Receptor for hyaluronan-mediated motility isoform B promotes liver metastasis in a mouse model of multistep tumorigenesis and a tail vein assay for metastasis Yi-Chieh Nancy Dua,b,1, Chen-Kung Chouc, David S. Klimstrad, and Harold Varmusa,e,1 aProgram in Cancer Biology and Genetics, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; bDepartment of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, NY 10065; cDepartment of Biomedical Sciences, Chang-Gung University, Tao-Yuan 333, Taiwan; dDepartment of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; and eCancer Biology and Genetics Section, Cancer Genetics Branch, National Human Genome Research Institute, Bethesda, MD 20892 Contributed by Harold Varmus, August 30, 2011 (sent for review July 14, 2011) The gene encoding the receptor for hyaluronan-mediated motility Using the RIP-Tag; RIP-tva model, we find that isoform B of B (RHAMM) is overexpressed in many human cancers. However, it RHAMM (RHAMM ) enhances the growth of mouse islet is unclear whether RHAMM plays a causal role in tumor initiation tumors and promotes metastasis exclusively to the liver and local or progression. Using somatic gene transfer in a mouse model of lymph nodes. Furthermore, we show that mouse pancreatic islet B islet cell tumorigenesis, we demonstrate that RHAMM isoform B tumor cells programmed to express RHAMM form hepatic me- (RHAMMB) promotes tumor growth and metastases to lymph tastasis when injected into the tail vein of mice in a traditional nodes and the liver. The propensity of RHAMMB-expressing cells assay for metastasis. The cells also show evidence that RHAMM fi has enhanced signaling via the epidermal growth factor receptor to metastasize to the liver was con rmed using an experimental B metastasis assay in which cells were injected into the tail vein of (EGFR). These observations and others suggest that RHAMM B may be an important factor in tumor growth and progression and immunodeficient mice. However, RHAMM did not increase cell that a better understanding of the RHAMM gene might offer migration or proliferation in culture. In initial efforts to identify insights into the organotropism of metastatic cancer cells. signaling pathways activated by RHAMMB, we found that B RHAMM induced phosphorylation of epidermal growth factor re- Results ceptor (EGFR), Erk1/2, and STAT3 and conferred susceptibility to RHAMMB Promotes Tumor Growth and Metastasis to Pancreatic apoptosis after treatment with an EGFR inhibitor, gefitinib. Taken Lymph Nodes and the Liver in a Mouse Model of Pancreatic Islet together, the results indicate that RHAMMB promotes hepatic Tumors. To evaluate the malignant potential of genes reported metastasis by islet tumor cells, perhaps through growth factor to be up-regulated both in HCC and during mouse liver re- receptor-mediated signaling. generation (2), we examined five candidate genes, including RHAMMB, paternally expressed 10 (PEG10), FLJ10540, FLJ11252, e have previously reported a bitransgenic mouse model, and FLJ11164. FLJ stands for the “full-length long Japan” col- WRIP-Tag; RIP-tva, in which the rat insulin promoter (RIP) lection of human cDNAs (8). drives production of both the SV40 T antigen (RIP-Tag) and The cDNAs of the candidates were cloned into avian retroviral the receptor for subgroup A avian leukosis virus (RIP-tva)in vector, RCASBP, with a FLAG epitope tag added to the N pancreatic β cells (1). Coding domains of genes suspected of terminus of PEG10, FLJ10540, and FLJ11252. We injected high 8 contributing to tumor progression can be introduced into pre- titer virus stocks (0.1 mL; >10 infectious units per milliliter) into malignant lesions by infection with the avian retroviral vector, 7-wk-old RIP-Tag; RIP-tva mice by the intracardiac route. At this RCASBP, after intracardiac injection. RIP-Tag transgenic mice point, many islets show evidence of hyperplasia, allowing in- develop islet tumors through well-defined stages that resemble fection with oncoretrovirus vectors, which are dependent on – the progression of several kinds of human cancers; for this rea- cell division for successful infection (1). RCASBP ALPP (Al- MEDICAL SCIENCES – son, we and others have used these mice, with or without addi- kaline Phosphatase) and RCASBP Bcl-xL were chosen as con- tional transgenes, to identify and validate mechanisms of trols. ALPP encodes a protein unlikely to contribute to tumori- genesis, serving as a negative control for effects of viral infection. tumorigenesis that may operate in multiple tissues. For instance, – we have used RIP-Tag; RIP-tva mice to show that RCASBP- We have previously shown that infection with RCASBP Bcl-xL promotes tumor growth and lymph node metastasis in RIP-Tag; mediated delivery of Bcl-xL or E-cadherin, factors implicated in RIP-tva mice (1), so infection with this virus provided a pos- various neoplasms, promotes tumorigenesis and invasion in islet itive control. cells (1). fi Nine weeks after infection, the pancreas and other organs High-throughput genomic technologies have identi ed many were harvested for histological staging and grading of the lesions. genes that may be critical in tumor initiation and progression. Human RHAMMB significantly increased pancreatic tumor However, it remains difficult to distinguish causative and pas- fi burden in 8 of 12 mice, but not all, compared with mice infected senger mutations and to assign speci c biological functions to with RCASBP–ALPP (Fig. 1, P = 0.0097, Wilcoxon rank sum altered genes in human cancers, and the RIP-Tag; RIP-tva mouse test). RCASBP–Bcl-xL induced a small increase in pancreatic model of multistage tumorigenesis offers an opportunity to ad- tumor burden (P = 0.0087), whereas none of the other vec- dress such issues. To that end, we have assessed the oncogenic functions of a small number of incompletely characterized genes that are up-regulated in human hepatocellular carcinomas Author contributions: Y.-C.N.D. and H.V. designed research; Y.-C.N.D. performed re- (HCC) and during mouse liver regeneration (2). One of the search; C.-K.C. provided new reagents; D.S.K. contributed pathology analysis; Y.-C.N.D. candidate genes, a gene encoding a receptor for hyaluronan- analyzed data; and Y.-C.N.D. and H.V. wrote the paper. mediated motility (RHAMM) is overexpressed in many types of The authors declare no conflict of interest. human cancers, including pancreatic ductal carcinoma, hepato- Freely available online through the PNAS open access option. cellular carcinoma, multiple myeloma, breast cancer, gliomas, 1To whom correspondence may be addressed. E-mail: [email protected] or colon cancer, and prostate cancer (2–7); but the functions of at [email protected]. least four proteins encoded by its alternatively spliced messenger This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. RNAs and their roles, if any, in tumorigenesis are unclear. 1073/pnas.1114022108/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1114022108 PNAS | October 4, 2011 | vol. 108 | no. 40 | 16753–16758 Downloaded by guest on September 28, 2021 tors (RCASBP–FLAG-PEG10, RCASBP–FLAG-FLJ10540, Table 1. Impact of candidate genes on tumorigenesis in vivo RCASBP–FLAG-FLJ11252, or RCASBP–FLJ11164) caused fi Tumor any signi cant increase in tumor burden (Table 1 and Table S1). Age burden Lymph node Liver To aid the search for metastasis of islet tumors in RIP-Tag; RCASBP– (wk) (mm3) metastasis (%) metastasis (%) RIP-tva mice, tissue sections were subjected to immunohisto- chemical staining for synaptophysin, a neuroendocrine marker, ALPP 12 8.1 ± 4.5 0/5 mice (0) 0/5 mice (0) and for insulin, a β-cell marker. Local lymph node metastases ALPP 16 99.7 ± 19.4 0/10 mice (0) 0/10 mice (0) – B were detected in mice infected with RCASBP RHAMM (8 of RHAMMB 12 16.2 ± 8.9 0/6 mice (0) 0/6 mice (0) 11 mice, P = 0.001, Fisher’s exact test), RCASBP–Bcl-xL (7 of 15 B ± – RHAMM 16 298.3 121.3 8/11 mice (73) 8/12 mice (67) mice, P = 0.013), RCASBP FLAG-PEG10 (2 of 8 mice, P = Bcl-xL 16 150.5 ± 23 7/15 mice (46) 0/6 mice (0) 0.183), RCASBP–FLAG-FLJ10540 (4 of 7 mice, P = 0.015), and ± – FLAG-PEG10 16 57.8 26.8 2/8 mice (25) 0/8 mice (0) RCASBP FLJ11164 (1 of 9 mice, P = 0.474) (Fig. 2, Table 1, FLAG-FLJ10540 16 126.1 ± 54.3 4/7 mice (57) 2/7 mice (28) and Table S1). The sizes of lymph node metastases in mice – – FLAG-FLJ11252 16 41.2 ± 30.9 0/7 mice (0) 0/7 mice (0) infected with RCASBP FLAG-PEG10,RCASBPFLAG- ± FLJ10540, and RCASBP–FLJ11164 were small and were not FLJ11164 16 43.8 25.1 1/9 mice (11) 0/6 mice (0)* easily detected by hematoxylin and eosin staining, unlike those in RCASBP retroviruses carrying indicated cDNAs were introduced to RIP- B mice infected with RCASBP–RHAMM and RCASBP–Bcl-xL. Tag; RIP-tva mice through intracardiac injection at 7 wk of age. Mice were No lymph node metastases were found in mice receiving the euthanized at 12 or 16 wk of age for measurement of tumor burden and for negative control virus or RCASBP–FLAG-FLJ11252 (Table 1 metastasis survey. A standard formula for tumor volume was applied (vol- and Table S1). ume [mm3] = 0.52 × width2 × length). Tumor burden is the sum of the tumor Importantly, liver metastases were found in 8 of 12 RCASBP– volume per mouse. RHAMMB infected mice (Fig. 2, Table 1, and Table S1, P = *Mice having micrometastases with fewer than five cells were excluded. 0.002, Fisher’s exact test), and the appearance of metastasis was not closely associated with the aggregated size of primary tumors.