Lysyl Oxidase Is Induced by Cell Density-Mediated Cell Cycle Suppression via RB-E2F1-HIF-1α Axis

Abstract

Remodeling of the matrix surrounding tumor cells plays a crucial role in the development and maintenance of cancer. Lysyl oxidase (LOX), a matrix remodeling factor, is induced by HIF-1α under hypoxic conditions and associated with tumor growth and metastasis. Here, we report that high cell density induces HIF-1α expression under normoxic condition, resulting in the promotion of LOX expression. This phenomenon was observed in the retinoblastoma tumor suppressor (RB)-proficient breast cancer cells but not in RB-deficient cells. In RB-proficient cancer cells, the cell cycle regulator E2F1 was down-regulated and cell cycle progression was inhibited at high density culture condition. Knockdown of E2F1 stabilized HIF-1α and promoted LOX expression, while knockdown of both E2F1 and HIF-1α prevented the up-regulation of LOX. These findings suggest that elevated cell density enhances cell cycle arrest and matrix remodeling via RB-E2F1-HIF-1α axis.

Introduction

It is known that cell proliferation is suppressed when the increased cell density induces a contact inhibition signal (Lieberman, 1981). Although it is often assumed that tumor cells grow without limitation due to deregulation of contact inhibition signals, the cell-cell contact of tumor cells can suppress cell growth in some degree and provide a survival signal which allows cells to become resistant to genotoxic stress (Day et al., 1999); however, the underlying mechanism is not fully understood.

Lysyl oxidase (LOX) is a matrix remodeling factor which plays a critical role in the formation and repair of the extra-cellular matrix. LOX covalently cross-links elastin and collagen by oxidizing lysine residues in these proteins (Kagan and Li, 2003). This LOX-mediated cross-linking stabilizes these fibrous proteins and increases the stiffness of the matrix. The stiffness of the matrix surrounding a tumor has recently been recognized to play an important role in tumor latency and malignancy, and the cross-linking of collagen by LOX plays its central role (Levental et al., 2009). The up-regulation of LOX is also associated with metastasis and poor clinical outcome in breast cancer (Erler et al., 2006). Thus, clarifying the mechanism through which LOX expression is regulated may provide insight into tumor progression and lead to the development of novel therapeutic approaches for cancer.

LOX expression has been shown to be induced under hypoxic conditions (Erler et al., 2006), and the stabilization of hypoxia inducible factor-1α (HIF-1α) directly enhances LOX transcription. A recent study reported that HIF-1α can be stabilized under conditions other than hypoxia (Hirota et al., 2009). Therefore, circumstances in which HIF-1α is induced under non-hypoxic conditions may lead to LOX expression.

We show here that increased cell density up-regulates LOX expression at normoxia. Our studies reveal that LOX expression is controlled by HIF-1α, which is in turn regulated by E2F1, a regulator of cell proliferation and apoptosis (Chen et al., 2009). These findings indicate a novel link between cell cycle regulation and extracellular matrix regulation.

Results

The expression of LOX increases in a cell density-dependent manner

A high cell density has previously been shown to create hypoxia-like conditions (Sheta et al., 2001). Therefore, the correlation between LOX expression and cell density in culture was examined. Various numbers of breast cancer cells [1×10⁵ (low density), 2×10⁵ (medium density), and 4×10⁵ (high density)] were seeded in 35 mm polystyrene culture dishes and cultured for 6 days. Cells were collected and the expression level of LOX was examined by western blot analysis (Fig. 1). In MDA-MB-231 cells and Hs578t cells, the expression of LOX gradually increased with increasing cell density (Fig. 1A, B). On the other hand, the expression level of LOX appeared to be unrelated to cell density in BT-549 cells (Fig. 1C). We have previously shown that the status of the retinoblastoma tumor suppressor (RB), which is a key regulator of cell cycle progression from the G1 phase to the S phase, differs among these three breast cancer cell lines (Arima et al., 2012a). MDA-MB-231 cells and Hs578t cells are RB-proficient and express wild-type RB, whereas BT-549 cells are RB-deficient. Therefore, we speculated that RB is involved in the induction of LOX at high cell densities.

Fig. 1

(A) MDA-MB-231 cells, (B) Hs578t cells, or (C) BT-549 cells were seeded at various numbers [1×10⁵ (low density; LD), 2×10⁵ (medium density; MD), or 4×10⁵ (high density; HD)], cultured for 2–6 days, and then subjected to immunoblot analysis of LOX, HIF-1α, RB, phospho-RB (phos RB), and α-tubulin. (D) MDA-MB-231 cells or (E) Hs578t cells were seeded at various densities [1×10⁵ (low density), 2×10⁵ (medium density), or 4×10⁵ (high density)], transfected with control or HIF-1α siRNAs, cultured for 2, 4, or 6 days, and then subjected to immunoblot analysis of LOX, HIF-1α, RB, phospho-RB (phos RB), and α-tubulin.

Given that HIF-1α is a strong inducer of LOX expression (Erler et al., 2006), the correlation between HIF-1α expression and LOX up-regulation at higher cell densities was examined. HIF-1α expression was examined in cells harvested at different cell densities. In accordance with previous reports, the expression of HIF-1α was increased at higher cell densities in the RB-proficient cell lines MDA-MB-231 (Fig. 1A). In Hs578t cells, HIF-1α expression was elevated at day 2 but returned to basal levels by day 4 (Fig. 1B). Then, to assess the contribution of HIF-1α to LOX induction at higher cell densities, HIF-1α was transiently depleted by siRNA in MDA-MB-231 cells and Hs578t cells. Depletion of HIF-1α suppressed the LOX up-regulation (Fig. 1D, E). These results suggest that LOX up-regulation at higher cell densities is mediated by HIF-1α.

The effect of cell density on proliferation and cell cycle progression of breast cancer cells

Cell density often affects cell cycle progression and cell proliferation, though the magnitude of this effect varies depending on the cell type. Accordingly, the growth of cells cultured at different cell densities was assessed. Cells were seeded at increasing densities, as described above, and cell numbers were counted after 2 and 4 days of culture. The rates of cell growth between day 0 and day 2, and between day 2 and day 4, were compared for cells seeded at different densities. Cells grew more slowly at higher culture densities (Fig. 2A, B). Cell cycle analysis showed that the G1 population of MDA-MB-231 cells, in which LOX expression is enhanced at a higher culture density, was increased in a cell density-dependent manner (Fig. 2C). These results suggest that in MDA-MD-231 cells, when cell density is increased, cell proliferation and cell cycle progression are suppressed. With respect to Hs578t cells, in which LOX was induced by cell density to a lesser extent than in MDA-MB-231 cells, the G1 population did not increase as in MDA-MD-231 cells cultured under high-density conditions (Fig. 2D). Therefore, the effect of high-density culture on cell cycle suppression correlates with the effect of cell density on HIF-1α and LOX expression.

Fig. 2

(A) MDA-MB-231 or (B) Hs578t cells were seeded at various densities [1×10⁵ (low density; LD), 2×10⁵ (medium density; MD), or 4×10⁵ (high density; HD)] and cultured for 2 or 4 days. The cell numbers at day 0, 2, and 4 were counted and the growth ratio was calculated by division. The growth ratios at each density were normalized to the growth ratio for each respective cell line at low density. Error bars represent the s.d. *, p<0.05 versus LD for that cell line. (C) MDA-MB-231 or (D) Hs578t cells were seeded at various numbers [1×10⁵(LD), 2×10⁵ (MD), 4×10⁵ (HD)] and cultured for 2 or 4 days. The extent of G1 at days 2 and 4 was determined by flow cytometry.

Depletion of E2F1 in RB-proficient breast cancer cells induces the up-regulation of LOX

Given that cell density-dependent growth suppression and LOX up-regulation were observed in RB-proficient cells, we hypothesized that RB-mediated cell cycle regulation is involved in this phenomenon. The E2F transcription factors are key downstream targets of RB, which controls cell cycle progression. Therefore, the role of E2F1, the major E2F subtype in epithelial tumor cells (Chen et al., 2009), in cell density-dependent LOX expression was determined. E2F1 expression decreased slightly as cell density increased (Fig. 3A, B). To characterize the effect of E2F1 down-regulation, MDA-MB-231 and Hs578t cells were transfected with siRNAs against E2F1 (Fig. 3C, D). Depletion of E2F1 markedly increased the expression of LOX in both cell lines. These results suggest that E2F1 inhibits the expression of LOX.

Fig. 3

(A) MDA-MB-231 or (B) Hs578t cells were seeded at various numbers [1×10⁵ (LD), 2×10⁵ (MD), and 4×10⁵ (HD)] and cultured for 2–6 days, then subjected to immunoblot analysis of LOX, E2F1, HIF-1α, and α-tubulin. (C) MDA-MB-231 cells or (D) Hs578t cells (2×10⁵ of each cell type) were transfected with control or E2F1 siRNAs for 2–6 days and were then subjected to immunoblot analysis of LOX, HIF-1α, phospho-RB (phos RB), and α-tubulin.

The effect of E2F1 depletion on LOX expression is HIF-1α-dependent

Lastly, the involvement of HIF-1α in the E2F1 depletion-induced up-regulation of LOX was examined. Indeed, E2F1 depletion induced the up-regulation of HIF-1α as well as LOX (Fig. 3C, D). The contribution of HIF-1α to the E2F1 knockdown-mediated up-regulation of LOX was then examined by depletion of E2F1 together with HIF-1α in the MDA-MB-231 and Hs578t cell lines. The effect of E2F1 depletion on LOX up-regulation was attenuated by HIF-1α depletion, indicating that HIF-1α is a mediator of the E2F1 depletion-induced LOX expression (Fig. 4A).

Fig. 4

(A) MDA-MB-231, Hs578t, or BT-549 cells (2×10⁵ of each cell type) were transfected with control or E2F1 siRNAs and/or HIF-1α siRNAs, as indicated, for 2–6 days and were then subjected to immunoblot analysis of LOX, HIF-1α, phospho-RB (phos RB), and α-tubulin. (B) Proposed mechanism of LOX expression induced by high cell density and impairment of cell cycle progression.

Discussion

This study demonstrated that high cell density increases LOX expression through the induction of HIF-1α. Furthermore, this cell density-induced LOX up-regulation is promoted by E2F1 down-regulation in RB-proficient basal-type breast cancer cells.

HIF-1α is stabilized at normoxia when cells are cultured at higher densities (Fig. 1). Given that HIF-1α is known to induce LOX expression (Erler et al., 2006), the density-dependent up-regulation of LOX is a consequence of HIF-1α stabilization at normoxia. The possibility cannot be ruled out that peripheral hypoxia occurs as cell density increases (Dayan et al., 2009); however, our results may indicate a condition other than hypoxia under which HIF-1α can be stabilized.

RB is a major cell cycle regulatory factor (Giacinti and Giordano, 2006; Khidr and Chen, 2006), and inactivation of RB is associated with aggressive characteristics in breast cancer cells (Arima et al., 2008, 2012b). In MDA-MB-231 cells and Hs578t cells, RB-proficient basal-like breast cancer cell lines, LOX expression, and growth suppression were induced by high-density culture. By contrast, BT-549 cells, an RB-deficient cell line, did not up-regulate LOX under high-density culture conditions, which is probably due to the constitutive cell cycle progression induced by RB inactivation. This hypothesis was supported by the finding that the adenovirus-mediated expression of phospho-deficient form of RB (Ad-RB Am), which is a constitutive active RB, tended to induce LOX expression (Supplementary figure S1). Therefore, we speculated that RB plays a critical role in the cell cycle arrest and HIF-1α-mediated LOX up-regulation induced by high density culture conditions.

RB is known to suppress the function of E2F. Therefore, we investigated the role of E2F in cell density-dependent LOX expression and found that E2F1 expression is markedly reduced when cells are cultured at a higher density (Fig. 3). E2F1 activity is well known to be dependent on the phosphorylation state of RB (Giacinti and Giordano, 2006; Khidr and Chen, 2006). Recent studies show that changes in the expression of E2F1 by microRNAs also affect its function (O’Donnell et al., 2005; Pickering et al., 2009). We have not uncovered the mechanism through which high cell density down-regulates E2F1, but there may be a relationship between cell-cell contact signals and specific mircoRNAs that target E2F1.

The attenuation of E2F1 promoted HIF-1α-mediated LOX expression. The mechanisms through which E2F1 regulates HIF-1α stabilization remain unknown. A recent study reports that HIF-1α is destabilized by members of the MCM family, which are well known to be E2F1 targets (Hubbi et al., 2011). It is possible that E2F1 attenuation results in MCM down-regulation, which leads to the stabilization of HIF-1α.

LOX has been reported to modify the tumor microenvironment and to enhance cancer invasion and metastasis (Erler et al., 2006, 2009; Levental et al., 2009). Thus, via the induction of LOX, increased cell density may have a significant effect on the tumor microenvironment, leading to tumor cell survival, heterogeneity, and metastasis (Fig. 4B). It was recently reported that a cyclin-dependent kinase inhibitor, which induces cell cycle arrest, promotes the production of matrix remodeling factors and leads to metastasis in pancreatic cancer cells (Liu and Korc, 2012). The management of the adverse effects of cell cycle arrest needs to be considered and investigated in future research.

In conclusion, cell cycle slowdown may both enhance cell cycle arrest and drive extracellular matrix remodeling via E2F1.

Experimental Procedures

Cell lines

The human breast cancer cell lines BT-549, MDA-MB-231, and Hs578T were obtained from the American Type Culture Collection (Manassas, VA).

Cell culture

MDA-MB-231 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS). BT-549 cells were cultured in RPMI 1640 supplemented with 10% FBS and human insulin (0.023 IU/ml; Sigma-Aldrich, St. Louis, MO). Hs578T cells were cultured in DMEM supplemented with 10% FBS and bovine insulin (0.01 mg/ml; Sigma-Aldrich). MDA-MB-231, BT-549, and Hs578T cells were maintained at 37°C in a humidified atmosphere of 5% CO₂. Culture media were changed every 2 days.

All cells were propagated for a short time after receipt and then frozen in batches as stock. A new batch was thawed every 2 months (corresponding to 15–20 passages).

Antibodies

The following antibodies were obtained from the indicated sources: mouse monoclonal antibodies to RB (Cell Signaling, Danvers, MA), α-tubulin (Sigma-Aldrich), and HIF-1α (BD Biosciences, San Jose, CA); and rabbit polyclonal antibodies to Ser807/811-phosphorylated Rb (Cell Signaling), LOX (Sigma-Aldrich), and E2F1(Cell Signaling).

SiRNAs and adenovirus

A negative control siRNA, as well as HIF-1α and E2F1 siRNA SMART pools, were obtained from Dharmacon/Thermo Scientific (Lafayette, CO). MDA-MB-231 and Hs578t cells were transfected using the Lipofectamine RNAiMAX reagent (Invitrogen/Life Technologies, Grand Island, NY). An adenovirus encoding a constitutively active mutant of human RB (Ad-RB Am) that is refractory to CDK-cyclin-mediated phosphorylation was kindly provided by E. Knudsen. Ad-RB obtained from VectoRBiolabs. Ad-GFP encoding the GFP gene was used as a control. Cells were cultured with adenovirus at a multiplicity of infection of 100 as described previously (Arima et al., 2012)

Statistical analysis

Data are presented as means±SD and were analyzed using Student’s paired t test. A P-value of <0.05 was considered statistically significant.

Cell cycle analysis and cell number counting

Cells were collected by exposure to trypsin, washed with PBS, fixed in ice-cold 70% ethanol, and stored at −20°C. They were subsequently washed twice with PBS, incubated for 30 min at room temperature with RNase A (100 μg/ml), and stained with propidium iodide PI (25 μg/ml) for 30 min. Flow cytometry was performed on a FACSCalibur instrument, and data were analyzed using FlowJo software (BD Biosciences).

Acknowledgments

The authors thank E. Knudsen for providing Ad-RB Am; the members of Division of Gene Regulation, Institute for Advanced Medical Research, School of Medicine, Keio University for insightful discussions. This study was supported in part by the Global COE Program from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan (T.M.G.); a Grant-in-Aid for Young Scientists (B) from MEXT (Y.A.); and a Grant-in-Aid from MEXT (H.S.).

References

• Arima, Y., Inoue, Y., Shibata, T., Hayashi, H., Nagano, O., Saya, H., and Taya, Y. 2008. Rb depletion results in deregulation of E-cadherin and induction of cellular phenotypic changes that are characteristic of the epithelial-to-mesenchymal transition. Cancer Research, 68: 5104–5112.
• Arima, Y., Hayashi, N., Hayashi, H., Sasaki, M., Kai, K., Sugihara, E., Abe, E., Yoshida, A., Mikami, S., Nakamura, S., and Saya, H. 2012a. Loss of p16 expression is associated with the stem cell characteristics of surface markers and therapeutic resistance in estrogen receptor-negative breast cancer. International Journal of Cancer, 130: 2568–2579.
• Arima, Y., Hayashi, H., Sasaki, M., Hosonaga, M., Goto, T.M., Chiyoda, T., Kuninaka, S., Shibata, T., Ohata, H., Nakagama, H., Taya, Y., and Saya, H. 2012b. Induction of ZEB by inactivation of RB is a key determinant of the mesenchymal phenotype of breast cancer. The Journal of Biological Chemistry, 287: 7896–7906.
• Chen, H.-Z., Tsai, S.-Y., and Leone, G. 2009. Emerging roles of E2Fs in cancer: an exit from cell cycle control. Nature reviews. Cancer, 9: 785–797.
• Day, M.L., Zhao, X., Vallorosi, C.J., Putzi, M., Powell, C.T., Lin, C., and Day, K.C. 1999. E-cadherin mediates aggregation-dependent survival of prostate and mammary epithelial cells through the retinoblastoma cell cycle control pathway. The Journal of Biological Chemistry, 274: 9656–9664.
• Dayan, F., Bilton, R.L., Laferrière, J., Trottier, E., Roux, D., Pouyssegur, J., and Mazure, N.M. 2009. Activation of HIF-1alpha in exponentially growing cells via hypoxic stimulation is independent of the Akt/mTOR pathway. Journal of Cellular Physiology, 218: 167–174.
• Erler, J.T., Bennewith, K.L., Nicolau, M., Dornhöfer, N., Kong, C., Le, Q.-T., Chi, J.-T.A., Jeffrey, S.S., and Giaccia, A.J. 2006. Lysyl oxidase is essential for hypoxia-induced metastasis. Nature, 440: 1222–1226.
• Erler, J.T., Bennewith, K.L., Cox, T.R., Lang, G., Bird, D., Koong, A., Le, Q.-T., and Giaccia, A.J. 2009. Hypoxia-induced lysyl oxidase is a critical mediator of bone marrow cell recruitment to form the premeta-static niche. Cancer Cell, 15: 35–44.
• Giacinti, C. and Giordano, A. 2006. RB and cell cycle progression. Oncogene, 25: 5220–5227.
• Hirota, S.A, Beck, P.L., and MacDonald, J.A. 2009. Targeting hypoxia-inducible factor-1 (HIF-1) signaling in therapeutics: implications for the treatment of inflammatory bowel disease. Recent Patents on Inflammation & Allergy Drug Discovery, 3: 1–16.
• Hubbi, M.E., Luo, W., Baek, J.H., and Semenza, G.L. 2011. MCM Proteins Are Negative Regulators of Hypoxia-Inducible Factor 1. Molecular Cell, 42: 700–712.
• Kagan, H.M. and Li, W. 2003. Lysyl oxidase: properties, specificity, and biological roles inside and outside of the cell. Journal of Cellular Biochemistry, 88: 660–672.
• Khidr, L. and Chen, P.-L. 2006. RB, the conductor that orchestrates life, death and differentiation. Oncogene, 25: 5210–5219.
• Levental, K.R., Yu, H., Kass, L., Lakins, J.N., Egeblad, M., Erler, J.T., Fong, S.F.T., Csiszar, K., Giaccia, A., Weninger, W., Yamauchi, M., Gasser, D.L., and Weaver, V.M. 2009. Matrix crosslinking forces tumor progression by enhancing integrin signaling. Cell, 139: 891–906.
• Lieberman, M.A. and Glaser, L. 1981. Density-Dependent Regulation of Cell Growth: An Example of a Cell-Cell Recognition Phenomenon. Journal of Membrane Biology, 63: 1–11.
• Liu, F. and Korc, M. 2012. Cdk4/6 Inhibition Induces Epithelial-Mesenchymal Transition and Enhances Invasiveness in Pancreatic Cancer Cells. Molecular Cancer Therapeutics, 11: 2138–2148.
• O’Donnell, K.A., Wentzel, E.A., Zeller, K.I., Dang, C.V., and Mendell, J.T. 2005. c-Myc-regulated microRNAs modulate E2F1 expression. Nature, 435: 839–843.
• Pickering, M.T., Stadler, B.M., and Kowalik, T.F. 2009. miR-17 and miR-20a temper an E2F1-induced G1 checkpoint to regulate cell cycle progression. Oncogene, 28: 140–145.
• Sheta, E.A., Trout, H., Gildea, J.J., Harding, M.A., and Theodorescu, D. 2001. Cell density mediated pericellular hypoxia leads to induction of HIF-1α via nitric oxide and Ras/MAP kinase mediated signaling pathways. Oncogene, 20: 7624–7634.