patients had significantly greater frequency of ipsilateral and contralateral breast chest wall recurrences (p=0.04 and p=0.046, respectively) while non-IBC young patients had the most frequent lung metastases (p=0.002). There were no significant differences in frequency of bone, brain, or liver metastases. cfDNA analyses showed that cfDNA 'tumor fraction' was highest in non-IBC young patients (ANOVA p=0.03 for maximum tumor fraction). Median overall survival from metastatic diagnosis was 22.9 months. IBC and non-IBC young patients had a worse prognosis relative to non-IBC patients over 45 yr (hazard ratio IBC=1.97, 95% CI 1.09-3.57; HR non-IBC young=1.60 95% CI 1.07-2.41; log-rank p=0.023). By subgroup, median overall survival from metastatic diagnosis for IBC was 15.2 months, non-IBC young 21.2 months, and non-IBC over 45 yr 31.2 months. Analyses of genome-wide copy number alterations from cell-free DNA will be presented. Conclusions: Among metastatic TNBCs, IBC patients and non-IBC young patients have a significantly worse overall survival compared with non-IBC patients over 45 yr of age. Young patients have more frequent lung metastases and higher 'tumor fraction' of cfDNA. Confirmation of the reported findings is limited due to cohort size and may reflect referral bias. Citation Format: Singh J, Asad S, Nock W, Zhang Y, Adams E, Damicis A, Parsons HA, Adalsteinsson VA, Winer EP, Lin NU, Partridge AH, Overmoyer B, Stover DG. Aggressive subgroups of metastatic triple-negative breast cancer: Inflammatory breast cancer and young patients in the Dana-Farber cell-free DNA cohort [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P4-01-17.

Tumor cell-targeted delivery of CRISPR/Cas9 by aptamer-functionalized lipopolymer for therapeutic genome editing of VEGFA in osteosarcoma.

Osteosarcoma (OS) is a highly aggressive pediatric cancer, characterized by frequent lung metastasis and pathologic bone destruction. Vascular endothelial growth factor A (VEGFA), highly expressed in OS, not only contributes to angiogenesis within the tumor microenvironment via paracrine stimulation of vascular endothelial cells, but also acts as an autocrine survival factor for tumor cell themselves, thus making it a promising therapeutic target for OS. CRISPR/Cas9 is a versatile genome editing technology and holds tremendous promise for cancer treatment. However, a major bottleneck to achieve the therapeutic potential of the CRISPR/Cas9 is the lack of invivo tumor-targeted delivery systems. Here, we screened an OS cell-specific aptamer (LC09) and developed a LC09-functionalized PEG-PEI-Cholesterol (PPC) lipopolymer encapsulating CRISPR/Cas9 plasmids encoding VEGFA gRNA and Cas9. Our results demonstrated that LC09 facilitated selective distribution of CRISPR/Cas9 in both orthotopic OS and lung metastasis, leading to effective VEGFA genome editing in tumor, decreased VEGFA expression and secretion, inhibited orthotopic OS malignancy and lung metastasis, as well as reduced angiogenesis and bone lesion with no detectable toxicity. The delivery system simultaneously restrained autocrine and paracrine VEGFA signaling in tumor cells and could facilitate translating CRISPR-Cas9 into clinical cancer treatment.

Correlation between KRAS mutation and 18F-FDG uptake in stage IV colorectal cancer.

The purpose of this study is to evaluate the correlation between KRAS mutation, 18F-FDG uptake, and metastatic pattern in advanced stage colorectal cancer (CRC) patients. Medical records of stage IV CRC patients who underwent 18F-FDG PET/CT for staging and KRAS mutation analysis were selected. On PET scans, a volume of interest (VOI) was drawn on the primary lesion. 18F-FDG indices (SUVmax, SUVmean, MTV, TLG) of the primary lesions were obtained and correlated with KRAS mutation of the primary lesion. Also, metastatic sites were recorded. Association between metastatic pattern and KRAS expression and FDG indices were analyzed. KRAS mutation was positive in 40 (43%) patients. Evaluation of FDG indices showed that higher SUVmax (14.0 vs. 11.2, p=0.004), higher SUVmean (5.3 vs. 4.7, p=0.005), and higher TLG (301.4 vs. 205.5, p=0.023) were predictive of KRAS mutation compared to wild-type (WT) KRAS. Lung metastasis was more frequently involved in patients with KRAS mutation (50.0% vs. 22.6%, p=0.006), and liver metastasis was more frequently involved in patients with WT KRAS (81.1% vs. 55.0%, p=0.007). Multivariate analysis showed that primary tumor location (OR 3.92, p=0.07) and KRAS mutation (OR 2.45, p=0.09)were significant factors in lung metastasis model. KRAS mutation patients had more frequent lung metastasis and had higher 18F-FDG uptake compared to WT KRAS in stage IV CRC.

ΔNp63α Silences a miRNA Program to Aberrantly Initiate a Wound-Healing Program That Promotes TGFβ-Induced Metastasis.

Primary cancer cell dissemination is a key event during the metastatic cascade, but context-specific determinants of this process remain largely undefined. Multiple reports have suggested that the p53 (TP53) family member p63 (TP63) plays an antimetastatic role through its minor epithelial isoform containing the N-terminal transactivation domain (TAp63). However, the role and contribution of the major p63 isoform lacking this domain, ΔNp63α, remain largely undefined. Here, we report a distinct and TAp63-independent mechanism by which ΔNp63α-expressing cells within a TGFβ-rich microenvironment become positively selected for metastatic dissemination. Orthotopic transplantation of ΔNp63α-expressing human osteosarcoma cells into athymic mice resulted in larger and more frequent lung metastases than transplantation of control cells. Mechanistic investigations revealed that ΔNp63α repressed miR-527 and miR-665, leading to the upregulation of two TGFβ effectors, SMAD4 and TβRII (TGFBR2). Furthermore, we provide evidence that this mechanism reflects a fundamental role for ΔNp63α in the normal wound-healing response. We show that ΔNp63α-mediated repression of miR-527/665 controls a TGFβ-dependent signaling node that switches off antimigratory miR-198 by suppressing the expression of the regulatory factor, KSRP (KHSRP). Collectively, these findings reveal that a novel miRNA network involved in the regulation of physiologic wound-healing responses is hijacked and suppressed by tumor cells to promote metastatic dissemination. Cancer Res; 76(11); 3236-51. ©2016 AACR.

Abstract 3421: Serial whole exome sequencing showed the genetic aggravation of refractory osteosarcoma

Abstract Osteosarcoma is the most common malignant bone tumor in children and adolescents with frequent lung metastasis. As the survival has not been improved with chemotherapy for the last two decades, novel therapeutic approaches are required to efficiently treat osteosarcoma. Genomic analyses have revealed biologically useful information and genetically altered therapeutic targets. The subject of this study was a 25 year old patient with conventional high-grade osteosarcoma with multiple metastasis of lung, lymph nodes and chest wall, who had failed from several palliative systemic chemotherapies and radiotherapy. We performed whole exome sequencing of the gDNA from peripheral blood mononucleated cells (PBMC) and formalin fixed paraffin embedded (FFPE) tissues of primary tumor, secondary (recurrence 1) and tertiary (recurrence 2) metastatic tumors. Samples were sequenced using one lane of paired-end, 100 bp reads on Illumina Hiseq for each sample. Here, we present an analysis result using VarScan and custom-made programs for the detection of somatic mutations and loss of heterozygosity (LOHs) in exome data from changes of genetic variations throughout the disease progression compared to blood germ-line sequencing data. As a result, we observed the 36 common somatic mutated genes, including p73 and TSHR, in three tumor types. And we indentified the somatic mutations affecting the functions of known cancer genes, RBM15, SYNE1, GNAQ and XPA, which were only present in the secondary and/or tertiary metastatic tumors but not in the primary tumor. Especially, GNAQ mutations which is found in the metastatic tumors, is known to be mutated in 50% of melanoma and driven constitutive activity of the MAPK pathway. Therefore, GNAQ mutation in recurrent osteosarcoma patient might be a potential marker for new therapeutic strategies of inhibiting MAPK pathway. In conclusion, the ability to track clonal evolution in cancers may provide new strategies and opportunities for drug development, especially in refractory and rare diseases. Citation Format: Su Jin Heo, Ji Woong Kim, Woo Sun Kwon, Hyo Ki Kim, Min Hee Hong, Woo Ick Yang, Se-Kyu Kim, Hyo Song Kim, Hyun Cheol Chung, Tae Hyun Hwang, Sun Young Rha. Serial whole exome sequencing showed the genetic aggravation of refractory osteosarcoma. [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 3421. doi:10.1158/1538-7445.AM2014-3421

Abstract B149: Yes-associated protein (YAP) is associated with aggressive characteristics of BRAFV600E thyroid cancer.

Abstract Up regulation of Yes-associated protein (YAP), a transcriptional co-activator regulated by Hippo (MST-LAST-YAP) pathway, has been reported to mediate oncogenic transformation in animal models of hepatocellular carcinomas. However, the expression status and molecular biological role of YAP in thyroid cancer remained to be investigated. We evaluated YAP expression and identified increased nuclear YAP expression in PTC and ATC. Interestingly, we could observe nuclear YAP staining more frequently in BRAFV600E(+) PTC. We also performed immunofluorescence staining to verify intracellular localization of YAP in TPC1 (harboring RET/PTC1), 8505C (harboring BRAFV600E) and BRAFV600E stably transfected HEK293T cells. Consistently, we could detect nuclear YAP in 8505C and BRAFV600E expressed cells, but not in TPC1. Although cell viability assays did not show any difference between YAP knockout cells and control cells generated by using shYAP in 8505C and shCTL (control) 8505C, YAP knockout cells showed remarkably decreased migration ability in the scratch assays. Using orthotopic mice models, shYAP 8505C injected mice showed decreased local invasion ability and less frequent lung metastasis. In conclusions, Nuclear YAP could be more frequently detected in BRAFV600E(+) thyroid cancer compared to BRAFV600E(−) thyroid cancer. Furthermore, nuclear YAP in BRAFV600E(+) thyroid cancer was associated with local invasiveness and distant metastasis, presenting that YAP could affect the aggressive phenotype of BRAFV600E(+) thyroid cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B149.

Abstract 2264: K-ras mutations in colorectal cancer may predict a pattern of organ-specific metastasis

Abstract Background: Activating mutations of the K-ras oncogene in colorectal cancer (CRC) are associated with resistance to EGFR antibodies and poor prognosis. We systematically analyzed clinical and pathological characteristics associated with K-ras mutations. Methods: Patients with CRC referred to the phase I program for targeted therapy were analyzed for K-ras mutations using PCR-based DNA sequencing. Patients with mutant K-ras were matched with wild-type (wt) K-ras controls. Multiple clinical and pathological characteristics were analyzed to determine, whether there is a phenotype associated with K-ras mutations. Results: A total of 151 patients with CRC were identified; 61 with K-ras mutations; 80 with wt K-ras and 10 with unknown K-ras status. Patients with K-ras mutations compared with wt K-ras had more frequently lung metastases (80% vs. 63%, p = 0.03). Patients whose tumors had K-ras mutations had higher number of metastatic sites of disease (3.18 vs. 2.65; p = 0.013). There were no associations between K-ras mutations and gender, age at diagnosis, liver metastases, peritoneal metastases, lymphatic involvement or Duke stage at diagnose. Patients with K-ras wt had a trend for longer median survival (283 vs. 222 weeks, p=0.09). There was no difference in the median survival from diagnosis in patients with different types of K-ras mutations (codon 12, codon 13, and codon 61). Dukes staging was the only statistically significant prognostic factor on multivariate analysis (p < 0.05). Conclusions: K-ras mutations in CRC in comparison with wt K-ras were associated with more frequent lung metastases and shorter survival. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2264. doi:10.1158/1538-7445.AM2011-2264

Retrospective study to elucidate a K-ras phenotype in patients with colorectal cancer.

462 Background: Activating mutations of the K-ras oncogene in colorectal cancer (CRC) are associated with resistance to EGFR antibodies and poor prognosis. Clinical and pathological characteristics associated with K-ras mutations have not been systematically analyzed. Methods: Patients with CRC referred to the phase I program for targeted therapy were analyzed for K-ras mutations using PCR-based DNA sequencing. Patients with mutant K-ras were matched with wild-type (wt) K-ras controls. Multiple clinical and pathological characteristics were analyzed to determine, whether there is a phenotype associated with K-ras mutations. Results: A total of 107 patients with CRC were identified; 58 with K-ras mutations; and 49 with wt K-ras. Patients with K-ras mutations compared with wt K-Ras had more frequently lung metastases (80% vs. 57%, p = 0.013). There was a trend towards higher number of metastatic sites in K-ras mutant patients compared with wt K-ras (3.16 vs. 2.69; p = 0.07). There were no associations between K-ras mutations and gender, age at diagnosis, liver metastases, peritoneal metastases, and lymphatic involvement. The median time from diagnosis to development of metastatic disease was similar in K-ras mutant and K-ras wt patients (194 vs. 196 weeks, p = 0.43). The median survival from diagnosis was also similar in K-ras mutant and K-ras wt patients (254 vs. 223 weeks, p = 0.32). There was no difference in the median survival from diagnosis in patients with different types of K-ras mutations (codon 12, codon 13, and codon 61). Dukes staging was the only statistically significant prognostic factor on multivariate analysis (p < 0.05). Conclusions: K-ras mutations in CRC in comparison with wt K-ras were associated with more frequent lung metastases. K-ras mutations did not impact survival. No significant financial relationships to disclose.

Anti-tumor effects of adenovirus containing human growth hormone sequences in a mouse model of human ovarian cancer

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31±1.2 to 40±1.4days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity.

Modification of an in vivo Lung Metastasis Model of Hepatocellular Carcinoma by Low Dose N-nitrosomorpholine and Diethylnitrosamine

Previously, we established the in vivo lung metastasis model of rat HCC induced by two hepatocarcinogens, diethylnitrosamine (DEN) and N-nitrosomorpholine (NMOR) at a dose of 120 ppm. This model allows us to investigate modifying factors leading to the inhibition of metastasis formation. However, low survival rates made the evaluation of metastasis formation difficult. The current experiments were conducted to modify the experimental protocol to improve survival and to establish a better animal metastasis model. Lower doses of NMOR (80 or 40 ppm in drinking water) were given to F344 rats for 14 weeks after DEN treatment. Survival rates in the 80 ppm group and in the 40 ppm group were 57% and 81%, respectively and these values were significantly higher than that in 120 ppm. Incidences of lung metastasis in the 40 ppm group steadily increased up to 67% by week 36 while that in the 80 ppm increased sharply up to 86% by week 24. Severity of lung metastases in the 40 ppm group at week 36 was mild compared with the 80 ppm group at week 24. In the second experiment, in order to characterize HCC development and lung metastasis in the 40 ppm group, rats given DEN and then followed with 40 ppm NMOR were killed sequentially. Development of HCC was observed at week 14 and reached 100% incidence at week 20. First lung metastatic lesions were evident at week 22, and incidence of lung metastasis reached 100%. Tumor cells were identified in the blood at week 20 by RT-PCR. The current study revealed that 40 ppm NMOR for 14 weeks after DEN treatment developed HCC without lung metastases at week 22, then HCC with a frequent lung metastasis at week 40. Thus, it can be said that this system is a more appropriate model for elucidation of mechanisms of metastasis and also for analysis of factors to inhibit natural metastasis.

Salivary Gland Tumors in Transgenic Mice with Targeted PLAG1 Proto-Oncogene Overexpression

Pleomorphic adenoma gene 1 (PLAG1) proto-oncogene overexpression is implicated in various human neoplasias, including salivary gland pleomorphic adenomas. To further assess the oncogenic capacity of PLAG1, two independent PLAG1 transgenic mouse strains were established, PTMS1 and PTMS2, in which activation of PLAG1 overexpression is Cre mediated. Crossbreeding of PTMS1 or PTMS2 mice with MMTV-Cre transgenic mice was done to target PLAG1 overexpression to salivary and mammary glands, in the P1-Mcre/P2-Mcre offspring. With a prevalence of 100% and 6%, respectively, P1-Mcre and P2-Mcre mice developed salivary gland tumors displaying various pleomorphic adenoma features. Moreover, histopathologic analysis of salivary glands of 1-week-old P1-Mcre mice pointed at early tumoral stages in epithelial structures. Malignant characteristics in the salivary gland tumors and frequent lung metastases were found in older tumor-bearing mice. PLAG1 overexpression was shown in all tumors, including early tumoral stages. The tumors revealed an up-regulation of the expression of two distinct, imprinted gene clusters (i.e., Igf2/H19 and Dlk1/Gtl2). With a latency period of about 1 year, 8% of the P2-Mcre mice developed mammary gland tumors displaying similar histopathologic features as the salivary gland tumors. In conclusion, our results establish the strong and apparently direct in vivo tumorigenic capacity of PLAG1 and indicate that the transgenic mice constitute a valuable model for pleomorphic salivary gland tumorigenesis and potentially for other glands as well.

Establishment and characterization of mouse mammary carcinoma cell lines expressing RET with a multiple endocrine neoplasia 2A mutation.

We recently generated transgenic mice expressing the RET proto-oncogene with a multiple endocrine neoplasia type 2A mutation (RET-MEN2A). Mammary tumors with frequent lung metastasis were developed in 22% of female transgenic mice in a stochastic fashion. In the current study, we established two cell lines (named MKK-f and MKK-s) from mammary tumors developed in RET-MEN2A transgenic mice. MKK-f and MKK-s were derived from well-differentiated ductal carcinoma and sarcomatous spindle cell carcinoma, respectively. MKK-f cells show epithelial-like morphology with a doubling time of 19 h, and MKK-s cells show spindle-shaped morphology with a doubling time of 15 h. When inoculated in immunodeficient mice, both cell lines were tumorigenic, metastasized to the lung and displayed histological features similar to those of the primary tumors. They maintained a high level of RET expression and activation of signaling molecules downstream of RET. Consistent with the histological phenotype, expression of E-cadherin was almost undetectable in MKK-s cells, whereas its expression was very high in MKK-f cells. When the difference of gene expression between the two cell lines was analyzed using cDNA microarrays including approximately 900 genes/ESTs, a total of 21 up- or down-regulated (> 2.0-fold) genes were identified. Differentially regulated genes included thymosin beta-10, fibroblast growth factor receptor 4, aldo-keto reductase and caspase 6 genes, which are known to be associated with tumor development and progression. These results may reflect the profiles of the transcriptional changes associated with dedifferentiation or progression of mammary carcinomas developed in genetically engineered mice.

Establishment of Two Rat Osteosarcoma Cell Lines (YROS-1 and YROS-2) Induced by Radioactive Phosphorus

Two rat osteosarcoma cell lines, YROS-1 and YROS-2, were established from two experimental osteosarcomas and induced by internal irradiation with radioactive phosphorus. Both cell lines formed a monolayer cell sheet in vitro with focal piling. The YROS-1 cells were refractile and spindle or polygonal in shape, whereas the YROS-2 cells were flat, spread and polygonal in shape. Ultrastructurally, the YROS-1 cells had well-developed rough-surfaced endoplasmic reticulum with focal pericellular deposition of calcified matrix, whereas YROS-2 had abundant polysomes and intracytoplasmic filaments. Both cell lines grew stably with population doubling times of 23 and 39 h, respectively. Flow cytometry revealed that YROS-1 was rich in proliferating cells compared to YROS-2, with a higher colony-forming efficiency. YROS-1 showed high alkaline phosphatase activity, while YROS-2 possessed low activity. When subcutaneously transplanted into lumbodorsal area of athymic nude mice, only YROS-1 formed tumors with frequent lung metastasis.