Three-dimensional (3D) cell culture techniques are frequently used to model alterations in tissue architecture critically important for tumor development. Here, we report on a detailed comparison of a spheroid model of human epidermal growth factor receptor (HER2) overexpressing cancer cells with the traditional monolayer culture. In 2D culture, HER2 and HER3 form heterodimers, whereas in multicellular spheroids HER2 homodimers are formed. These homodimers localize in membrane rafts, resulting in enhanced inhibition of the proliferation of cancer cells with trastuzumab (Herceptin), a monoclonal antibody specifically targeting HER2. Within the tumor spheroids, HER2 homodimerization leads to enhanced activation of HER2 and results in a switch in signaling pathways from phosphoinositide 3-kinase (PI3K) to mitogen-activated protein kinase (MAPK). Diminished PI3K signaling is accompanied by the activation of the integrin β4/Rac1/PAK 2 signaling cascade. We propose that the described 3D culture system may better reflect some in vivo aspects of HER signaling and can be used to further improve the understanding of the molecular mechanisms of trastuzumab action. Furthermore, the described human multicellular tumor spheroids may allow identification of new targets for the treatment of HER2-positive breast cancer patients who currently benefit suboptimally from trastuzumab treatment.
The human epidermal growth factor receptor (HER) family consists of four receptor tyrosine kinases (epidermal growth factor receptor (EGFR), HER2, HER3 and HER4), which regulate cell survival, proliferation, differentiation and migration, and have been implicated in oncogenesis (Yarden, 2001). Several studies have shown that HER2-mediated signal transduction is induced by homodimerization or heterodimerization with other HER family members (Yarden, 2001).
HER2 gene amplification and protein overexpression have been identified in different tumor types (Wang and Hung, 2001), including 15–20% of breast tumors in which it is associated with poor prognosis (Owens et al., 2004; Yaziji et al., 2004; Wolff et al., 2007). Trastuzumab (Herceptin) is a humanized recombinant monoclonal antibody that offers a significant clinical benefit for patients with HER2-positive disease when administered either as monotherapy or in combination with chemotherapy (Hudis, 2007).
When investigating the role of HER2 in oncogenesis, the in vitro model system used is of fundamental importance, as studies have shown that the molecular behavior of cells can be altered in three-dimensional (3D) compared with two-dimensional (2D) culture (Shaw et al., 2004; Lee et al., 2007). 3D cell culture models of breast neoplasm were developed to investigate the aspects of cell–cell, cell–matrix interaction and loss of attachment to substratum in cancer development, and thus resemble a physiologically relevant in vitro model to investigate tumor development and behavior (Petersen et al., 1992; Yamada and Cukierman, 2007). These spheroids have since been used to investigate cellular signaling mechanisms and the effects of agent such as trastuzumab on neoplastic cell proliferation (Shaw et al., 2004; Lee et al., 2007; Ritter et al., 2007). Traditionally, HER2 signaling pathways and the effects of trastuzumab have been dissected using 2D culture systems. Earlier 3D culture systems used to investigate cell signaling in tumor cells have relied on an exogenously provided extracellular matrix (ECM) (Petersen et al., 1992; Lee et al., 2007). Our 3D model relied on anchorage-independent growth, in which tumor cells aggregate spontaneously, without supplying an exogenous ECM (Shin et al., 1975). Here, we use, for the first time, an anchorage-independent 3D model in comparison with traditional monolayer culture to specifically investigate endogenous HER2 signaling in cancer cells and elucidate the molecular action of trastuzumab on breast cancer cells in vitro.
In 2D culture of SKBR-3 cells, treatment with trastuzumab (10 μg/ml) for 7 days induced a slight reduction in proliferation, whereas in 3D, spheroid proliferation was inhibited by 48% (Figure 1a), mirroring the clinical benefit of trastuzumab for HER2-positive breast cancer patients (Slamon et al., 1989). This inhibition was not because of stress conditions induced by the culture system, as data from an independent proliferation assay showed that SKBR-3 cells grew well in 3D culture (data not shown). Similar results were observed for the ovarian tumor line SKOV-3, indicating that these effects are not restricted to a specific cell line (Figure 1a). Inhibition of the proliferation in 2D culture depends on the confluency of the monolayer (data not shown). Furthermore, Hoechst 33342 staining of SKBR-3 cells in multicellular spheroids showed that the antiproliferative effects of trastuzumab were evident across the total cell population (Figure 1b). To further investigate the effects of trastuzumab on 3D culture of SKBR-3 cells, morphologic analysis of cell aggregation in the presence or absence of trastuzumab (10 μg/ml) was performed at different time points after treatment. Despite inhibiting the proliferation of SKBR-3 cells, trastuzumab did not interfere with spheroid formation (Figures 1b and c). Similar results were obtained using the SKOV-3 cell line, although a comparison of the cell morphology of both cell types grown in 2D and 3D undergoing trastuzumab treatment showed that in contrast to SKBR-3 cells, SKOV-3 cells formed very tight aggregates in 3D culture (Figure 1c).
As a significantly enhanced response to trastuzumab treatment in spheroids was observed, we investigated whether the HER2 signaling pathway differed in 2D culture. Interestingly, we detected a significantly enhanced phosphorylation of HER2, which was induced simply by culturing SKBR-3 cells in polyHEMA-coated plates. Levels of phosphorylated HER3 and EGFR were increased in multicellular spheroids, whereas total levels of HER2 and HER3 were comparable under both culture conditions. Total EGFR was slightly reduced in 3D culture (Figure 2a). mRNA expression of HER-family ligands did not differ significantly (data not shown). Treatment with trastuzumab substantially reduced HER2 and HER3 phosphorylation (Figure 2a). As HER2-mediated signaling has been associated with both the alpha serine/threonine protein kinase (Akt)/phosphoinositide 3-kinase (PI3K) (Zhou et al., 2004) and mitogen-activated protein kinase (MAPK) (Timms et al., 2002) signaling pathways, we evaluated the activation of these pathways after enhanced HER phosphorylation. Activated Akt was detected in 2D culture, but downregulated in 3D culture (Figure 2b). In contrast to the downregulation of Akt, levels of activated MEK1/2 and MAPK were enhanced in tumor spheroids (Figure 2b). Increased HER2 phosphorylation in 3D and a similar shift in signaling pathway activities between 2D and 3D cultures were observed in several other cell lines, including SKOV-3 (Figure 2c), BT474, Calu-3 and KPL-4 (Supplementary Figure). Treatment with trastuzumab reduced phosphorylation of MEK1/2 and MAPK in 3D culture (Figure 2b) compared with earlier studies, performed with 2D monolayer cultures, which focused on trastuzumab effects on the Akt/PI3K-mediated pathway (Yakes et al., 2002; Longva et al., 2005). The revelation that the Akt signaling pathway, is less activated in 3D spheroids, may have profound implications for future research and confirms the importance of this model system. Consistent with earlier data (Longva et al., 2005), Akt phosphorylation was not inhibited by trastuzumab in SKOV-3 cells, suggesting that a mechanism exists to reduce cellular response to trastuzumab (Nahta and Esteva, 2006).
Integrins regulate different cellular functions by mediating adhesion to an ECM, inducing cytoskeletal rearrangements and activating intracellular signaling pathways, including p21-activated kinase (PAK) (Schoenwaelder and Burridge, 1999). Integrin β4 phosphorylation was significantly increased in SKBR-3 cells grown in 3D culture for 48 h without the presence of an ECM (Figure 2d). Guo et al., (2005) reported earlier that integrin β4 signaling was necessary for tumor cell proliferation and suppression of apoptosis at the onset of HER2-initiated mammary tumorigenesis, but that these effects were not solely the result of ECM ligand binding to integrin β4 . Mammary tumors expressing wild-type integrin β4 lacked signs of tissue organization, which is similar to our in vitro 3D culture model. This is in contrast to the study by Weaver et al. (2002), in which the focus was on polarized 3D tumor models . Analysis of the integrin β4 downstream targets Rac1 and PAK showed that in 2D culture, Rac1 was highly phosphorylated at serine 71, a target phosphorylation site by which Akt kinase inactivates Rac1 (Kwon et al., 2000). No phosphorylation of Rac1 was detectable in the spheroids; instead, there was a significant phosphorylation of PAK2 at serine 141, which was not inhibited by trastuzumab (Figure 2d). This residue is required for full PAK2 activation and is consistent with Rac1 activity (Jung and Traugh, 2005). These observations indicate that the Rac1/PAK2 signaling pathway could serve as a compensating survival pathway for SKBR-3 cells in 3D culture, in which only low levels of phosphorylated Akt are present (Figure 2b).
Next, the nature of the HER2 signaling complex in 3D culture was investigated by analyzing proteins that co-immunoprecipitated with HER2. Significantly lower HER3 levels and concordantly lower levels of PI3K co-precipitated with HER2 in 3D when compared with 2D culture (Figure 3a). As HER3 is a major adaptor protein for recruiting PI3K to HER2 heterodimers, these results indicate a reduction in HER2/HER3 heterodimerization. In contrast, increased levels of Grb2 and Shc (MAPK pathway upstream signaling molecules) were co-precipitated with HER2 in 3D cultures (Figure 3a), providing further evidence of a switch in signaling pathways downstream of HER2. This switch is likely to be associated with the reduction or loss of HER2/HER3 heterodimerization. A time-dependent loss of Shc and Grb2 co-precipitates from 3D culture samples was observed after trastuzumab treatment (Figure 3b). These results suggest that HER2 forms homodimers in 3D culture. Trastuzumab binds to HER2 homodimers and inhibits the activation of these pathways, thus inhibiting cell proliferation (Hudziak et al., 1989). A transient increase in EGFR co-precipitation with HER2 was observed at earlier time points (4–8 h) in 3D culture, but after 24 h this difference was not discernable (Figures 3a and b). Transient HER2 co-precipitation with EGFR coincided with cell aggregation, suggesting that HER2/EGFR heterodimerization resulted from reseeding and reattachment during aggregation.
The mechanism by which HER2 overexpression contributes to oncogenesis has been the subject of several studies. The physical proximity of HER2 molecules on the cell surface is a significant factor and HER2-rich domains have been shown to colocalize with membrane rafts in the SKBR-3 breast cancer cell line (Nagy et al., 2002; Mocanu et al., 2005; Sottocornola et al., 2006). Membrane rafts are small, dynamic and lipid-rich regions that compartmentalize cellular processes (Pike, 2006). These are stabilized by gangliosides (glycophospholipids), which play a key role in modulating transmembrane signaling (Miljan and Bremer, 2002). When HER2 was activated by the HER3 ligand EGF and heregulin in SKBR-3 cells, an increase in the number of HER2 proteins recruited to membrane rafts was observed (Nagy et al., 2002). There is also an evidence that HER2 activity can be modulated by the ganglioside GM3 (Sottocornola et al., 2003) and by caveolin 1, a protein found in specialized plasma membrane-associated membrane rafts called caveolae (Engelman et al., 1998).
An earlier study showed that HER2 is weakly associated with membrane rafts in 2D culture of SKBR-3 cells, as crosslinking with cholera toxin B-chain (CT-B) diminished the membrane raft association of HER2 (Nagy et al., 2002; Mocanu et al., 2005). CT-B binds selectively to the GM1 ganglioside, a structural component of lipid rafts and caveolae. Both GM1 and GM3 are known to modulate HER2 homodimerization and heterodimerization activity (Nagy et al., 2002; Sottocornola et al., 2003). It is currently unknown how gangliosides modulate the dimerization of HER2, but earlier studies suggest that they may retain HER2 within the lipid raft, thereby modulating the local density of HER proteins and increasing the likelihood of HER heterodimerization and homodimerization and activation (Nagy et al., 2002; Sottocornola et al., 2003). To determine whether HER2 colocalized with membrane rafts in 3D culture, SKBR-3 cells were labeled in parallel with CT-B conjugated with the Alexa 488 fluorochrome and trastuzumab. Confocal microscopy of stained cells showed that HER2 colocalized with GM1 ganglioside in 3D cell culture but there was no significant colocalization in 2D cell culture after crosslinking with CT-B (Figure 4a). These experiments indicate that HER2 is more tightly associated with membrane rafts in 3D than in 2D culture.
To interfere with the assembly of lipid rafts, we used threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride, an inhibitor of ganglioside biosynthesis. Threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol hydrochloride treatment depleted cell surface GM1 by 50% in 3D culture (data not shown) and reduced the levels of phosphorylated HER2 in a dose-dependent manner (Figure 4b). HER2 phosphorylation was restored with addition of exogenous ganglioside GM3, corroborating the relationship between HER2 activation and membrane raft association (Figure 4b). It is unclear how trastuzumab affects HER2 localization in these lipid rafts. Caveolin 1 was detected in cell extracts from 2D but not from 3D SKBR-3 cultures (Figure 4b) and was unaffected by trastuzumab (data not shown). Furthermore, caveolin 1 mRNA levels were downregulated in spheroids compared with 2D culture of SKBR-3 cells and caveolin-1 protein levels were reduced to differing degrees in 3D culture of additional cancer cell lines (data not shown). Caveolin 1, a main structural component of caveolae, has also been implicated in tumorigenesis, and various human tumors and cancer cell lines exhibit a decrease or loss of caveolin 1 expression (Razani et al., 2001). Caveolin 1 is potentially a key modulator of HER2 activity, possibly by interacting directly with HER2 or by localizing with HER2 in the caveolae. However, in tumor cells, caveolin 1 downregulation coupled with the localization of HER2 within membrane rafts may diminish the regulatory ability of caveolin 1 in relation to HER2.
Our results show a clear difference in HER2 molecular signaling in cells cultured in 2D versus 3D, which correlates with other studies using 3D cell culture models. These have highlighted differences in the behavior of cells cultured in this manner (Shaw et al., 2004; Lee et al., 2007), possibly as a result of the different architectural phenotype of the culture system (Shaw et al., 2004). We suggest that the reorganization of the cell surface that occurs in 3D culture induces changes in protein association and localization, offering alternative signaling platforms that may not be available in 2D culture. These analyses of 2D versus 3D culture underline the flexibility of oncogene switching between HER receptor family members, which may play an important role in the development of drug resistance.
Engelman JA, Lee RJ, Karnezis A, Bearss DJ, Webster M, Siegel P et al. (1998). Reciprocal regulation of neu tyrosine kinase activity and caveolin-1 protein expression in vitro and in vivo. Implications for human breast cancer. J Biol Chem 273: 20448–20455.
Guo H, Wei B, Zhang HY, Liu GJ, Bu H, Lang ZQ et al. (2005). [HER2 expression and its prognostic implication in lymph node negative breast carcinoma: a meta-analysis]. Zhonghua Bing Li Xue Za Zhi 34: 140–146.
Hudis CA . (2007). Trastuzumab—mechanism of action and use in clinical practice. N Engl J Med 357: 39–51.
Hudziak RM, Lewis GD, Winget M, Fendly BM, Shepard HM, Ullrich A . (1989). p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol Cell Biol 9: 1165–1172.
Jung J-H, Traugh JA . (2005). Regulation of the interaction of Pak2 with Cdc42 via autophosphorylation of serine 141. J Biol Chem 280: 40025–40031.
Kwon T, Kwon DY, Chun J, Kim JH, Kang SS . (2000). Akt protein kinase inhibits Rac1-GTP binding through phosphorylation at serine 71 of Rac1. J Biol Chem 275: 423–428.
Lee GY, Kenny PA, Lee EH, Bissell MJ . (2007). Three-dimensional culture models of normal and malignant breast epithelial cells. Nat Methods 4: 359–365.
Longva KE, Pedersen NM, Haslekås C, Stang E, Madshus IH . (2005). Herceptin-induced inhibition of ErbB2 signaling involves reduced phosphorylation of Akt but not endocytic down-regulation of ErbB2. Int J Cancer 116: 359–367.
Miljan EA, Bremer EG . (2002). Regulation of growth factor receptors by gangliosides. Sci STKE 2002: RE15.
Mocanu M-M, Fazekas Z, Petrás M, Nagy P, Sebestyén Z, Isola J et al. (2005). Associations of ErbB2, β1-integrin and lipid rafts on Herceptin (Trastuzumab) resistant and sensitive tumor cell lines. Cancer Lett 227: 201–212.
Nagy P, Vereb G, Sebestyén Z, Horváth G, Lockett SJ, Damjanovich S et al. (2002). Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2. J Cell Sci 115: 4251–4262.
Nahta R, Esteva FJ . (2006). Herceptin: mechanisms of action and resistance. Cancer Lett 232: 123–138.
Owens MA, Horten BC, Da Silva MM . (2004). HER2 amplification ratios by fluorescence in situ hybridization and correlation with immunohistochemistry in a cohort of 6556 breast cancer tissues. Clin Breast Cancer 5: 63–69.
Petersen OW, Rønnov-Jessen L, Howlett AR, Bissell MJ . (1992). Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proc Natl Acad Sci USA 89: 9064–9068.
Pike LJ . (2006). Rafts defined: a report on the Keystone symposium on lipid rafts and cell function. J Lipid Res 47: 1597–1598.
Razani B, Schlegel A, Liu J, Lisanti MP . (2001). Caveolin-1, a putative tumour suppressor gene. Biochem Soc Trans 29: 494–499.
Ritter CA, Perez-Torres M, Rinehart C, Guix M, Dugger T, Engelman JA et al. (2007). Human breast cancer cells selected for resistance to trastuzumab in vivo overexpress epidermal growth factor receptor and ErbB ligands and remain dependent on the ErbB receptor network. Clin Cancer Res 13: 4909–4919.
Schoenwaelder SM, Burridge K . (1999). Bidirectional signaling between the cytoskeleton and integrins. Curr Opin Cell Biol 11: 274–286.
Shaw KRM, Wrobel CN, Brugge JS . (2004). Use of three-dimensional basement membrane cultures to model oncogene-induced changes in mammary epithelial morphogenesis. J Mammary Gland Biol Neoplasia 9: 297–310.
Shin S-I, Freedman VH, Risser R, Pollack R . (1975). Tumorigenicity of virus-transformed cells in nude mice is correlated specifically with anchorage independent growth in vitro. Proc Natl Acad Sci USA 72: 4435–4439.
Slamon DJ, Godolphin W, Jones LA, Holt JA, Wong SG, Keith DE et al. (1989). Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 244: 707–712.
Sottocornola E, Berra B, Colombo I . (2003). GM3 content modulates the EGF-activated p185c-neu levels, but not those of the constitutively activated oncoprotein p185neu. Biochim Biophys Acta 1635: 55–66.
Sottocornola E, Misasi R, Mattei V, Ciarlo L, Gradini R, Garofalo T et al. (2006). Role of gangliosides in the association of ErbB2 with lipid rafts in mammary epithelial HC11 cells. FEBS J 273: 1821–1830.
Timms JF, White SL, O’Hare MJ, Waterfield MD . (2002). Effects of ErbB-2 overexpression on mitogenic signalling and cell cycle progression in human breast luminal epithelial cells. Oncogene 21: 6573–6586.
Wang SC, Hung MC . (2001). HER2 overexpression and cancer targeting. Semin Oncol 28: 115–124.
Weaver VM, Lelièvre S, Lakins JN, Chrenek MA, Jones JCR, Giancotti F et al. (2002). β4 integrin-dependent formation of polarized three-dimensional architecture confers resistance to apoptosis in normal and malignant mammary epithelium. Cancer Cell 2: 205–216.
Wolff AC, Hammond MEH, Schwartz JN, Hagerty KL, Allred DC, Cote RJ et al. (2007). American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. J Clin Oncol 25: 118–145.
Yakes FM, Chinratanalab W, Ritter CA, King W, Seelig S, Arteaga CL . (2002). Herceptin-induced inhibition of phosphatidylinositol-3 kinase and Akt is required for antibody-mediated effects on p27, cyclin D1, and antitumor action. Cancer Res 62: 4132–4141.
Yamada KM, Cukierman E . (2007). Modeling tissue morphogenesis and cancer in 3D. Cell 130: 601–610.
Yarden Y . (2001). The EGFR family and its ligands in human cancer: signalling mechanisms and therapeutic opportunities. Eur J Cancer 37 (Suppl 4): S3–S8.
Yaziji H, Goldstein LC, Barry TS, Werling R, Hwang H, Ellis GK et al. (2004). HER-2 testing in breast cancer using parallel tissue-based methods. JAMA 291: 1972–1977.
Zhou X, Tan M, Hawthorne VS, Klos KS, Lan KH, Yang Y et al. (2004). Activation of the Akt/mammalian target of rapamycin/4E-BP1 pathway by ErbB2 overexpression predicts tumor progression in breast cancers. Clin Cancer Res 10: 6779–6788.
We thank Leopoldo Luistro for providing the SKOV-3 cell line. KPL-4 cells were kindly provided by Professor J Kurebayashi, Kawasaki Medical School, Kurashiki, Okayama, Japan. We are grateful to Olaf Mundigl and Heike Seul for their assistance with confocal microscopy, and Ute Baer for technical assistance with gene expression profiling. We gratefully acknowledge Manfred Kubbies for fruitful discussions and expert advice, Max Hasmann and Mark Sliwkowski for helpful discussions, Stefan Ries for critical review of the paper and Lynda Chang for medical writing support.
About this article
Cite this article
Pickl, M., Ries, C. Comparison of 3D and 2D tumor models reveals enhanced HER2 activation in 3D associated with an increased response to trastuzumab. Oncogene 28, 461–468 (2009). https://doi.org/10.1038/onc.2008.394
- HER2 signaling
- membrane raft
- 3D cell culture
Dual-Targeted Delivery of Nanoparticles Encapsulating Paclitaxel and Everolimus: a Novel Strategy to Overcome Breast Cancer Receptor Heterogeneity
Pharmaceutical Research (2020)
Organoid and Assembloid Technologies for Investigating Cellular Crosstalk in Human Brain Development and Disease
Trends in Cell Biology (2020)
Molecular Biology Reports (2020)
Understanding and Modeling Metastasis Biology to Improve Therapeutic Strategies for Combating Osteosarcoma Progression
Frontiers in Oncology (2020)
Effects of microtubule-inhibiting small molecule and antibody-drug conjugate treatment on differentially-sized A431 squamous carcinoma spheroids
Scientific Reports (2020)