Receptor for Hyaluronan-Mediated Motility Isoform B Promotes Liver Metastasis in a Mouse Model of Multistep Tumorigenesis and a Tail Vein Assay for Metastasis

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 tumorigenesis, 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 immunohistochemical 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. Several RIP-Tag; RIP-tva mice infected with RCASBP– Some islet tumors and the metastases in pancreatic lymph nodes B and the livers in RIP-Tag; RIP-tva mice infected with RCASBP– RHAMM with less or similar pancreatic tumor burden than B control mice still developed metastasis (Table S1), suggesting RHAMM stained positive for RHAMM (Fig. 2E). Although that RHAMMB does not merely promote metastasis as a direct RHAMM is negatively regulated by p53 in cell lines (9) and T- consequence of promoting tumor growth. In addition, 2 of 7 antigen represses p53 transactivation in RIP-Tag; RIP-tva mice, mice infected with RCASBP–FLAG-FLJ10540 developed small endogenous mouse RHAMM was not detectable by immuno- liver metastases, and 3 of 9 mice infected with RCASBP– histochemical staining in most of the pancreatic islets and islet FLJ11164 developed liver micrometastases with fewer than five tumors from uninfected RIP-Tag; RIP-tva mice (Fig. 2 C and D). Taken together, our results suggest that ectopic expression of tumor cells on the histological sections (Table 1 and Table S1). B These findings suggest that PEG10, FLJ10540, and FLJ11164 human RHAMM increases tumor burden and independently have a modest effect on the promotion of metastasis in this promotes metastasis to pancreatic lymph nodes and the liver in model, but only RHAMMB had a dramatic effect on both tumor our model of mouse pancreatic islet tumors. size and metastasis to lymph nodes and the liver. No metastases B were found at other sites, including lungs, heart, thymus, kidney, RHAMM Promotes Liver-Specific Metastasis in a Tail Vein Assay of and spleen. Cancer Metastasis. The portal vein brings blood from the pan- In view of the profound effects of RHAMMB on tumor size creas, spleen, stomach, duodenum, and colon to the liver. Thus, B and metastasis 9 wk after infection, we asked whether such it seemed possible that RHAMM promotes metastasis of pan- effects could be detected at earlier times. RIP-Tag; RIP-tva mice creatic islet tumor cells to the liver because it promotes metas- were euthanized 5 wk after being infected with RCASBP– tasis in general and the liver is the first target organ in the path of RHAMMB at 7 wk of age. Although these RIP-Tag; RIP-tva mice blood drainage from the primary tumors. To distinguish more had twofold more tumor burden than did the mice infected with rigorously between enhancement of liver-specific versus gener- RCASBP–ALPP at the same age, no metastases were found after alized metastasis, we asked whether mouse islet tumor cell lines microscopic examination of tissue slices stained with reagents expressing RHAMMB metastasized preferentially to the liver that detect insulin and synaptophysin (Table 1), indicating that after introduction into the general circulation. For this experi- more than 5 wk were required to observe any effects on ment, we injected established cell lines into the tail vein of re- metastasis. cipient mice. Cells entering the venous circulation will first To verify that RHAMM was indeed produced in the tumors encounter the capillaries of the lungs. If they are able to pass and metastases of RCASBP–RHAMMB infected mice, we gen- through the lungs, they will then enter the arterial system and erated rabbit polyclonal antibodies against a region entirely eventually pass through the portal circulation. Assuming that conserved among human, mouse, and rat RHAMM proteins, promotion of metastasis by RHAMMB affects steps following and present in all four human isoforms (Table S2 and Fig. S1). intravasation, this approach should distinguish between liver- specific tropism and dependency on the circulatory route as an explanation for the observed liver metastases. We infected a β-cell tumor cell line βTC-N134, (N134 for brevity) (1) derived from an islet tumor in a RIP-Tag; RIP-tva A control infection B RCAS-RHAMMB mouse with RCASBP–Luciferase, RCASBP–RHAMMB,or RCASBP–Bcl-xL in vitro. To verify the expression of human RHAMMB in N134 cells infected with RCASBP–RHAMMB,we performed reverse transcription–PCR using primers specific for exon 16 of human RHAMM and readily detected human RHAMM mRNA in cells infected with RCASBP–RHAMMB, but not in uninfected cells (Fig. 3A). A total of 1 × 106 tumor cells were introduced into the tail vein Fig. 1. Significantly increased pancreatic islet tumor burden in 67% of RIP- of immunodeficient mice, NOD/scid-lL2Rgc knockout (NSG). Tag; RIP-tva mice infected with RCASBP–RHAMMB (n = 12). Representative The mice receiving tumor cells infected with RCASBP–Lucifer- pancreas and spleen from RIP-Tag; RIP-tva mice infected with RCASBP–ALPP ase were monitored by in vivo bioluminescence imaging (Fig. (A) or RCASBP–RHAMMB (B). Mouse islet tumors are red due to angiogenesis. 3B). We observed signals from the thoracic cavity during the first

16754 | www.pnas.org/cgi/doi/10.1073/pnas.1114022108 Du et al. Downloaded by guest on September 28, 2021 -RT +RT ABsynaptophysin insulin A B 1d B B HeLa uninfected RHAMM HeLa uninfected RHAMM lymph node lymph node Human RHAMM 2d Human actin 5x 20x Mouse actin

3d liver liver

5x 20x C RHAMM E RHAMM 5d

islet tumor 7d

20x 20x D RHAMM

Fig. 3. Detection of human RHAMM mRNA or luciferase activity in the mouse islet tumor cell line infected with RCASBP–RHAMMB or RCASBP–Lu- B lymph node ciferase.(A) The expression of human RHAMM in N134 cells infected with RCASBP–RHAMMB was conﬁrmed by reverse transcription–PCR. mRNAs from HeLa cell, N134 cells (uninfected), and N134 cells infected with RCASBP– B B

islet tumor (control) normal pancreas RHAMM (RHAMM ) were isolated for RT-PCR. Speciﬁc primers for human 20x RHAMM (exon 16) and human/mouse β-actin (as a control) were used. (B)A

20x total of 1 × 106 N134 cells infected with RCASBP–Luciferase were injected into the tail vein of five recipient mice. The locations of tumor cells overexpressing luciferase were monitored by in vivo bioluminescent imaging 1, 2, 3, 5, and 7 d after injection. RT, reverse transcriptase. liver RHAMMB-expressing tumor cells continued to express insulin as indicated by low blood glucose levels and by immunohisto- 20x chemical staining for insulin (Fig. 4 D and E). To survey for MEDICAL SCIENCES Fig. 2. Detection of metastases in RIP-Tag; RIP-tva mice infected with micrometastases, immunohistochemical staining for synapto- – B physin was performed. Mice receiving uninfected tumor cells had RCASBP RHAMM .(A and B) Representative lymph node and liver metastases ± – found in RCASBP–RHAMMB infected mice. Immunohistochemical staining of 1.8 0.8 liver micrometastases and mice receiving Bcl-xL synaptophysin (A) and insulin (B) is shown. (C–E) Immunohistochemical expressing tumor cells had 26.6 ± 5 liver micrometastases (Fig. staining of RHAMM in pancreas from wild-type mouse (C), islet tumor from 4F). Immunohistochemical staining for synaptophysin and in- a control RIP-Tag; RIP-tva mice (D), and islet tumor, lymph node metastases, sulin in sections of liver, lung, heart, thymus, brain, pancreas, and liver metastases found in RCASBP–RHAMMB infected mice (E). spleen, kidney, bone marrow, and mammary gland revealed that the liver is the only organ with metastases (Fig. 4E). Our results indicate that overexpression of RHAMMB significantly promotes few days postinjection and the signals gradually became un- the pancreatic islet tumor cells to establish large liver-specific detectable within a week, suggesting that the tumor cells are metastases after injection into the general venous circulation. initially trapped in the capillary beds of the lungs. In addition, we examined histological sections of several After 5 wk, organs of the recipient mice were harvested to organs for the existence of islet tumor cells at earlier time points survey for metastases. The findings were dramatic: all five mice (i.e., 1.5 h and 1, 3, 5, 7, and 14 d after tail vein injection). B receiving RHAMM -expressing tumor cells exhibited macro- Consistent with in vivo bioluminescent results using Luciferase- metastases in the livers, with an average of 57 macrometastases expressing N134 cells in Fig. 3B, parental and RHAMMB- per mouse (Fig. 4 A and B). In contrast, three of five mice re- expressing N134 cells in clusters of various sizes were found ceiving Bcl-xL–expressing tumor cells had liver macrometastases, within pulmonary vessels associated with fresh fibrin during the with an average of one macrometastases per mouse. No mac- first few days. Over the course of 7 d, we observed increasing rometastases were found in mice receiving uninfected tumor organization of fibrin, infiltrated neutrophils centered on pul- cells or Luciferase-expressing tumor cells (Fig. 4 A and B). The monary vessels, and gradual clearance of tumor cells. By 14 d, liver weight was increased about twofold in the mice receiving tumor cells were no longer found in pulmonary vessels. Im- RHAMMB-expressing tumor cells compared with that in other munohistochemical staining for synaptophysin in liver sections mice (Fig. 4C). Moreover, the liver metastases in recipients of revealed a few positive cells. Thus, RHAMMB did not appear to

Du et al. PNAS | October 4, 2011 | vol. 108 | no. 40 | 16755 Downloaded by guest on September 28, 2021 B Bcl-xL uninfected Luciferase RHAMM A A 3000 B

100 2500

2000 90

160 B 70 C 2500 D 1500 140 60 80 2000 120 50 1000 100 # of migratory cells 40 1500 80 70 30 500 % of Ki67 positive cells 1000 60 liver weight (mg) 20

blood glucose (mg/dL) 40 500 0

# of macromets in the liver 0 10 20

0 0 0

Fig. 5. RHAMMB does not promote migration in a Transwell assay in vitro. E F (A) Uninfected N134 cells and N134 cells infected with RCASBP–RHAMMB or insulin synaptophysin – RHAMMB uninfected RHAMMB Bcl-xL RCASBP Bcl-xL were plated in the upper chambers of Transwell plates. After 72 h, cells were counted in the lower chambers. Data are presented as the mean numbers of cells in five fields under 20× magnification, and are representative of three independent experiments. (B) Proliferative indices of the two cell lines. Cultures grown on chamber slides from uninfected N134 B 10x 4x 4x 4x cells and N134 cells infected with RCASBP–RHAMM were stained with antisera against Ki67. Data shown are the mean percentage ± SD from B Fig. 4. RHAMM greatly promotes liver metastasis of pancreatic islet tu- triplicate experiments. mor cells in the tail vein metastasis assay. A total of 1 × 106 parental mouse pancreatic islet tumor N134 cells and tumor cells overexpressing Luciferase, B RHAMM , or Bcl-xL were injected into the tail vein of recipient mice. Five RHAMMB Increases Phosphorylation of EGFR, Erk, and STAT3. Little is weeks later, the recipient mice (n = 5 for each group) were euthanized to known about the biochemical properties of the RHAMMB.It survey for metastatic sites and incidence (A and B), to record liver weight − − has been shown that Erk1/2 activity is aberrant in RHAMM / (C), and to measure the blood glucose (D). (E) The liver metastases in mice fi receiving RHAMMB-expressing cells continued to express insulin. Liver broblasts (10) and activation of EGFR is required for wound- sections from recipient mice with N134 tumor cells or tumor cells over- ing-induced hyaluronan synthesis (11). As an initial effect to expressing RHAMMB or Bcl-xL were subjected to immunohistochemical explain the physiological consequences of overexpression of B staining of synaptophysin (F) to reveal the presence of metastases. Original RHAMM , we measured the relative amounts and phosphory- magnification is indicated. lation status of EGFR, Erk1/2, and STAT3 by Western blot analysis using antibodies against total and phosphorylated EGFR, Erk, and STAT3. We observed increased phosphoryla- enhance infiltration of the lung by circulating tumor cells or by tion of EGFR (on Tyr1068), Erk1/2 (on Thr202/Tyr204), and outgrowth of micrometastasis in the liver at these early time STAT3 (on Ser727) in RHAMMB-expressing tumor cells com- points. pared with uninfected N134 cells (Fig. 6A), suggesting that overexpression of RHAMMB activates signaling pathways that RHAMMB Does Not Enhance Cell Migration in a Transwell Assay or include EGFR, Erk1/2, and STAT3. In addition, we observed Proliferation in Vitro. To examine whether RHAMMB enhanced slightly reduced levels of the cell adhesion molecule E-cadherin the ability of mouse pancreatic tumor cells to migrate in vitro, we in RHAMMB-expressing tumor cells; reduced E-cadherin could performed a two-chamber cell migration assay. Uninfected N134 contribute to invasiveness of the tumor cells (12). cells, RHAMMB-expressing tumor cells, or Bcl-xL–expressing To gauge whether the EGFR signaling pathway might be re- B tumor cells were seeded in the upper chambers of Transwell quired for the survival of N134 cells overexpressing RHAMM , fi inserts. Bcl-xL–expressing tumor cells was used as a positive we treated cells with ge tnib, a small molecule inhibitor of the fi control, as we have previously demonstrated that these cells have EGFR kinase (13). Two days later, ge tnib-treated and untreated cells were labeled with Alexa Fluor 488 annexin V and greater ability to migrate in the two-chamber migration assay fl than do parental cells (1). We measured cell migration along propidium iodide and then subjected to ow cytometry to identify apoptotic cells. We found that (i) overexpression of a serum gradient through the membrane to the bottom of the B B RHAMM in untreated tumor cells provided modest protection chambers. Although RHAMM , but not Bcl-xL, strongly pro- against apoptosis, and (ii)gefitnib induced an approximately moted liver metastasis of mouse islet tumors in mice, the number B B fourfold increase in apoptosis of RHAMM -expressing cells, but of migratory RHAMM -expressing tumor cells was similar to did not induce significant apoptosis in parental N134 cells that of uninfected cells (Fig. 5A), suggesting that expression of fi B in vitro (Fig. 6B). Ge tinib treatment also reduced phosphory- RHAMM does not affect migration in vitro. This result indi- fi B lation of both Erk and EGFR, without signi cant changes in cates that the effect of RHAMM on hepatic metastasis is un- phosphorylation of STAT3 or Bcl-xL protein levels, in related mechanistically to cell migration or that the Transwell RHAMMB-expressing tumor cells (Fig. 6C). These results sug- assay does not mimic the in vivo microenvironment sufficiently gest that RHAMM uses the EGFR signaling pathway to promote well to demonstrate an effect of RHAMMB on migration. B tumor cell survival, although this effect cannot explain the spe- To determine whether RHAMM promoted cell proliferation cific increase in hepatotropic metastasis. in vitro, we performed immunocytochemistry using antisera against a proliferation marker, Ki67. The frequency of Ki67- Discussion positive cells was slightly reduced in RHAMMB-expressing tu- Metastasis is the major cause of cancer mortality and appears to mor cells compared with the uninfected cells (Fig. 5B). These occur in an orderly sequence of general steps: local invasion, findings indicate that RHAMMB does not stimulate cell pro- intravasation, survival in the circulation, extravasation, and col- liferation in vitro and may even have a slight inhibitory effect. onization. Two in vivo assays, “spontaneous metastasis” in RIP-

16756 | www.pnas.org/cgi/doi/10.1073/pnas.1114022108 Du et al. Downloaded by guest on September 28, 2021 ABWCE The functions and subcellular localizations of the various products of the RHAMM locus are controversial (16). RHAMM 60 proteins have been implicated in multiple functions due to their association with hyaluronan, BRCA1, BARD1, CD44, Erk1/2, 50 mitotic spindle, microtubules, and microﬁlaments (17). They p-EGFR have been reported to act as cell-surface receptors but have also 40 been reported in the cytoplasm or the cell nucleus. These total EGFR observations may be attributed to the existence of different iso- 30 p-Erk forms generated by alternative splicing, as it is not clear which isoform(s) was examined in many reports due to the uncertain 20 % of apoptotic cells ﬁ total Erk speci city of the antibodies and nucleic acid probes. Even though RHAMM proteins have been shown to be overexpressed in 10 p-STAT3 various cancer cells, their oncogenic potentials and growth promoting signals have not been rigorously demonstrated either total STAT3 0 B in vitro or in vivo. One possible explanation is that the oncogenic RHAMM : - - + + B E-cadherin activity of RHAMM is too modest to be observed in conven- Gefitnib: - + - + tional cell transformation assays but becomes evident in a more sensitive context, such as our RIP-Tag; RIP-tva mouse model. C Gefitnib ( M): 0 0.5 1 10 10 Further investigation is needed to decipher the mechanism by Time (hr): 2 2 2 2 24 which metastasis is enhanced and to determine whether other p-Erk RHAMM isoforms have similar effects. Because hepatotropic metastasis promoted by RHAMMB is total Erk also observed with tail vein injection, the effect cannot be mediated directly through intravasation or be dependent on access p-STAT3 to the liver through the portal vein. The effect is not a conse-

total STAT3 quence of size of the primary tumors, because abundant hepatic metastases developed even in RCASBP–RHAMMB infected RIP- p-EGFR Tag; RIP-tva mice that had relatively small tumors. Increased proliferation also appears not to explain the enhanced hepatic B total EGFR metastasis, because RCASBP–RHAMM infected N134 cells had a lower Ki67 index than uninfected cells. Furthermore, the effect BcL-xL is unlikely to be attributable to enhanced migration, because RHAMMB does not promote tumor cell migration in a two- B Fig. 6. Overexpression of RHAMMB activates EGFR in pancreatic tumor cells, chamber Transwell assay. Our findings suggest that RHAMM and EGFR small molecule inhibitor induced apoptosis of mouse pancreatic may allow islet tumor cells to invade and grow specifically in the islet tumor cells in vitro. (A) Western blot analysis revealed the elevated microenvironment of the liver. It will be important to determine phosphorylated EGFR (p-EGFR), phosphorylated Erk (p-Erk), and phosphor- the cell specificity of this phenomenon and to identify the factor(s) ylated STAT3 (p-STAT3), and decreased E-cadherin in N134 cells over- in the liver that might be recognized by hepatotropic cancer cells expressing RHAMMB (lane 2) compared with uninfected N134 cells (lane 1). and thus serve as targets for interventions that prevent metastasis. B (B) N134 cells and N134 cells overexpressing RHAMM were cultured in the In a preliminary effort to characterize the biochemical effects μ fi presence of DMSO (vehicle control) or 10 Mge tnib. Two days later, cells of RHAMMB on pancreatic islet tumor cells, we showed that it were harvested and labeled with Alexa Fluor 488 annexin. The apoptotic fl enhances phosphorylation of EGFR, Erk1/2, and STAT3, im- cells were distinguished using a ow cytometer with a 488-nm laser to excite plying activation of signaling pathways that are often affected in the dye. Data shown are the mean percentage ± SD from triplicate experi- B cancers. Furthermore, treatment with an inhibitor of EGFR ments. (C) N134 cells overexpressing RHAMM were cultured in the presence fi B of DMSO, 0.5, 1, or 10 μMgefitnib, and whole cell extracts were prepared 2 signaling, ge tnib, induced apoptosis in RHAMM -expressing

or 24 h later for Western blot analysis of indicated antibodies. WCE, whole cells, although we cannot exclude the possibility that this effect MEDICAL SCIENCES cell extracts. may be mediated by inhibition of other uncharacterized kinases. Additionally, it has been shown that EGFR signaling contributes to tumorigenesis in RIP-Tag mice (18). The RHAMM full-length A Tag; RIP-tva mouse model and “tail vein assay of cancer me- isoform, RHAMM , enhances serum-induced Erk1/2 phos- tastasis,” provide complementary information to dissect the phorylation in embryonic fibroblasts from RHAMM knockout fi mice (10), and here we showed that overexpression of speci c metastatic steps. Using the avian retroviral vector to B deliver five candidate genes that are up-regulated in human HCC RHAMM phosphorylates Erk1/2 in mouse pancreatic islet tu- into mouse pancreatic hyperplastic cells in RIP-Tag; RIP-tva mor cells. Thus, Erk1/2 seem to be common targets of two mice, we have shown that RHAMMB among the five genes RHAMM isoforms. Conditional STAT3 knockout mice have dramatically promotes metastasis in an organ-specific pattern, been used to show that STAT3 is important for the function of with the appearance of liver metastasis regardless of whether interleukin-6 (IL-6) in liver regeneration (19). Because IL-6 is enriched in the liver, tumor cells with activated STAT3 may have tumor cells enter the portal circulation endogenously in a mouse a growth advantage in the liver. model or whether they are injected into a tail vein to enter the We previously showed that normal, untransformed mammary major venous circulation. B cells, when introduced to the tail vein of the recipient mice, In addition to RHAMM , we found that FLJ10540 promotes colonize to the lungs, a major organ site for breast cancer me- metastasis to lymph nodes and the liver, but the sizes and num- B B tastasis (20). Although RHAMM promotes hepatotropic me- bers of metastases are less profound than RHAMM . PEG10, tastasis of islet tumor cells in the tail vein assay, it remains FLJ11252, and FLJ11164 were not able to promote liver metas- possible that the hepatotropism is an inherent property of the tasis in the RIP-Tag; RIP-tva model. FLJ10540 has been shown to islet tumor cells rather than determined by RHAMM protein. be up-regulated in HCC and oral cavity squamous cell carcinoma Further studies are required to determine whether uninfected and is associated with poor survival (14, 15). Overexpression of pancreatic islet tumor cells also reach the liver, but remain FLJ10540 in 3T3 cells was also shown to promote survival in soft dormant or are unable to survive and/or grow. Although agar and in low serum medium and induces tumor formation in RHAMMB does not provide a proliferation advantage in vitro, nude mice (14). the slight protection against apoptosis observed in RHAMMB-

Du et al. PNAS | October 4, 2011 | vol. 108 | no. 40 | 16757 Downloaded by guest on September 28, 2021 expressing tumor cells might help them survive better in ectopic RCASBP is a replication-competent avian leukosis virus with a splice acceptor and hemodynamically stressful sites, such as the liver. and the Bryan-RSV pol gene. RCASBP–ALPP has been described previously The liver is the most common organ for the metastases in (23). RCASBP–Luciferase was generated by Yi Li in the Varmus laboratory. human pancreatic neuroendocrine tumors and cancers of the RCASBP–RHAMMB, RCASBP–FLAG-PEG10, RCASBP–FLAG-FLJ10540, RCASBP– intestines, and almost all patients will succumb to liver failure FLAG-FLJ11252, and RCASBP–FLJ11164 were generated in the Chou labora- from the metastases. Because RIP-Tag mice (21) develop islet tory. Viral propagation and titer determination were described (1). In- tumors that do not metastasize, whereas such tumors in RIP-Tag; tracardiac injection was performed as described (24). RIP-tva mice infected with vectors encoding genes like RHAMMB form liver metastases readily, the model described here should Tail Vein Injection of Tumor Cells and in Vivo Bioluminescent Imaging. Single be useful for preclinical studies of cancers that preferentially cell suspension of tumor cells was prepared before tail vein injection. Mice metastasize to the liver. were i.v. injected in the tail vein using insulin syringes with 1 × 106 tumor cells Numerous microarray-based screens and immunohistochemi- in 150 μL PBS. Mice were subjected to in vivo bioluminescent imaging as cal screens have been performed to identify genes differentially described previously (25). All other experimental details are provided in SI expressed in human tumors and normal cells. Some have Materials and Methods. reported overexpression of RHAMM in various tumors and a negative prognostic signiﬁcance for its expression in breast ACKNOWLEDGMENTS. We thank members in the H.V. laboratory, especially cancer (4), multiple myeloma (22), and colon cancer (7). Our Jennifer Demers, Mary Ann Melnick, Andreas Giannakou, Gabriela Sanchez, and Raymond Dematteo for technical assistance; Katrina Podsypanina and study provides support for these observations by demonstrating B Romel Somwar for insightful discussions; Levi Beverly for 293T cell line and a causal role for RHAMM in liver metastasis in a mouse model reagents. We thank Danny Huang for mouse database design; and Irina of multistep tumorigenesis. Linkov for protocols. We thank Leigh Selesner in the Y.-C.N.D. laboratory for technical assistance; Hua-Chien Chen at Chang-Gung University for RHAMM Materials and Methods expression data from HCC samples; Mouse Genetics Core Facility and Research Animal Resource Center for foster service and animal husbandry; Generation of RIP-Tag; RIP-tva mice and N134 cell line has been described and Memorial Sloan-Kettering Cancer Center’s Molecular Cytology, Flow (1). NSG mice were generated by The Jackson Laboratory. All mice were Cytometry, and Small-Animal Imaging Cores for technical assistance. This housed in accordance with institutional guidelines. All procedures involving work was funded in part by a grant from the National Institutes of mice were approved by the institutional animal care and use committee. Health (5P01CA094060).

1. Du YC, Lewis BC, Hanahan D, Varmus H (2007) Assessing tumor progression factors by 13. Ciardiello F, et al. (2000) Antitumor effect and potentiation of cytotoxic drugs activity somatic gene transfer into a mouse model: Bcl-xL promotes islet tumor cell invasion. in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptor-se- – PLoS Biol 5:e276. lective tyrosine kinase inhibitor. Clin Cancer Res 6:2053 2063. 2. Yang CW, et al. (2005) Integrative genomics based identification of potential human 14. Chen C-H, et al. (2007) FLJ10540-elicited cell transformation is through the activation – hepatocarcinogenesis-associated cell cycle regulators: RHAMM as an example. Bio- of PI3-kinase/AKT pathway. Oncogene 26:4272 4283. 15. Chen C-H, et al. (2009) Expression of FLJ10540 is correlated with aggressiveness of oral chem Biophys Res Commun 330:489–497. cavity squamous cell carcinoma by stimulating cell migration and invasion through 3. Akiyama Y, et al. (2001) Hyaluronate receptors mediating glioma cell migration and increased FOXM1 and MMP-2 activity. Oncogene 28:2723–2737. proliferation. J Neurooncol 53:115–127. 16. Hofmann M, et al. (1998) Problems with RHAMM: A new link between surface ad- 4. Assmann V, et al. (2001) The pattern of expression of the microtubule-binding protein hesion and oncogenesis? Cell 95:591–592, author reply 592–593. RHAMM/IHABP in mammary carcinoma suggests a role in the invasive behaviour of 17. Maxwell CA, McCarthy J, Turley E (2008) Cell-surface and mitotic-spindle RHAMM: tumour cells. J Pathol 195:191–196. Moonlighting or dual oncogenic functions? J Cell Sci 121:925–932. 5. Grützmann R, et al. (2004) Gene expression profiling of microdissected pancreatic 18. Nolan-Stevaux O, et al. (2010) Differential contribution to neuroendocrine tumori- ductal carcinomas using high-density DNA microarrays. Neoplasia 6:611–622. genesis of parallel Egfr signaling in cancer cells and pericytes. Genes Cancer 1: 6. Gust KM, et al. (2009) RHAMM (CD168) is overexpressed at the protein level and may con- 125–141. stitute an immunogenic antigen in advanced prostate cancer disease. Neoplasia 11:956–963. 19. Li W, Liang X, Kellendonk C, Poli V, Taub R (2002) STAT3 contributes to the mitogenic 7. Zlobec I, Baker K, Terracciano LM, Lugli A (2008) RHAMM, p21 combined phenotype response of hepatocytes during liver regeneration. J Biol Chem 277:28411–28417. identifies microsatellite instability-high colorectal cancers with a highly adverse 20. Podsypanina K, et al. (2008) Seeding and propagation of untransformed mouse – prognosis. Clin Cancer Res 14:3798–3806. mammary cells in the lung. Science 321:1841 1844. 8. Ota T, et al. (2004) Complete sequencing and characterization of 21,243 full-length 21. Hanahan D (1985) Heritable formation of pancreatic beta-cell tumours in transgenic mice expressing recombinant insulin/simian virus 40 oncogenes. Nature 315:115–122. human cDNAs. Nat Genet 36:40–45. 22. Maxwell CA, et al. (2004) RHAMM expression and isoform balance predict aggressive 9. Sohr S, Engeland K (2008) RHAMM is differentially expressed in the cell cycle and disease and poor survival in multiple myeloma. Blood 104:1151–1158. downregulated by the tumor suppressor p53. Cell Cycle 7:3448–3460. 23. Fekete DM, Cepko CL (1993) Replication-competent retroviral vectors encoding al- 10. Tolg C, et al. (2006) Rhamm-/- fibroblasts are defective in CD44-mediated ERK1,2 mo- kaline phosphatase reveal spatial restriction of viral gene expression/transduction in – togenic signaling, leading to defective skin wound repair. J Cell Biol 175:1017 1028. the chick embryo. Mol Cell Biol 13:2604–2613. 11. Monslow J, Sato N, Mack JA, Maytin EV (2009) Wounding-induced synthesis of hya- 24. Kang Y, et al. (2005) Breast cancer bone metastasis mediated by the Smad tumor luronic acid in organotypic epidermal cultures requires the release of heparin-binding suppressor pathway. Proc Natl Acad Sci USA 102:13909–13914. egf and activation of the EGFR. J Invest Dermatol 129:2046–2058. 25. Du YC, Klimstra DS, Varmus H (2009) Activation of PyMT in beta cells induces irre- 12. Perl AK, Wilgenbus P, Dahl U, Semb H, Christofori G (1998) A causal role for E-cad- versible hyperplasia, but oncogene-dependent acinar cell carcinomas when activated herin in the transition from adenoma to carcinoma. Nature 392:190–193. in pancreatic progenitors. PLoS ONE 4:e6932.

16758 | www.pnas.org/cgi/doi/10.1073/pnas.1114022108 Du et al. Downloaded by guest on September 28, 2021