Increased Expression of the E3 Ubiquitin Ligase RNF5 Is Associated with Decreased Survival in Breast Cancer

Research Article

Kenneth D. Bromberg,2 Harriet M. Kluger,3 Agnes Delaunay,1 Sabiha Abbas,1 Kyle A. DiVito,3 Stan Krajewski,1 andZe’ev Ronai 1

1Signal Transduction Program, The Burnham Institute for Medical Research, La Jolla, California; 2Department of Oncological Sciences, Mount Sinai School of Medicine, New York, New York; and 3Department of Pathology, Yale University School of Medicine, New Haven, Connecticut

Abstract cancer development. For example, the MDM2 oncogene, which The selective ubiquitination of proteins by ubiquitin E3 ligases targets p53 for ubiquitination-dependent degradation, is overex- plays an important regulatory role in control of cell pressed in human tumors (6). In contrast, the tumor suppressors differentiation, growth, and transformation and their dysre- BRCA1 and BRCA2 and von Hippel-Lindau are frequently mutated gulation is often associated with pathologic outcomes, or deleted in human tumors (7, 8). RNF5 is an 18-kDa RING including tumorigenesis. RNF5is an E3 ubiquitin ligase that finger E3 ligase that is important for development in Caeno- has been implicated in motility and endoplasmic reticulum rhabditis elegans by regulating proper formation of muscle stress response. Here, we show that RNF5expression is up- attachment sites (9). In mammalian cells, RNF5 regulates cell regulated in breast cancer tumors and related cell lines. motility by ubiquitinating the focal adhesion protein paxillin (10), which triggers the exclusion of paxillin from the focal adhesions. Elevated expression of RNF5was seen in breast cancer cell lines that became more sensitive to cytochalasin D– and RNF5 (a.k.a. RMA1) was also shown to serve as a quality control paclitaxel-induced apoptosis following its knockdown with protein in the endoplasmic reticulum, through which it contributes to the clearance of malfolded proteins, including mutant specific short interfering RNA. Inhibition of RNF5expression D markedly decreased cell proliferation and caused a reorgani- CFTR ( 108; ref. 11). RNF5/RMA1 was also shown to affect the zation of the actin cytoskeleton in response to stress in MCF-7 Salmonella protein SOP-A by ubiquitination that facilitates its but not in p53 mutant breast cancer cells, suggesting a p53- trafficking from the endosomes/vacuoles to the cytosol (12), dependent function. Significantly, high levels of RNF5were further substantiating the role of this ligase in control of protein associated with decreased survival in human breast cancer localization in the cell. In this study, we show that RNF5 specimens. Similarly, RNF5levels were higher in metastatic modulates breast cancer cell sensitivity to cytoskeletal-targeting melanoma specimens and in melanoma, leukemia, ovarian, anticancer agents and that RNF5 expression inversely correlates and renal tumor-derived cell lines, suggesting that increased with survival of breast cancer patients. Our findings suggest that RNF5expression may be a common event during tumor through the dynamic rearrangement of the actin cytoskeleton and increased cell proliferation, RNF5 plays an important role in progression. These results indicate that RNF5is a novel regulator of breast cancer progression through its effect on neoplastic progression of breast cancer and possibly other human actin cytoskeletal alterations, which also affect sensitivity of tumors. breast cancer cells to cytoskeletal targeting antineoplastic agents. [Cancer Res 2007;67(17):8172–9] Materials and Methods Tumor tissue mRNA array. The BioChain Human Adult Tumor/Normal Introduction Tissues mRNA Array (BioChain Institute, Inc.) was used to analyze RNF5 expression. This array contains equal amounts of mRNA from 47 tumor Ubiquitin E3 ligases regulate a vast number of cellular tissues and their corresponding normal tissues. Biotinylated DNA probes processes, including development, proliferation, cell cycle control, recognizing either RNF5 or glyceraldehyde-3-phosphate dehydrogenase transcription, apoptosis, and DNA repair (1–3). It is evident that (GAPDH) transcripts were generated by random labeling using the NEBlot many really interesting new gene (RING) finger proteins, which Phototope kit (New England Biolabs). Hybridization was carried out have been implicated in cell growth and development, possess E3 overnight using FastHyb-Hybridization Solution (BioChain Institute) ligase activity and mediate the ubiquitination of their substrates according to the manufacturer’s instructions. Levels of RNF5 or GAPDH (4). A growing number of RING E3 ligases are aberrantly expressed mRNA were detected by enhanced chemiluminescence (ECL) using the or regulated during tumorigenesis (4, 5). Depending on the Phototope-Star detection kit (New England Biolabs) and analyzed on a specific substrate, an E3 ligase can either promote or inhibit Bio-Rad GS800 scanning densitometer. The RNF5 expression level for each tumor and corresponding normal tissue was normalized to GAPDH and then compared. Cell lines and cell culture. Breast carcinoma MCF-7, SKBR3, BT-24, BT-474, MDA-MB-231, MDA-MB-435, and ZR-75-1 cells were cultured in Note: Supplementary data for this article are available at Cancer Research Online growth medium [DMEM and 10% fetal bovine serum (FBS) and supple- (http://cancerres.aacrjournals.org/). Current address for K.D. Bromberg: Department of Pharmacology and Systems mented with 100 units penicillin/mL and 100 units streptomycin/mL]. Therapeutics, Mount Sinai School of Medicine, New York, NY 10029. Human mammary epithelial cells (HMEC) were cultured in MEGM Requests for reprints: Ze’ev Ronai, Signal Transduction Program, The Burnham mammary epithelial cell medium supplemented with singlequots (Clonetics). Institute, 10901 North Torrey Pines Road, La Jolla, CA 92037. Phone: 858-646-3185; RNA isolation and reverse transcription-PCR analysis. Total RNA was Fax: 815-366-8003; E-mail: [email protected]. I2007 American Association for Cancer Research. purified with the Qiagen RNeasy kit according to the manufacturer’s doi:10.1158/0008-5472.CAN-07-0045 instructions. cDNA synthesis was done with the Moloney murine leukemia

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. RNF5 in Breast Cancer Progression virus reverse transcriptase (Invitrogen) using 2 Ag RNA and a polydeox- Immunohistochemistry of dewaxed tissue sections was done as described ythymidine oligonucleotide as a template. Semiquantitative PCR was previously (15). Antisera specific for RNF5 were applied followed by a goat carried out for 20 cycles using [a-32P]dCTP (Amersham Biosciences) and anti-rabbit secondary antibody (16). The immunostaining was scored as 10 ng cDNA. Reaction products were resolved by 5% PAGE and visualized per Krajewska et al. (17). The immunoscore was determined by visual and quantified on a Bio-Rad FX Molecular Imager. inspection (intensity X% = 300 as a maximal score translated as 3+ intensity Western blot. Cells were lysed in radioimmunoprecipitation assay in 100% of cells). (RIPA) buffer (PBS, 1% NP40, 0.5% deoxycholate, 0.1% SDS, 1 mmol/L EDTA) RNF5short interfering RNA and retroviral infections. The pSUPER. supplemented with protease and phosphatase inhibitors. Western blotting retro vector was used to introduce short interfering RNA (siRNA) targeting was done using standard procedures with antibodies directed against RNF5 RNF5 and its corresponding scramble control into MCF-7, MDA-MB-231, and visualized by ECL. Antibodies directed against h-actin and a-tubulin MDA-MB-435, and BT-474 cells. pSUPER.retro.shRnf5 or pSUPER.retro. were used as loading controls. scramble infectious retroviral stock was generated in human 293T cells. The National Cancer Institute 60-tumor cell line panel microarray. above indicated cells were infected for 6 h with the addition of 5 Ag/mL Established tumor cell lines were cultured as described previously (13), polybrene. After 48 h, cells stably expressing RNF5 siRNA were selected by harvested after fixation, and embedded in paraffin to be spotted onto a treatment with 2 Ag/mL puromycin. Alternatively, MCF-7 cells were tissue microarray block for immunohistochemical screening. The cell lines transfected with an oligonucleotide siRNA targeting RNF5 or a scramble included the National Cancer Institute (NCI) 60-tumor cell line panel (14). siRNA (Ambion) using LipofectAMINE 2000 (Invitrogen) in Opti-MEM.

Figure 1. Increased RNF5 expression in human cancers. A, mRNA array of 47 tumors and their corresponding normal tissues. The array was probed for RNF5, stripped,and then reprobed for GAPDH. Tumor mRNA is spotted in duplicate in odd-numbered rows ( T) and mRNA from the corresponding normal tissues is spotted to the right of the tumor in the even-numbered rows (N). Lane 1, positive control; lane 2, negative control. B, tumors that displayed greater than a 2.5-fold increase in RNF5 expression compared with normal tissue. The colors of the rectangles enclosing the spots quantified in (A) correspond to the matching colors representing RNF5 expression levels in (B). C, increased RNF5 mRNA expression in breast carcinoma cell lines. RNA was isolated from the indicated cell lines and semiquantitative RT-PCR was done with primers specific for RNF5 or GAPDH. Left, representative gel of three independent experiments; right, quantification. Error bars, SD of three independent experiments. D, increased RNF5 protein expression in breast carcinoma cell lines. The indicated cell lines were lysed in RIPA buffer and the level of endogenous RNF5 in the whole-cell lysates was analyzed by Western blot. h-Actin was used as a loading control. www.aacrjournals.org 8173 Cancer Res 2007; 67: (17). September 1, 2007

Figure 2. RNF5 protein expression profile in a NCI 60-tumor cell line panel. A cell line microarray slide was immunostained with an antibody directed against RNF5. The histogram depicts percentage of RNF5 immunopositivity in these cell lines,as evaluated by visual inspection done by two independent readers,as detailed in Materials and Methods and ref. 17. Inset, positive RNF5 staining for the OVCAR-3 ovarian cancer cell line.

Three oligonucleotides targeting different sequences in the RNF5 gene were a-tubulin (Sigma) for 1 h at room temperature followed by washing and used. The medium was changed 6 h after transfection and experiments were further incubation with a goat anti-mouse Alexa Fluor 568–conjugated done 48 h after transfection. secondary antibody and Alex Fluor 488–phalloidin (Invitrogen) for 1 h. Fluorescence-activated cell sorting cell cycle analysis. Cells at The coverslips were mounted on glass slides in ProLong Gold (Invitrogen) approximately 50% to 60% confluence were treated for 24 h with DMSO, and images were acquired on a Zeiss LSM 510 Meta confocal microscope 5 Amol/L cytochalasin D, or 1 Amol/L paclitaxel (Sigma) for 24 h. Cells were at Â63 magnification. harvested, washed twice in PBS, and fixed in ice-cold 70% ethanol overnight Tissue microarrays. The breast cancer and melanoma tissue micro- at 4jC. Cells were washed twice in PBS and then labeled with propidium arrays were constructed as described previously (18). Immunohistochemical iodide solution (50 Ag/mL propidium iodide, 10 Ag/mL RNase A, 0.1% staining of the arrays with polyclonal antibodies against RNF5 was carried sodium citrate, 0.1% Triton X-100) for 30 min at room temperature. according to the protocol of Chung et al. (19). The regions of most intense Fluorescent-stained cells were then transferred to polystyrene tubes with staining were scored by eye for each spot. The staining was graded using the cell strainer caps (BD Falcon) and subjected to fluorescence-activated cell following scale: 0, no staining; 1, weak staining; 2, moderate staining; and sorting using a Becton Dickinson FACScan. Acquisition and analysis of 3, intense staining. See Supplementary Materials and Methods for tissue cells containing sub-G1 DNA content (apoptotic cells) were done using microarray construction and details. CellQuest 3.2 software (Becton Dickinson). Cell proliferation. MCF-7 cells were plated in 96-well plates at a density of 5,000 cells per well. After an overnight incubation, cells were treated with Results 20 AL CellTiter 96 Aqueous One Solution Reagent (Promega). The cells were RNF5expression is elevated in human cancers. A growing further incubated at 37jC for 1 h and cell growth was quantified at an absorbance of 480 nm on an Envision 2101 multilabel reader (Perkin-Elmer). number of ubiquitin ligases that are important regulators of Confocal microscopy. MCF-7 cells were grown on glass coverslips, fixed, development and cell growth are found to be aberrantly expressed and permeabilized according to the protocol of Didier et al. (10). After in human tumors. To evaluate the expression pattern of RNF5 in blocking in 5% bovine serum albumin for 30 min, cells were incubated human tumors, we used a mRNA array that contained mRNA from with 100 AL of monoclonal antibodies directed against paxillin (Upstate) or 47 different tumors and their corresponding normal tissues

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(Fig. 1A). For each tumor, the mRNA was spotted in duplicate. After to knock down RNF5 expression in several breast cancer cell lines, probing the array for RNF5 expression, the two duplicate spots including MCF-7, MDA-MB-231, MDA-MB-435, and BT-474 cells. were averaged and then normalized to the expression levels of The efficiency of siRNA targeting RNF5 was >80%, which was the housekeeping gene GAPDH. As seen in Fig. 1B, seven different confirmed in each experiment by Western blot (Fig. 3A and B). tumors displayed at least a 2.5-fold increase in RNF5 mRNA levels. Because RNF5 has been shown to regulate cell motility (10), we RNF5 expression was markedly increased in several breast cancers, assessed whether RNF5 altered breast cancer cell migration using with an f10-fold increase in breast intraductal carcinoma and an a pipette scratch assay. RNF5 siRNA did not have a significant f3-fold increase in breast adenocarcinoma. Marked increases in effect on the migratory capacity of any of the cell lines compared RNF5 expression were also observed in lung cancer (f5-fold) and with scramble siRNA (Supplementary Fig. S1; data not shown). esophageal cancer (f4-fold). Consistent with this result, RNF5 knockdown did not affect paxillin Next, we sought to confirm the results of the mRNA array by localization in MCF-7 cells (data not shown). These data suggest examining RNF5 expression in breast cancer cell lines by reverse that unlike nontransformed cultures, RNF5 does not affect paxillin transcription-PCR (RT-PCR) analysis. As shown in Fig. 1C, RNF5 or cell migration in the breast cancer cells. Next, we examined mRNA levels were increased f3.5-fold in several breast cancer cell whether loss of RNF5 expression altered breast cancer cell death. lines compared with HMECs, including MCF-7, BT-20, and BT-474 Knockdown of RNF5 expression did not induce apoptosis in cells. These results were confirmed on the protein level via Western MCF-7, MDA-MB-231, MDA-MB-435, or BT-474 cells (data not blotting with antibodies directed against endogenous RNF5 shown). However, MCF-7, MDA-MB-231, and BT-474 cells express- (Fig. 1D). To confirm RNF5 protein expression in other cancers, ing RNF5 siRNA displayed increased apoptosis on treatment with we generated and probed a cell microarray of the NCI 60-tumor cell the actin-disrupting agent cytochalasin D compared with scramble- line panel. Consistent with the mRNA tumor array, strong RNF5 expressing cells (Fig. 3C). A similar enhancement of cytochalasin expression was seen in breast, colon, and lung cancer cell lines D–induced apoptosis was observed for MCF-7 cells transfected (Fig. 2). In addition, RNF5 was strongly expressed in several with three other siRNAs targeting different RNF5 sequences ovarian, renal, melanoma, and leukemia cell lines. Taken together, (Supplementary Fig. S2). In addition, MDA-MB-435 cells expressing these findings indicate that RNF5 is strongly expressed in many RNF5 siRNA, which were less affected by cytochalasin, exhibited different cancers and that RNF5 is up-regulated on the transcrip- greater sensitivity to treatment with the microtubule-targeting tional level in several human breast cancers. drug paclitaxel compared with scrambled siRNA-expressing cells RNF5siRNA enhances breast cancer cell apoptosis. To (Fig. 3D). RNF5 siRNA also sensitized BT-474 cells, albeit to a lesser examine the role of RNF5 in breast tumorigenesis, siRNA was used degree, to paclitaxel treatment (Fig. 3D). These results indicate that

Figure 3. Loss of RNF5 expression enhances breast cancer apoptosis induced by cytochalasin D and paclitaxel. A, MCF-7 cells stably expressing scramble siRNA (SC) or RNF5 siRNA were generated by retroviral infection and selection in puromycin. Cells were lysed and processed as per Fig. 1D. h-Actin was used as a loading control. B, MDA-MB-231,MDA-MB-435,and BT-474 cells stably expressing scramble siRNA or RNF5 siRNA were generated and processed as in ( A). a-Tubulin was used as a loading control. C and D, knockdown of RNF5 enhances cytochalasin D– and paclitaxel-induced apoptosis. The indicated cell line expressing scramble or RNF5 siRNA was treated with either 5 Amol/L cytochalasin D (C)or1Amol/L paclitaxel (D) for 24 h. Cells were fixed and stained with propidium iodide,and DNA content was analyzed with a fluorescent-activated cell sorter. The sub-G 1 DNA content indicates apoptotic cells. Error bars, SE of two to three independent experiments. *, P < 0.05; **, P < 0.04; ***, P < 0.02,difference between scramble siRNA and RNF5 siRNA for MCF-7,MDA-MB-231,and BT-474 cell apoptosis,respectively. www.aacrjournals.org 8175 Cancer Res 2007; 67: (17). September 1, 2007

Figure 4. Loss of RNF5 expression reorganizes MCF-7 cell actin cytoskeleton. A, representative confocal microscopic images showing the cellular localization of actin in MCF-7 cells stably expressing scramble siRNA or RNF5 siRNA. Asynchronously growing (Grow),serum-starved ( Starve),and serum-starved MCF-7 cells that had recovered for 2 h in serum (Recover) were fixed,permeabilized,and stained with phalloidin for the actin cytoskeleton. B, quantification of serum-starved scramble and RNF5 siRNA-expressing MCF-7 cells that displayed the actin morphology in (Starve, A). Approximately 70 cells from several random fields were counted per experiment. Error bars, SE of two to three independent experiments. *, P < 0.02,difference between scramble siRNA and RNF5 siRNA MCF-7 cell actin morphology. C, RNF5 siRNA inhibits MCF-7 cell proliferation. Cell proliferation was measured as described in Materials and Methods. Each experiment was done in triplicate. Error bars, SE of three sets of experiments. *, P < 0.01; **, P < 0.05,difference between scramble siRNA and RNF5 siRNA MCF-7 cell proliferation,10% FBS and 2% FBS,respectively.

RNF5 contributes to the degree of sensitivity of breast cancer cells microtubule cytoskeletal organization under the above conditions to cytoskeletal-targeting anticancer agents. (Supplementary Fig. S3; data not shown). Of interest, analysis of Loss of RNF5causes rearrangement of the actin cytoskele- cytoskeletal proteins in melanoma cells that were subjected to ton and decreased cell proliferation in MCF-7 cells. Because elevated RNF5 expression also revealed altered pattern of actin loss of RNF5 expression sensitized breast cancer cells to expression (Supplementary Fig. S4), further noting the effect on cytoskeletal-targeting agents, we examined the actin and microtu- RNF5 on select cytoskeletal proteins. These data suggest that RNF5 bule architecture of breast cancer cells expressing scramble or contributes to cytoskeletal organization in MCF-7 cells. RNF5 siRNA. For MCF-7 cells growing asynchronously, no differ- To examine the effect of RNF5 on MCF-7 cell proliferation, ences between the two cell lines were observed in the actin we used a colorimetric 3-(4-5-dimethylthiazol-2-yl)-5-(3-carboxy- cytoskeleton (Fig. 4A) or paxillin (data not shown) localization. methoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium salt (MTS) assay. However, marked differences in the organization of actin As shown in Fig. 4C, siRNA of RNF5 markedly decreased MCF-7 cell cytoskeleton were observed after serum starvation. Approximately proliferation compared with scrambled siRNA in both low (2% 55% of scrambled siRNA-expressing MCF-7 cells retained their FBS) and high (10% FBS) serum. Knockdown of RNF5 in MCF-7 network of actin fibers (Fig. 4A and B). In contrast, only 21% of cells transfected with three other siRNAs targeting different RNF5 RNF5 siRNA-expressing MCF-7 cells were able to maintain their sequences also decreased cell proliferation (Supplementary Fig. S5). actin cytoskeleton and displayed mainly peripheral actin staining In contrast to these results in MCF-7 cells, RNF5 siRNA did not (Fig. 4A and B). After 2-h recovery in serum, both cell types began affect the actin cytoskeletal architecture or cell proliferation of to elongate back to their original morphology and RNF5 siRNA MDA-MB-231, MDA-MB-435, or BT-474 cells (Supplementary cells were able to reestablish their actin cytoskeletal architecture. Figs. S6 and S7). However, consistent with the increased apoptosis Similar results were observed for MCF-7 cells transfected with in MDA-MB-435 cells induced by paclitaxel, MDA-MB-435 cells three other siRNAs targeting different RNF5 sequences (Supple- displayed a reorganization of the microtubule architecture in asyn- mentary Fig. S3). RNF5 siRNA did not affect MCF-7 cell chronously growing cells (Supplementary Fig. S6B). No differences

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. RNF5 in Breast Cancer Progression in microtubule architecture were observed for MDA-MB-231 or and expression of E6 in these cells caused efficient decrease in BT-474 cells (Supplementary Fig. S6A and S6C). The differences p53 levels (Fig. 5A). Decrease of p53 levels in the MCF-7 cells between cell lines may be due to the fact that MCF-7 cells express ameliorated the effects of RNF5 siRNA on MCF-7 cell proliferation wild-type p53 and MDA-MB-231, MDA-MB-435, or BT-474 cells and actin cytoskeletal architecture (Fig. 5B and C). These data either have mutations in or do not express p53 (20, 21). suggest that RNF5 contributes to cytoskeletal organization and To test the possibility that functional p53 is required for RNF5- affects cell proliferation in part via functional p53. dependent effect on cytoskeletal architecture, we have inactivated Decreased survival in breast cancers expressing RNF5. To p53 in the MCF-7 cells and tested whether the changes seen above further investigate the significance of dysregulated RNF5 in human would be attenuated. To this end, we have expressed the human cancers, we evaluated RNF5 expression in human breast cancer papilloma virus E6 protein, which is among the potent ligases for specimens. Of the 655 breast cancer tumors on the tissue p53. Knockdown of RNF5 in MCF-7 caused an increase in p53 levels microarrays, 479 (73%) were interpretable for cytoplasmic RNF5

Figure 5. Inhibition of p53 attenuates the decrease in cell proliferation and actin cytoskeleton reorganization in response to knockdown of RNF5 expression in MCF-7 cells. A, MCF-7 cells stably expressing scramble siRNA (SC) or RNF5 siRNA were transfected with human papilloma virus E6 protein (E6) or pCDNA3 as a control (Con). Cells were lysed in RIPA buffer and the level of p53 in the whole-cell lysates was analyzed by Western blot with a p53 antibody. a-Tubulin was used as a loading control. B, MCF-7 cells stably infected with scramble (SC) or RNF5 siRNA were transfected with human papilloma virus E6 protein (E6) or pCDNA3 as a control (Con). Cell proliferation was measured as described in Materials and Methods. Each experiment was done in triplicate. Error bars, SE of three sets of experiments. *, P < 0.03,difference between pCDNA3- and E6-transfected RNF5 siRNA-expressing MCF-7 cells for cell proliferation. C, representative confocal microscopic images showing the cellular localization of actin and a-tubulin in MCF-7 cells stably expressing scramble siRNA or RNF5 siRNA that were transfected with pCDNA3 or E6. Cells were processed as per Fig. 4A. www.aacrjournals.org 8177 Cancer Res 2007; 67: (17). September 1, 2007

In the melanoma arrays, high RNF5 expression was seen more frequently in metastatic than in primary specimens (m2 = 5.7; P = 0.017). No association was found with survival or other clinical variables.

Discussion In the current study, we show that the RING finger E3 ligase RNF5 is up-regulated on the transcription level in breast cancer and that loss of RNF5 expression sensitizes breast cancer cells to cytochalasin D– and paclitaxel-induced apoptosis. RNF5 siRNA also causes a reorganization of the actin cytoskeleton in response to stress and a marked inhibition of cell proliferation in MCF-7 cells. Moreover, increased RNF5 expression in human breast cancer specimens is associated with decreased survival. It is of interest to note that noninvasive breast cancer (intraductal) has higher RNF5 expression than invasive breast cancer, suggesting changes in RNF5 expression (and likely activity) during select stages of breast cancer development. These results suggest that RNF5 is a novel regulator of breast cancer development and suggest that increased expression of RNF5 may contribute to breast tumor progression. Previously, we have shown that expression of RNF5 in nontransformed mouse fibroblast NIH-3T3 cells excludes paxillin from the focal adhesions and inhibits cell migration (10). Here, we show that RNF5 can modulate breast cancer apoptosis and plays a role in promoting breast cancer progression. Whereas RNF5 is involved in organization of the actin cytoskeleton, it does not seem to affect cell migration or paxillin localization in the breast cancer cell lines. Thus, RNF5 substrate(s) in the breast cancer and possibly other tumor cells are different than those identified in the nontransformed cells. The latter could be explained by different post-translational modification of paxillin in the tumor cells or the modification and activity of RNF5, aspects that deserve further studies. In addition to the tumors with increased RNF5 expression Figure 6. Increased RNF5 expression in human breast cancer and melanoma (Fig. 1B), we have identified several other tumors with decreased tissue samples. A, representative histospots showing positive and negative RNF5 expression, which we are presently studying. In this manner, Â RNF5 staining for breast cancer (top) and melanoma (bottom). Insets, 60 RNF5 is expected to affect different cellular targets, which play an magnification of the spots. B, Kaplan-Meier survival curves for RNF5 staining for the entire breast cancer cohort over a 20-y period comparing high and low important role in cytoskeletal organization and function. Because cytoplasmic RNF5 staining. RNF5 was also implicated in the clearance of malfolded proteins, it is likely that RNF5 affects other targets in breast cancer cells than those studied thus far. staining. Spots that were deemed uninterpretable had insufficient It is important to delineate the mechanisms by which RNF5 tumor cells in the spot, loss of tissue in the spot, or an abundance modulates drug-induced apoptosis and regulates the actin of necrotic tissue. Survival information was available for 424 (90%) cytoskeleton and cell proliferation to promote tumor progression. tumor cores. Examples of scores of positive and negative spots To this end, RNF5 may function somewhat similar to TRIM 32, are shown in Fig. 6A. High RNF5 staining was associated with which has been shown to interact with myosin and to ubiquitinate decreased survival (P = 0.0286). Kaplan-Meier survival curves, actin (22). Alternatively, RNF5 activity may affect Rho family of using 20-year follow-up, are shown in Fig. 6B. Using the Cox GTPases, which are among key regulators of actin and microtubule proportional hazards model, we did multivariate analyses. The cytoskeleton (23) and which are often overexpressed or hyperactive prognostic variables used included tumor size, nodal status, in human breast cancers (24). Along these lines, the ubiquitin E3 estrogen receptor staining, progesterone receptor staining, Her2/ ligase Smurf1 targets RhoA for degradation at lamellipodia and neu staining, nuclear grade, and patient age at diagnosis. RNF5 was filopodia and is required for the morphology of transformed 293T not an independent predictor of survival, and the only three cells (25). independent predictors of survival were nodal status, nuclear The tyrosine kinase Src also can alter cell morphology and the grade, and tumor size. actin cytoskeleton during tumorigenesis. A previous study has Correlation between clinicopathologic variables and RNF5 shown that the ubiquitin E3 ligase Hakai ubiquitinates E-cadherin staining was further examined. RNF5 staining was associated with and disrupts cell-cell contacts in epithelial cells in response to a nuclear grade of 2 or 3. Strong RNF5 staining was found in 66% Src activation (26). A recent study has also indicated that Src can of high nuclear grade and 42% of low nuclear grade specimens modulate sensitivity to paclitaxel-induced cytotoxicity by phosphor- (m2 = 13; P = 0.0003) and had no significant associations with the ylating caveolin-1 (27). Whether RNF5 is a regulator or an effector other variables. of a Rho GTPase or a Src signaling network that reorganizes

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. RNF5 in Breast Cancer Progression cytoskeletal architecture and enhances cell proliferation during Because RNF5 was strongly expressed in several different cancers tumorigenesis remains to be determined. and RNF5 levels were higher in metastatic melanoma specimens The loss of RNF5 expression in MCF-7 cells led to a decrease in than primary tumors, increased RNF5 expression may be a cell proliferation and alterations in actin cytoskeleton in response common event during tumor progression. Given its role in to stress but did not affect in either MDA-MB-231, MDA-MB-435, modulating anticancer drug-induced apoptosis and its association or BT-474 cells. This may be attributable to the differences in p53 with clinical prognosis, RNF5 should be further assessed as a status in these cell lines, as only MCF-7 cells express a functional marker and possible drug target in breast cancer. p53 (20, 21). Further supporting this hypothesis was the finding that reducing p53 levels in MCF-7 cells by the expression of E6 Acknowledgments attenuated the effects of RNF5 siRNA on actin cytoskeleton and cell proliferation. MCF-7 cells were also the most sensitized cell line to Received 1/4/2007; revised 5/31/2007; accepted 6/19/2007. Grant support: NIH grants CA97105 (Z. Ronai) and T32 CA88796 (K.D. Bromberg). cytochalasin D–induced apoptosis of the RNF5 siRNA-expressing NIH-NCI shared resources grant 5R24 CA095823-04, NSF Major Research Instrumen- cells. Similar to this effect in breast cancer, p53 status can tation grant DBI-9724504, and NIH shared instrumentation grant 1 S10 RRO 9145-01 modulate the sensitivity of non–small cell lung cancer cells to (Mount Sinai School of Medicine Microscopy Shared Resource Facility). The costs of publication of this article were defrayed in part by the payment of page gefitinib, an epidermal growth factor receptor tyrosine kinase charges. This article must therefore be hereby marked advertisement in accordance inhibitor (28). RNF5 may signal through the above-mentioned Rho with 18 U.S.C. Section 1734 solely to indicate this fact. We thank members of the Ronai laboratory for helpful discussions and Mount Sinai GTPase or Src networks or ERAD to inhibit p53, as loss of RNF5 School of Medicine-Microscopy Shared Resource Facility for confocal laser scanning expression also increased p53 expression in MCF-7 cells (Fig. 5A). microscopy.

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2007 American Association for Cancer Research. Increased Expression of the E3 Ubiquitin Ligase RNF5 Is Associated with Decreased Survival in Breast Cancer

Kenneth D. Bromberg, Harriet M. Kluger, Agnes Delaunay, et al.

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