Metastasis of cancer cells to distant organ systems is a complex process that is initiated with the programming of cells in the primary tumor. The formation of distant metastatic foci is correlated with poor prognosis and limited effective treatment options. We and others have correlated Mouse double minute 2 (Mdm2) with metastasis; however, the mechanisms involved have not been elucidated. Here, it is reported that shRNA-mediated silencing of Mdm2 inhibits epithelial–mesenchymal transition (EMT) and cell migration. In vivo analysis demonstrates that silencing Mdm2 in both post-EMT and basal/triple-negative breast cancers resulted in decreased primary tumor vasculature, circulating tumor cells, and metastatic lung foci. Combined, these results demonstrate the importance of Mdm2 in orchestrating the initial stages of migration and metastasis.

Implication: Mdm2 is the major factor in the initiation of metastasis. Mol Cancer Res; 15(11); 1598–607. ©2017 AACR.

Metastatic foci arise after primary tumor cells successfully transition through various phases of the metastatic process. These phases include invasive migration from the primary tumor, intravasation into the blood stream, extravasation into a distant organ, and colonization in conducive tissues. In response to avascular hypoxic conditions, primary tumor cells may produce VEGF, which stimulates angiogenesis, recruits endothelial cells for neovascularization, and/or permeabilizes existing vasculature through which tumor cells can intravasate. The dissemination of tumor cells to distant organs can be initiated through an intracellular program called epithelial–mesenchymal-transition (EMT). The programming of tumor cells to migrate requires the upregulation of transcription factors, such as Snail Family Zinc Finger 1 (SNAI1), and increased levels of adhesion proteins and filaments, including N-cadherin and vimentin. Conversely, E-cadherin is decreased as cells transition into a mesenchymal phenotype. Moreover, we have shown that the oncogene Mouse double minute 2 (Mdm2) is also positively correlated with EMT (1–5). Clinically, mdm2 gene amplification occurs in approximately 10% of all human cancers, yet more importantly, several studies have detected the Mdm2 protein in 40% to 80% of high-grade human tumors (6, 7). Increased levels of Mdm2 are correlated with poor prognosis, especially in breast cancer (8), and several correlative studies have inferred its involvement in metastasis (9–12). Similarly, we have previously shown that Mdm2 is elevated in 73% of breast carcinomas with known metastases (1).

Although several reports have asserted that Mdm2 promotes metastasis, they were either correlative studies using human patient samples (9, 10, 12), or in vitro studies with or without a correlative component (1, 3–5). Only two studies thus far have explored the importance of Mdm2 in metastasis in vivo. One study, conducted in pancreatic carcinoma cells, found that silencing Mdm2 resulted in decreased proliferation (2). This study, along with another that used tail vein injection of transiently transfected KHT-C murine cells (11), employed approaches that did not directly assess the initiation or other phases of metastasis to define the role of Mdm2. The authors of both studies attributed the differences in metastasis to the ability of Mdm2 to downregulate wild-type p53. Early reports show that Mdm2 can facilitate the destabilization of p53 in response to genotoxic stress; however, 20% to 40% of breast cancers subtypes (13), and 80% of basal-like breast cancers (14) maintain mutant p53 (mutp53). Considering the gain-of-function properties of mutant p53, it might be expected that any downregulation of mutant p53 would result in decreased metastasis. Regardless, unlike p53, mutp53 is unable to induce mdm2 gene expression. We recently demonstrated that the TGFβ1–Smad3/4 axis transcriptionally activates the mdm2 gene independently of p53. Furthermore, the activated TGFβ1 pathway and elevated Mdm2 levels were correlated with human invasive/metastatic breast cancer specimens (1). Here, we show that Mdm2 is necessary for upregulating essential markers of EMT, and consequently, migration/invasion. In addition, we show that Mdm2 is important in upregulating MMP-2 (matrix metalloproteinase 2), an indicator of highly metastatic cancers that colonize the lung. Our orthotopic experiments show that Mdm2 is a key mediator of metastatic lung foci through regulation of angiogenesis and intravasation. These events are independent of mutp53 and demonstrate a distinct role for Mdm2 at specific stages of the metastatic process.

Cell lines, culture conditions, induction of EMT, and Western blotting

shMdm2 was obtained in pLKO from Open Biosystems (TRCN0000003380, which targets exon 12). Additional cell lines expressing shMdm2 constructs (TRCN0000003376 and TRCN0000003377) were also examined to confirm the phenotypes were due to knockdown of Mdm2. Previously described vectors include shp53 (15, 16), MCHERRY (17), and EGFP (17). Lentivirus was produced as described previously (16). Briefly, Lentiviral vectors were packaged in 293T cells using second-generation packaging constructs: pCMV- dR8.74 and pMD2G. Supernatant media containing virus were collected at 36 to 48 hours, supplemented with 4 μg/mL polybrene, filtered through a 0.45-μm filter, and added to MDA-MB-468 (R273H p53) and TMD231 cells [a derivative of MDA-MB-231 cells, which metastasize to the lung (18) and have R280K p53]. Cells were selected for puromycin (2 μg/mL) resistance. All cells were grown as described previously (1). Cells were incubated under normoxia (21% oxygen in complete DMEM), hypoxia (1% oxygen in complete DMEM/F-12 1:1 HyClone), and/or with 10 ng/mL TGFβ1 for the times indicated. Western blot analysis was performed as described by Waning and colleagues (19) using antibodies: Mdm2 [IF2 (Ab-1; OP46), and 4B2 (OP145) from Calbiochem and 2A10 (ab16895) from Abcam; N-cadherin (610920) from BDBioSci]; p53 (DO-1; sc-126), GAPDH (6C5; sc-32233), vinculin (N-19; sc-7649 or E1E9V-13901p from Cell Signaling Technology), E-cadherin (G-10; sc-8426 and 67A4; sc-2179), vimentin (RV202; sc-32322), VEGF (147; sc-507), tubulin (TU-02; sc-8035), and PAI (H-135-sc-8979) from Santa Cruz Biotechnology; Snail (F.31.8- MA5-14801 from Thermo Fisher Scientific); actin (A1978) from Sigma; HIF1α (NBP1-02160) from Novus; and MMP2 (MAB-3308) from Millipore.

Cell line verification.

All cells were tested for mycoplasma with the Mycoplasma Detection Kit (InvivoGen) during the experiments and before injection into animals; TMD231 cells have a mesenchymal morphology and MDA-MB-468 have an epithelial-like morphology, which was verified by microscopy. Molecularly, TMD231 do not express E-cadherin, and MDA-MB-468 express E-cadherin, which is verified by Western blot analysis. The TMD231 cells were generated in-house from MDA-MB-231 cells by Dr. Nakshatri, Indiana University School of Medicine, Indianapolis. MDA-MB-468 cells were obtained from ATCC. All cells were passaged for no more than 3 months between thawing and use in experiments.

Wound healing and invasion assays

Migration of MDA468 cells was assessed using culture inserts (IBIDI) according to the manufacturer's instructions. Briefly, 2.45 × 104 MDA468 shControl cells and 4.9 × 104 MDA468 shMdm2 cells were placed into both wells of the culture insert (separate insert for each cell line). After a 48-hour incubation, inserts were removed, and images were obtained at the indicated time points. Gap area was quantified using the wound healing tool for imageJ (http://dev.mri.cnrs.fr/projects/imagej-macros/wiki/Wound_Healing_Tool). Inserts were manufactured to result in a 500-μm gap. Time 0 hour was imaged at ×5 magnification, and all subsequent time points were imaged at ×10 magnification.

TMD231 mCherry shControl and GFP shMdm2 cells were plated onto coverslips in a 35-mm plate (64,000 cells/plate). Coverslips were then transferred to a 60-mm plate and secured with paramount the next day. Images were obtained immediately and at the times indicated; the number of cells of each color in three separate fields of view were counted and averaged.

Boyden chamber assays were conducted with BD Matrigel Invasion chamber 8.0-μm pore size according to the manufacturer's instructions. Briefly, 2.5 × 104 cells were seeded into each well of Matrigel chambers, with 5% serum used as the chemoattractant. After a 22-hour incubation under normal growth conditions, the media were removed, and the surface of the membrane was wiped with a cotton tip swab to remove noninvading cells. Membranes were then removed from the chambers and stained by incubating in methanol for 2 minutes and then 1% toluidine blue for 2 minutes. Cells were counted in three random fields at a magnification of ×20, and the experiment was done in triplicate. Percent invasion was calculated by dividing the mean number of cells invading through the Matrigel insert membrane by the mean number of cells migrating through the control insert membrane.

Orthotopic implantation in mice and assessment

All animal studies have been approved by Indiana University School of Medicine IACUC (protocol #11190) and were performed by the Indiana University In Vivo Therapeutics Core. A total of 7.5 × 105 TMD 231 shGFP, or shMdm2 cells or 1 × 106 MDA468 shControl or shMdm2 cells were injected into the fourth mammary fat pad of NOD/SCID gamma mice (n = 7/group) obtained from the In Vivo Therapeutics Core of the Indiana University Simon Cancer Center (Indianapolis, IN). Tumor growth was calculated by volumetric analysis using calipers. Tumors were allowed to grow to an average volume of 600 mm3 (TMD231 and MDA468 shControl), 200 mm3 (MDA468 shMdm2), or 900 mm3 (TMD231 in the shp53 experiment).

Circulating tumor cell analysis

Cardiac punctures with 1 mL TB syringe and a 27 g × 0.5 needle (BD Biosciences) were performed at necropsy and temporarily stored in vacutainers coated with sodium heparin (BD Biosciences) prior to peripheral blood mononuclear cells (PBMC) processing. PBMCs were isolated by Ficoll–Paque Plus (GE Healthcare) separation and cells were stained with 1 μg PE-conjugated mouse anti-human EGF receptor antibody (#555997; BD Pharmigen) per 1 × 106 cells. FACS analysis was conducted and data analysis was performed using FlowJo 10.0.8r1. The percentage of circulating tumor cell (CTC) population of each shControl and shMdm2 sample was determined by positive human-specific EGFR staining of CTCs in mouse blood.

Tissue processing and immunostaining

Tissues were fixed overnight at room temperature in 10% neutral-buffered formalin, after which they were transferred through graded concentrations of alcohol to xylene inside a Leica Automated Vacuum Tissue Processor. Samples were embedded in paraffin before being cut into 5-μm thick sections, mounted onto positively charged slides, and baked at 60°C.

The Indiana University Pathology Core performed all tumor preparation, hematoxylin and eosin (H&E) and CD31 staining, and quantitation. Slides were then deparaffinized in xylene and rehydrated through graded alcohols to water. Antigen retrieval was performed by immersing the slides in Target Retrieval Solution (Dako) for 20 minutes at 90°C, cooling at room temperature for 10 minutes, washing in water, and then proceeding with immunostaining. Slides were blocked with protein blocking solution (Dako) for 30 minutes. All subsequent staining steps were performed using the Dako FLEX SYSTEM on an automated Immunostainer; incubations were done at room temperature and Tris buffered saline plus 0.05% Tween 20, pH 7.4 (TBS; Dako Corp.) was used for all washes and diluents. Thorough washing was performed after each incubation. Standard H&E staining was then performed. Alternatively, slides were stained with anti-mouse CD31 (DIA 310; Dianova). Control sections were treated with an isotype control using the same concentration as primary antibodies to verify the staining specificity.

Image capturing

Aperio's whole slide digital imaging system, ScanScope CS, was used for imaging. All slides were imaged at 20×. Digital images were obtained using Aperio's software, ImageScope. For CD31 analysis, whole primary tumor cross-sections were analyzed, with the exception of the excluded necrotic tissue. Vessels less than 125 μm and/or having a diameter less than 30 μm were considered immature. Data from 5 independent hand counts per tumor were confirmed by a pathologist, averaged, and presented as the average percent of immature vessels (the average number of immature vessels divided by the average total vessels in each primary tumor).

Lung metastases quantification

H&E evaluation of lung metastases was performed by a pathologic assistant. All hand counts matched pathological reads performed by a board-certified pathologist. Metastases from all lungs were classified as small (1–25 cells), medium (26–100 cells), large (101–500 cells), or very large (501+). Represented values are averages from each corresponding metastatic size group.

The entire lung of each mouse was scored for metastases and divided by the area of each lung.

The EMT program is regulated by Mdm2

To confirm that Mdm2 is required for metastasis, MDA468 and TMD231 cells were transduced with control and Mdm2-specific shRNA lentiviruses. MDA468 cells, a basal-like cell line, can undergo EMT while TMD231 cells, a derivative of MDA231 cells, are post-EMT and resemble mesenchymal cells. Once the cell lines were confirmed for decreased levels of Mdm2 (Fig. 1A; Supplementary Fig. S1), we analyzed levels of mutp53 as Mdm2 can regulate p53, and many studies have demonstrated that mutp53 promotes tumorigenesis and metastasis (20–38). Interestingly, knockdown of Mdm2 in the MDA468 cells resulted in an increase in mutp53 levels, while no discernable difference was observed when TMD231 shMdm2 and shControl cells were compared (Fig. 1A). We next explored whether shMdm2 cell lines would undergo EMT. The mesenchymal marker, SNAI1, was decreased in both shMdm2 cell lines (Fig. 1B). EMT markers in shControl and shMdm2 MDA468 cells were further examined, and shMdm2 cells were found to molecularly resemble epithelial cells with regards to E-cadherin, N-cadherin, and vimentin (Fig. 1C; Supplementary Fig. S1). Silencing Mdm2 also resulted in a more epithelial-like morphology compared with the shControl cells (Fig. 1D), which is consistent with the low level of EMT markers. MDA468 cells were treated with TGFβ1 and hypoxia to induce EMT. While TGFβ1 treatment of shControl cells resulted in increased levels of various mesenchymal markers and a more mesenchymal morphology, it did not affect the markers or alter the epithelial-like morphology of shMdm2 cells. TGFβ1 treatment of TMD231 cells did not cause EMT, as these cells are morphologically (spindle-shaped) and molecularly (no E-cadherin, high snail, vimentin, N-cadherin) mesenchymal like (Supplementary Fig. S1). Interestingly, despite having increased mutp53 levels, shMdm2 MDA468 cells did not have a mesenchymal phenotype, suggesting a mutant p53-independent role for Mdm2 in EMT. Taken together, these results indicate that Mdm2 plays an important role in regulating key intracellular programing for EMT.

Figure 1.

Silencing of Mdm2 results in decreased levels of EMT markers and mesenchymal morphology. A, Knockdown of Mdm2 was confirmed by Western blot analysis in MDA468 cells and resulted in increased p53 levels. Although silencing Mdm2 in TMD231 cells resulted in decreased Mdm2 levels, p53 levels were not affected. B, SNAI1 levels were also decreased in the shMdm2 cell lines. C, SNAI1, vimentin, and N-cadherin levels were correlated with Mdm2 protein levels, while E-cadherin (an epithelial marker) was inversely correlated with Mdm2 levels in MDA468. TGFβ treatment (for 48 hours with normoxia and 72 hours with hypoxia) resulted in increased EMT marker levels in control cells, but not in shMdm2 MDA468 cells. D, Phase-contrast images showing the morphology of MDA468 cells after 48-hour TGFβ treatment with or without hypoxia as indicated (scale bar, 100 μm). All panels: shControl, shC; shMdm2, sh2; Norm, 21% oxygen; Hyp, 1% oxygen.

Figure 1.

Silencing of Mdm2 results in decreased levels of EMT markers and mesenchymal morphology. A, Knockdown of Mdm2 was confirmed by Western blot analysis in MDA468 cells and resulted in increased p53 levels. Although silencing Mdm2 in TMD231 cells resulted in decreased Mdm2 levels, p53 levels were not affected. B, SNAI1 levels were also decreased in the shMdm2 cell lines. C, SNAI1, vimentin, and N-cadherin levels were correlated with Mdm2 protein levels, while E-cadherin (an epithelial marker) was inversely correlated with Mdm2 levels in MDA468. TGFβ treatment (for 48 hours with normoxia and 72 hours with hypoxia) resulted in increased EMT marker levels in control cells, but not in shMdm2 MDA468 cells. D, Phase-contrast images showing the morphology of MDA468 cells after 48-hour TGFβ treatment with or without hypoxia as indicated (scale bar, 100 μm). All panels: shControl, shC; shMdm2, sh2; Norm, 21% oxygen; Hyp, 1% oxygen.

Close modal

Migration and invasion are impaired in cells with decreased Mdm2

As migration is coupled with EMT, we tested whether Mdm2 affected cellular migration. Initially, we conducted traditional wound healing assays and quantified gap closure (Fig. 2A). Gap closure was dramatically delayed in shMdm2 MDA468 cells (50% at 70 hours) compared with shControl cells (completed at 40 hours). To confirm these data, we also conducted a slight variation of the wound healing assay by examining the migration of fluorescent cells off of coverslips. A GFP-shMdm2 plated coverslip and a mCherry shControl plated coverslip were transferred and fixed on a new plate. Cells that migrated into the void between the coverslips were then counted. This latter technique allowed us to compare shMdm2 and shControl cells under the same conditions. shControl cells were more effective at migration than shMdm2 cells (Fig. 2B). Finally, using a Boyden chamber assay, we demonstrated that shControl cells were able to invade through Matrigel more effectively than shMdm2 cells (Fig. 2C). Thus, using three independent methods, our results show that migration and invasion are diminished with decreased Mdm2.

Figure 2.

shMdm2 cells have impaired migration and invasion. A, Traditional wound healing assay showing that MDA468 shControl cells (left) were able to close the gap in less time compared with shMdm2 cells (right). Note: 0-hour time points were imaged at 5×; all following time points were imaged at 10× (scale bar, 200 μm). Gap area was measured using a wound healing macro for ImageJ (bottom). B, mCherry fluorescent shControl TMD231 cells and GFP fluorescent shMdm2 TMD231 cells were plated on coverslips and transferred to a new plate. Migrating cells of each color were counted in three separate fields of view and averaged. C, shControl and shMdm2 cells were plated in 8-μm Matrigel chambers and allowed to invade for 22 hours using serum as a chemoattractant. Cells were counted in three random ×25 magnification fields and experiments were done in triplicate. Percent invasion was determined as defined in Materials and Methods. All panels: Error bars, ± SEM (n = 3). Significance of differences between shControl and shMdm2 values was determined using unpaired, two-tailed, Student t tests (*, P < 0.05; **, P < 0.01).

Figure 2.

shMdm2 cells have impaired migration and invasion. A, Traditional wound healing assay showing that MDA468 shControl cells (left) were able to close the gap in less time compared with shMdm2 cells (right). Note: 0-hour time points were imaged at 5×; all following time points were imaged at 10× (scale bar, 200 μm). Gap area was measured using a wound healing macro for ImageJ (bottom). B, mCherry fluorescent shControl TMD231 cells and GFP fluorescent shMdm2 TMD231 cells were plated on coverslips and transferred to a new plate. Migrating cells of each color were counted in three separate fields of view and averaged. C, shControl and shMdm2 cells were plated in 8-μm Matrigel chambers and allowed to invade for 22 hours using serum as a chemoattractant. Cells were counted in three random ×25 magnification fields and experiments were done in triplicate. Percent invasion was determined as defined in Materials and Methods. All panels: Error bars, ± SEM (n = 3). Significance of differences between shControl and shMdm2 values was determined using unpaired, two-tailed, Student t tests (*, P < 0.05; **, P < 0.01).

Close modal

MDA468 and TMD231 shMdm2 cells have delayed tumor growth

As cell growth and migration can be interrelated, and shMdm2 cells exhibit a defect in migration, we performed cell-cycle analysis by FACS. Interestingly, although both cell lines have mutp53 (elevated in shMdm2 MDA468), there were no differences in cell-cycle distribution between shControl and shMdm2 (Supplementary Table S1). These data eliminate the role of Mdm2 in regulating gain-of-function mutp53 in cell cycle and migration. To garner insight into the role of Mdm2 in in vivo tumor growth, we orthotopically injected shControl or shMdm2 MDA468 or TMD231 into the mammary fat pad of 6-week-old NSG female mice (n = 7 for each group). The average tumor volumes and weights of shMdm2 MDA468 tumors were lower than shControl tumors (Fig. 3A and B, left), despite allowing them to develop for 30 days longer. This result is notable because mutp53 is thought to enhance tumorigenicity, and its levels are higher in the shMdm2 MDA468 cell line. There were no significant differences between the final TMD231 tumor volumes or weight at the time of euthanasia (Fig. 3A and B, right).

Figure 3.

Knockdown of Mdm2 slows tumor growth. A, Tumor volumes of MDA468 (left) and TMD231 (right) shControl orthotopically implanted animals increased faster than that of shMdm2 implanted mice. B, The final tumor weights of MDA468 shControl tumors were significantly greater than those in the shMdm2 orthotopically implanted animals (left), while the TMD231 set was not significantly different (right). All panels: Error bars, ± SEM (n = 7). Significance of differences between shControl (shC) and shMdm2 (sh2) values was determined using unpaired, two-tailed, Student t tests (ns = not significant; ***, P < 0.001).

Figure 3.

Knockdown of Mdm2 slows tumor growth. A, Tumor volumes of MDA468 (left) and TMD231 (right) shControl orthotopically implanted animals increased faster than that of shMdm2 implanted mice. B, The final tumor weights of MDA468 shControl tumors were significantly greater than those in the shMdm2 orthotopically implanted animals (left), while the TMD231 set was not significantly different (right). All panels: Error bars, ± SEM (n = 7). Significance of differences between shControl (shC) and shMdm2 (sh2) values was determined using unpaired, two-tailed, Student t tests (ns = not significant; ***, P < 0.001).

Close modal

Mdm2 promotes vascularization

Considering the delay in tumor growth in both TMD231 and MDA468 shMdm2 cells, we examined in vitro levels of key factors associated with angiogenesis. We and others have shown that Mdm2 can play a role in stabilizing hypoxia-inducible factor 1α (HIF1α) and upregulating VEGF (39–41). Mdm2 can also stabilize VEGF mRNA levels to promote angiogenesis (42). In accordance with these published findings, there was a substantial decrease in the levels of HIF1α and VEGF in both the cultured TMD231 and MDA468 lines grown under normoxia or hypoxia when Mdm2 was knocked down compared with the shControl (Fig. 4A). On the basis of these in vitro results, primary tumors were sectioned and the endothelial cells were stained with cluster of differentiation 31 (CD31). In the presence of Mdm2, the percentage of newly formed, immature vessels in both MDA468 and TMD231 shControl tumors was significantly higher than shMdm2 tumors (Fig. 4B). We next analyzed lysates from MDA468 and TMD231 shMdm2 tumor sets to confirm the in vitro results. In agreement with the levels seen in cell culture (Fig. 4A), Figure 4C demonstrates diminished HIF1α and VEGF levels in shMdm2 tumors compared with shControl tumors. Taken together, our in vitro and in vivo data are congruent in showing that Mdm2 is integrated into the angiogenesis pathway.

Figure 4.

Mdm2 is important for angiogenesis. A, Western blots of cell lysates from cells grown in either normoxia or hypoxia showing that despite culture conditions, when Mdm2 is knocked down, HIF1α and VEGF levels are decreased. B, Representative images of tumor sections stained brown with CD31 (PECAM-1). The percentages of immature vessels in tumors of both shMdm2 cell lines were significantly lower compared with the respective shControl tumors. The percentages in shp53 tumors, on the other hand, were significantly higher than the shControl tumors (scale bar, 100 μm). C, HIF1α and VEGF levels were lower in the shMdm2 tumors compared with shControl tumors as assessed by Western blot analysis. All panels: shControl, shC; shMdm2, sh2; Norm, 21% oxygen; Hyp, 1% oxygen. Error bars, ± SEM (n = 7). Significance of differences was determined using unpaired, two-tailed, Student t tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 4.

Mdm2 is important for angiogenesis. A, Western blots of cell lysates from cells grown in either normoxia or hypoxia showing that despite culture conditions, when Mdm2 is knocked down, HIF1α and VEGF levels are decreased. B, Representative images of tumor sections stained brown with CD31 (PECAM-1). The percentages of immature vessels in tumors of both shMdm2 cell lines were significantly lower compared with the respective shControl tumors. The percentages in shp53 tumors, on the other hand, were significantly higher than the shControl tumors (scale bar, 100 μm). C, HIF1α and VEGF levels were lower in the shMdm2 tumors compared with shControl tumors as assessed by Western blot analysis. All panels: shControl, shC; shMdm2, sh2; Norm, 21% oxygen; Hyp, 1% oxygen. Error bars, ± SEM (n = 7). Significance of differences was determined using unpaired, two-tailed, Student t tests (*, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

Although our data suggest Mdm2 is an important driver of angiogenesis, previous reports implicate mutp53 is also important for this phenomenon (27, 43–45). Considering that our cells maintain mutp53, we tested the importance of this protein in angiogenesis by implanting TMD231 cells that had been transduced with either shControl or shp53 plasmid (Supplementary Fig. S2). Surprisingly, shp53 tumors had a significantly higher percentage of immature vessels compared with shControl tumors (Fig. 4B). Analysis of tumor extracts revealed that Mdm2 levels were slightly elevated in shp53 tumors (Supplementary Fig. S2). Thus, Mdm2 promotes vascularization of the tumor via regulation of the HIF1α–VEGF axis and possibly permits intravasation of tumor cells into the blood stream.

Abrogation of Mdm2 results in diminished metastasis

Tumor cells undergo intracellular reprograming to signal for cellular migration. To assess whether these changes were evident in our in vivo metastasis models, we examined EMT markers from shMdm2 MDA468 tumor extracts. The results support our in vitro findings of decreased levels of N-cadherin and SNAI1 and increased levels of E-cadherin compared with the shControl tumors (Fig. 5A). Two additional proteins that are associated with metastasis, plasminogen activator inhibitor-1 (PAI-1), an important tumor angiogenic factor involved in metastasis (46), and MMP2, a protein that acts to break down extracellular matrix and is associated with metastatic lung foci (47) were examined by Western blot analysis of tumor extracts. Both PAI-1 and MMP2 were found to be decreased in shMdm2 primary tumors (Fig. 5A).

Figure 5.

Mdm2 is ultimately critical for metastasis. A, Western blots showing that EMT markers and MMP2 are higher in lysates from shControl tumors compared with shMdm2 tumors. E-Cadherin and p53 are more abundant in shMdm2 MDA468 tumors compared with shControl tumors. B, FACS analysis using human-specific EGFR revealed that there was less shMdm2 and more shp53 CTC compared with the respective controls. C, H&E-stained lung sections (left) demonstrating decreased metastasis with the shMdm2 implanted mice compared with those implanted with shControl cells. Only the foci composed of greater than 25 cells were counted for the TMD231 shControl and shp53 implanted animals. All panels: shControl, shC; shMdm2, sh2. Error bars, ± SEM (n = 7). Significance of differences was determined using unpaired, two-tailed, Student t tests (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Figure 5.

Mdm2 is ultimately critical for metastasis. A, Western blots showing that EMT markers and MMP2 are higher in lysates from shControl tumors compared with shMdm2 tumors. E-Cadherin and p53 are more abundant in shMdm2 MDA468 tumors compared with shControl tumors. B, FACS analysis using human-specific EGFR revealed that there was less shMdm2 and more shp53 CTC compared with the respective controls. C, H&E-stained lung sections (left) demonstrating decreased metastasis with the shMdm2 implanted mice compared with those implanted with shControl cells. Only the foci composed of greater than 25 cells were counted for the TMD231 shControl and shp53 implanted animals. All panels: shControl, shC; shMdm2, sh2. Error bars, ± SEM (n = 7). Significance of differences was determined using unpaired, two-tailed, Student t tests (ns, not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001).

Close modal

These data indicate that the tumor cells are primed to intravasate and, with our angiogenesis data in Fig. 4, suggest that shControl tumor cells would enter into the blood stream. We extracted blood from the euthanized animals harboring shControl and shMdm2 tumors and stained for human EGFR to detect CTCs by FACS analysis. We found a greater than 4-fold increase in CTC in animals implanted with MDA468 shControl cells compared with those implanted with the shMdm2 cells (Fig. 5B). Conversely, a greater number of shp53 CTC versus shControl CTC was observed. This result is in accordance with the vasculature data shown in Fig. 4, but contradicts published findings that suggest mutant p53 is driving metastasis (20–23, 25–37, 48–50). Thus, in the presence of Mdm2, tumor cells are priming the vasculature and entering the blood stream.

After establishing that the numbers of tumor cells entering the blood stream were dependent on Mdm2, we determined whether a corresponding change in lung metastatic foci was evident. MDA468 shControl cells readily metastasized to the lung with an average of 1 foci/mm2, whereas the shMdm2 cells averaged only 0.15 foci in the same area. When Mdm2 was silenced, an 85% decrease in lung foci was observed even with elevated mutp53 (Fig. 5C, top). Similarly, the TMD231 shControl cells were significantly more likely to metastasize to the lung than shMdm2 cells (Fig. 5C, middle). Surprisingly, considering the higher number of CTCs in shp53 TMD231 cells, there were less metastatic foci in the lungs compared with control animals (P = 0.0046; Fig. 5C, bottom). These results are supported by the majority of publications implicating a role for mutp53 in metastasis (20–23, 25–37, 48–50); however, mutp53 is dispensable for metastatic initiation, which requires Mdm2.

Here, we demonstrate that Mdm2 is extremely important in breast cancer metastases to the lung. Specifically, we show that Mdm2 is important to promote cancer invasiveness via cell migration, angiogenesis, and intravasation (Fig. 6). Molecularly, Mdm2 stabilizes HIF1α, which increases VEGF and transcription factors associated with intracellular metastatic programming (such as SNAI1; ref. 11). This intracellular pathway increases angiogenesis, and the decrease of vasculature observed with decreased Mdm2 provides one possible explanation for the small tumor size in the MDA468 shMdm2 tumors as well as the delay in tumor growth of TMD231 shMdm2 tumors. Therefore, the abundance of CTCs, and lung metastatic foci is reliant on the presence of Mdm2. Several studies have reported a role for Mdm2 in migration or EMT in vitro. In contrast, our experiments were conducted in vivo, which is significant in light of recent publications delineating the importance of the microenvironment in tumor growth and invasion.

Figure 6.

Mdm2 impacts metastasis in distinct ways. Schematic representing major steps of metastasis and the involvement of Mdm2.

Figure 6.

Mdm2 impacts metastasis in distinct ways. Schematic representing major steps of metastasis and the involvement of Mdm2.

Close modal

In addition to being essential in early metastasis, Mdm2 may also be important in later stages as well. Although extravasation and colonization was not directly assessed, at least one study has shown that tail vein injection (bypassing the initial stages of metastasis) of transiently overexpressing Mdm2 in murine cells results in increased metastatic potential. This suggests that Mdm2 may have a role in extravasation and/or foci formation. Not surprisingly, this study also demonstrated increased metastatic potential with tail vein–injected murine cells that were transiently knocked down for p53. The main difference between this study and our study is that we knocked down gain-of-function p53 and not wild-type p53. The only other in vivo study that demonstrated a role for Mdm2 in metastasis was also in a wild-type p53 background and credited the decreased metastasis on the activation of p53 genes. Furthermore, the knockdown of Mdm2 by RNAi resulted in decreased proliferation and tumor growth, which also contributed to the decrease in metastasis in these wild-type p53 cells.

Our results provide a significant advancement in our understanding of how Mdm2 is regulating the cellular programs to promote tumor metastasis independent of gain-of-function mutp53. If mutp53 was driving tumorigenesis, one would expect the MDA468 shMdm2 tumors (which have significantly more mutp53 than the controls) to be increased in size. However, these cells grew much slower in the implanted animals. Furthermore, when mutp53 was knocked down, the tumors actually had increased neovascularization than the controls. Importantly, we show that the animals implanted with the shp53 cells had higher numbers of CTCs, suggesting that mutp53 is actually a disadvantage in the early stages of metastasis. However, the abundance of shp53 CTC did not correlate with increased numbers of metastatic foci, signifying that mutp53 is important for extravasation or other steps of metastasis. Finally, knocking down mutp53 by shRNA in shMdm2 cells did not alter EMT-related responses, demonstrating that the effects of Mdm2 knockdown are independent of mutp53 levels (Supplementary Fig. S3). Thus, we show there are independent and distinct yet dynamic roles of mutp53 and Mdm2 during the different phases of metastasis.

A major roadblock in the ability to treat metastatic disease is delineating the basic signaling pathways involved in the genesis, survival, drug resistance, migration, and growth of metastatic cells. The effectiveness of new therapies will require a comprehensive approach to target multiple pathways, preferably those that are important before tumor cells reach circulation. On the basis of our work, future drugs should be designed to interrupt the p53-independent functions of Mdm2.

No potential conflicts of interest were disclosed.

Conception and design: P.M. Hauck, C.N. Batuello, L.D. Mayo

Development of methodology: P.M. Hauck, C.N. Batuello, G.E. Sandusky

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): P.M. Hauck, E.R. Wolf, D.J. Olivos III, C.P. McAtarsney, R.M. Cournoyer, G.E. Sandusky, L.D. Mayo

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): P.M. Hauck, E.R. Wolf, D.J. Olivos III, C.N. Batuello, K.C. McElyea, R.M. Cournoyer, G.E. Sandusky, L.D. Mayo

Writing, review, and/or revision of the manuscript: P.M. Hauck, E.R. Wolf, D.J. Olivos III, C.N. Batuello, R.M. Cournoyer, L.D. Mayo

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): K.C. McElyea

Study supervision: L.D. Mayo

The authors thank In Vivo Therapeutics Core for implantation and collection of in vivo data.

This study was supported by NIH/NCI CA172256 and a diversity supplement to this RO1, and Riley Children's Foundation (L.D. Mayo). This work was also supported by NIH: In Vivo Therapeutics Core IUSCC P30 CA082709, T32HL007910 (D.J. Olivos III), and T35HL110854 (R.M. Cournoyer).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Araki
S
,
Eitel
JA
,
Batuello
CN
,
Bijangi-Vishehsaraei
K
,
Xie
XJ
,
Danielpour
D
, et al
TGF-beta1-induced expression of human Mdm2 correlates with late-stage metastatic breast cancer
.
J Clin Invest
2010
;
120
:
290
302
.
2.
Shi
W
,
Meng
Z
,
Chen
Z
,
Hua
Y
,
Gao
H
,
Wang
P
, et al
RNA interference against MDM2 suppresses tumor growth and metastasis in pancreatic carcinoma SW1990HM cells
.
Mol Cell Biochem
2014
;
387
:
1
8
.
3.
Chen
X
,
Qiu
J
,
Yang
D
,
Lu
J
,
Yan
C
,
Zha
X
, et al
MDM2 promotes invasion and metastasis in invasive ductal breast carcinoma by inducing matrix metalloproteinase-9
.
PLoS One
2013
;
8
:
e78794
.
4.
Polanski
R
,
Warburton
HE
,
Ray-Sinha
A
,
Devling
T
,
Pakula
H
,
Rubbi
CP
, et al
MDM2 promotes cell motility and invasiveness through a RING-finger independent mechanism
.
FEBS Lett
2010
;
584
:
4695
702
.
5.
Yang
JY
,
Zong
CS
,
Xia
W
,
Wei
Y
,
Ali-Seyed
M
,
Li
Z
, et al
MDM2 promotes cell motility and invasiveness by regulating E-cadherin degradation
.
Mol Cell Biol
2006
;
26
:
7269
82
.
6.
Onel
K
,
Cordon-Cardo
C
. 
MDM2 and prognosis
.
Mol Cancer Res
2004
;
2
:
1
8
.
7.
Rayburn
E
,
Zhang
R
,
He
J
,
Wang
H
. 
MDM2 and human malignancies: expression, clinical pathology, prognostic markers, and implications for chemotherapy
.
Curr Cancer Drug Targets
2005
;
5
:
27
41
.
8.
Turbin
DA
,
Cheang
MC
,
Bajdik
CD
,
Gelmon
KA
,
Yorida
E
,
De Luca
A
, et al
MDM2 protein expression is a negative prognostic marker in breast carcinoma
.
Mod Pathol
2006
;
19
:
69
74
.
9.
Ladanyi
M
,
Cha
C
,
Lewis
R
,
Jhanwar
SC
,
Huvos
AG
,
Healey
JH
. 
MDM2 gene amplification in metastatic osteosarcoma
.
Cancer Res
1993
;
53
:
16
8
.
10.
Ladanyi
M
,
Lewis
R
,
Jhanwar
SC
,
Gerald
W
,
Huvos
AG
,
Healey
JH
. 
MDM2 and CDK4 gene amplification in Ewing's sarcoma
.
J Pathol
1995
;
175
:
211
7
.
11.
Zhang
L
,
Hill
RP
. 
Hypoxia enhances metastatic efficiency by up-regulating Mdm2 in KHT cells and increasing resistance to apoptosis
.
Cancer Res
2004
;
64
:
4180
9
.
12.
Zhang
P
,
Wu
SK
,
Wang
Y
,
Fan
ZX
,
Li
CR
,
Feng
M
, et al
p53, MDM2, eIF4E and EGFR expression in nasopharyngeal carcinoma and their correlation with clinicopathological characteristics and prognosis: a retrospective study
.
Oncol Lett
2015
;
9
:
113
8
.
13.
Tennis
M
,
Krishnan
S
,
Bonner
M
,
Ambrosone
CB
,
Vena
JE
,
Moysich
K
, et al
p53 Mutation analysis in breast tumors by a DNA microarray method
.
Cancer Epidemiol Biomarkers Prev
2006
;
15
:
80
5
.
14.
The Cancer Genome Atlas Research Network
. 
Comprehensive molecular portraits of human breast tumours
.
Nature
2012
;
490
:
61
70
.
15.
Brummelkamp
TR
,
Bernards
R
,
Agami
R
. 
A system for stable expression of short interfering RNAs in mammalian cells
.
Science
2002
;
296
:
550
3
.
16.
Patton
JT
,
Mayo
LD
,
Singhi
AD
,
Gudkov
AV
,
Stark
GR
,
Jackson
MW
. 
Levels of HdmX expression dictate the sensitivity of normal and transformed cells to Nutlin-3
.
Cancer Res
2006
;
66
:
3169
76
.
17.
Shannon
HE
,
Fishel
ML
,
Xie
J
,
Gu
D
,
McCarthy
BP
,
Riley
AA
, et al
Longitudinal bioluminescence imaging of primary versus abdominal metastatic tumor growth in orthotopic pancreatic tumor models in NSG Mice
.
Pancreas
2015
;
44
:
64
75
.
18.
Burnett
RM
,
Craven
KE
,
Krishnamurthy
P
,
Goswami
CP
,
Badve
S
,
Crooks
P
, et al
Organ-specific adaptive signaling pathway activation in metastatic breast cancer cells
.
Oncotarget
2015
;
6
:
12682
96
.
19.
Waning
DL
,
Lehman
JA
,
Batuello
CN
,
Mayo
LD
. 
c-Abl phosphorylation of Mdm2 facilitates Mdm2-Mdmx complex formation
.
J Biol Chem
2011
;
286
:
216
22
.
20.
Arjonen
A
,
Kaukonen
R
,
Mattila
E
,
Rouhi
P
,
Hognas
G
,
Sihto
H
, et al
Mutant p53-associated myosin-X upregulation promotes breast cancer invasion and metastasis
.
J Clin Invest
2014
;
124
:
1069
82
.
21.
Coffill
CR
,
Muller
PA
,
Oh
HK
,
Neo
SP
,
Hogue
KA
,
Cheok
CF
, et al
Mutant p53 interactome identifies nardilysin as a p53R273H-specific binding partner that promotes invasion
.
EMBO Rep
2012
;
13
:
638
44
.
22.
Dong
P
,
Karaayvaz
M
,
Jia
N
,
Kaneuchi
M
,
Hamada
J
,
Watari
H
, et al
Mutant p53 gain-of-function induces epithelial-mesenchymal transition through modulation of the miR-130b-ZEB1 axis
.
Oncogene
2013
;
32
:
3286
95
.
23.
Dong
P
,
Tada
M
,
Hamada
J
,
Nakamura
A
,
Moriuchi
T
,
Sakuragi
N
. 
p53 dominant-negative mutant R273H promotes invasion and migration of human endometrial cancer HHUA cells
.
Clin Exp Metastasis
2007
;
24
:
471
83
.
24.
Dong
P
,
Xu
Z
,
Jia
N
,
Li
D
,
Feng
Y
. 
Elevated expression of p53 gain-of-function mutation R175H in endometrial cancer cells can increase the invasive phenotypes by activation of the EGFR/PI3K/AKT pathway
.
Mol Cancer
2009
;
8
:
103
.
25.
Hsiao
M
,
Low
J
,
Dorn
E
,
Ku
D
,
Pattengale
P
,
Yeargin
J
, et al
Gain-of-function mutations of the p53 gene induce lymphohematopoietic metastatic potential and tissue invasiveness
.
Am J Pathol
1994
;
145
:
702
14
.
26.
Kogan-Sakin
I
,
Tabach
Y
,
Buganim
Y
,
Molchadsky
A
,
Solomon
H
,
Madar
S
, et al
Mutant p53(R175H) upregulates Twist1 expression and promotes epithelial-mesenchymal transition in immortalized prostate cells
.
Cell Death Differ
2011
;
18
:
271
81
.
27.
Lenfert
E
,
Maenz
C
,
Heinlein
C
,
Jannasch
K
,
Schumacher
U
,
Pantel
K
, et al
Mutant p53 promotes epithelial-mesenchymal plasticity and enhances metastasis in mammary carcinomas of WAP-T mice
.
Int J Cancer
2015
;
136
:
E521
33
.
28.
Mak
AS
. 
p53 in cell invasion, podosomes, and invadopodia
.
Cell Adh Migr
2014
;
8
:
205
14
.
29.
Morton
JP
,
Timpson
P
,
Karim
SA
,
Ridgway
RA
,
Athineos
D
,
Doyle
B
, et al
Mutant p53 drives metastasis and overcomes growth arrest/senescence in pancreatic cancer
.
Proc Natl Acad Sci U S A
2010
;
107
:
246
51
.
30.
Muller
PA
,
Caswell
PT
,
Doyle
B
,
Iwanicki
MP
,
Tan
EH
,
Karim
S
, et al
Mutant p53 drives invasion by promoting integrin recycling
.
Cell
2009
;
139
:
1327
41
.
31.
Muller
PA
,
Vousden
KH
,
Norman
JC
. 
p53 and its mutants in tumor cell migration and invasion
.
J Cell Biol
2011
;
192
:
209
18
.
32.
Neilsen
PM
,
Noll
JE
,
Mattiske
S
,
Bracken
CP
,
Gregory
PA
,
Schulz
RB
, et al
Mutant p53 drives invasion in breast tumors through up-regulation of miR-155
.
Oncogene
2013
;
32
:
2992
3000
.
33.
Noll
JE
,
Jeffery
J
,
Al-Ejeh
F
,
Kumar
R
,
Khanna
KK
,
Callen
DF
, et al
Mutant p53 drives multinucleation and invasion through a process that is suppressed by ANKRD11
.
Oncogene
2012
;
31
:
2836
48
.
34.
Subramanian
M
,
Francis
P
,
Bilke
S
,
Li
XL
,
Hara
T
,
Lu
X
, et al
A mutant p53/let-7i-axis-regulated gene network drives cell migration, invasion and metastasis
.
Oncogene
2015
;
34
:
1094
104
.
35.
Weissmueller
S
,
Manchado
E
,
Saborowski
M
,
Morris
JP
,
Wagenblast
E
,
Davis
CA
, et al
Mutant p53 drives pancreatic cancer metastasis through cell-autonomous PDGF receptor beta signaling
.
Cell
2014
;
157
:
382
94
.
36.
Yeudall
WA
,
Vaughan
CA
,
Miyazaki
H
,
Ramamoorthy
M
,
Choi
MY
,
Chapman
CG
, et al
Gain-of-function mutant p53 upregulates CXC chemokines and enhances cell migration
.
Carcinogenesis
2012
;
33
:
442
51
.
37.
Adorno
M
,
Cordenonsi
M
,
Montagner
M
,
Dupont
S
,
Wong
C
,
Hann
B
, et al
A mutant-p53/Smad complex opposes p63 to empower TGFbeta-induced metastasis
.
Cell
2009
;
137
:
87
98
.
38.
Lang
GA
,
Iwakuma
T
,
Suh
YA
,
Liu
G
,
Rao
VA
,
Parant
JM
, et al
Gain of function of a p53 hot spot mutation in a mouse model of Li-Fraumeni syndrome
.
Cell
2004
;
119
:
861
72
.
39.
Bardos
JI
,
Chau
NM
,
Ashcroft
M
. 
Growth factor-mediated induction of HDM2 positively regulates hypoxia-inducible factor 1alpha expression
.
Mol Cell Biol
2004
;
24
:
2905
14
.
40.
Carroll
VA
,
Ashcroft
M
. 
Regulation of angiogenic factors by HDM2 in renal cell carcinoma
.
Cancer Res
2008
;
68
:
545
52
.
41.
LaRusch
GA
,
Jackson
MW
,
Dunbar
JD
,
Warren
RS
,
Donner
DB
,
Mayo
LD
. 
Nutlin3 blocks vascular endothelial growth factor induction by preventing the interaction between hypoxia inducible factor 1alpha and Hdm2
.
Cancer Res
2007
;
67
:
450
4
.
42.
Zhou
S
,
Gu
L
,
He
J
,
Zhang
H
,
Zhou
M
. 
MDM2 regulates vascular endothelial growth factor mRNA stabilization in hypoxia
.
Mol Cell Biol
2011
;
31
:
4928
37
.
43.
Kieser
A
,
Weich
HA
,
Brandner
G
,
Marme
D
,
Kolch
W
. 
Mutant p53 potentiates protein kinase C induction of vascular endothelial growth factor expression
.
Oncogene
1994
;
9
:
963
9
.
44.
Koura
A
,
Vangolen
K
,
Tsan
R
,
Radinsky
R
,
Price
J
,
Ellis
L
. 
Regulation of genes associated with angiogenesis, growth, and metastasis by specific p53 point mutations in a murine melanoma cell line
.
Oncol Rep
1997
;
4
:
475
9
.
45.
Perryman
LA
,
Blair
JM
,
Kingsley
EA
,
Szymanska
B
,
Ow
KT
,
Wen
VW
, et al
Over-expression of p53 mutants in LNCaP cells alters tumor growth and angiogenesis in vivo
.
Biochem Biophys Res Commun
2006
;
345
:
1207
14
.
46.
Chen
H
,
Peng
H
,
Liu
W
,
Sun
Y
,
Su
N
,
Tang
W
, et al
Silencing of plasminogen activator inhibitor-1 suppresses colorectal cancer progression and liver metastasis
.
Surgery
2015
;
158
:
1704
13
.
47.
Minn
AJ
,
Gupta
GP
,
Siegel
PM
,
Bos
PD
,
Shu
W
,
Giri
DD
, et al
Genes that mediate breast cancer metastasis to lung
.
Nature
2005
;
436
:
518
24
.
48.
Ali
A
,
Shah
AS
,
Ahmad
A
. 
Gain-of-function of mutant p53: mutant p53 enhances cancer progression by inhibiting KLF17 expression in invasive breast carcinoma cells
.
Cancer Lett
2014
;
354
:
87
96
.
49.
Roger
L
,
Jullien
L
,
Gire
V
,
Roux
P
. 
Gain of oncogenic function of p53 mutants regulates E-cadherin expression uncoupled from cell invasion in colon cancer cells
.
J Cell Sci
2010
;
123
:
1295
305
.
50.
Wang
SP
,
Wang
WL
,
Chang
YL
,
Wu
CT
,
Chao
YC
,
Kao
SH
, et al
p53 controls cancer cell invasion by inducing the MDM2-mediated degradation of Slug
.
Nat Cell Biol
2009
;
11
:
694
704
.