IL-6 triggers malignant features in mammospheres from human ductal breast carcinoma and normal mammary gland (original) (raw)
High levels of IL-6 mRNA are present in MS from aggressive ductal breast carcinoma and in basal-like breast carcinoma tissues. T-MS were generated from the tumor tissues of 3 patients with ductal breast carcinoma (samples 1–3; Table 1 and Figure 1A).
IL-6 mRNA is expressed in MS and in basal-like breast carcinoma tissues. (A) Phase-contrast microscopy of day 10 primary T-MS generated from samples listed in Table 1. Scale bars: 100 μm. (B) RT-PCR analysis of IL-6, Bmi-1, CK-5, CD44, Oct-4, and β2μ mRNA in T-MS and in tumor tissues from which T-MS had been obtained. (C) Day 10 primary N-MS and T-MS were obtained from the same patient (see Table 1). RT-PCR analysis of IL-6, Bmi-1, CK-5, BCRP-1, and CD133 and quantitation of IL-6, Bmi-1, and CK-5 mRNA, first normalized onto β2μ mRNA and then expressed as a ratio of N-MS to T-MS. *P = 0.031, Mann-Whitney test. NA, not available. (D) Breast carcinoma tissues from patients affected by basal-like or ductal breast carcinoma (see Table 2) were subjected to RT-PCR analysis. Shown is the ratio of IL-6 to β2μ mRNA. #P = 0.001, Mann-Whitney test.
Clinical-pathologic parameters of 17 breast ductal carcinomas used for T-MS and N-MS generation
T-MS were characterized by immunohistochemistry (IHC). We found that T-MS were composed almost entirely of cells that were CD44+ (97% ± 3%) and CD24– (<1% CD24+; Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI32533DS1), suggesting that the majority of cells in T-MS present a CD44+CD24– cancer stem cell phenotype (28). Further, cells in T-MS expressed Oct-4 (88% ± 7%), which has been previously reported to hyperexpress T-MS (27), and cytokeratin 5 (CK-5; 22% ± 7%), which identifies the mammary gland basal cell compartment (ref. 20 and Supplemental Figure 1). IHC showed also that T-MS were composed of E-cadherin+ (97% ± 2%), CK-14+ (99% ± 1%), and CK-18+ (24% ± 7%) cells, revealing that T-MS are composed of epithelial cells showing ductal (CK-18) and luminal (CK-14) markers (Supplemental Figure 2).
RT-PCR analysis revealed that T-MS, but not the tumor tissues from which T-MS were obtained, expressed detectable levels of IL-6 mRNA (Figure 1B). RT-PCR analysis also revealed that, compared with tumor tissues, T-MS expressed high levels of Bmi-1 mRNA, a gene associated with stem cell renewal (23); CD44 mRNA, a gene whose expression has been associated with cancer stem cell phenotype in different organs (28, 29); and CK-5 and Oct-4 mRNA (Figure 1B).
T-MS were then obtained from a set of samples (n = 14) in which the normal mammary gland tissue was also available to generate N-MS (Table 1, samples 4–17).
Similar to T-MS, N-MS lacked CD24 expression and contained cells expressing CD44 (95% ± 3%), CK-5 (14% ± 3%), CK-14 (78% ± 7%), and CK-18 (75% ± 9%; Supplemental Figure 3). The availability of N-MS and T-MS from the same patient allowed us to assess the level of IL-6 mRNA, accounting for variability caused by genetic makeup and age (30). We found that, compared with matched N-MS, T-MS from node-invasive tumors (pN3/pN2) expressed increased levels of IL-6 mRNA (Figure 1C). The same comparison performed on T-MS generated from scarcely node-invasive tumors (pN0/pN1) of ductal carcinomas revealed a negligible difference in IL-6 mRNA level between N-MS and T-MS (Figure 1C). Notably, compared with matched N-MS, T-MS obtained from patients affected by pN3/pN2 invasive tumors expressed similar levels of Bmi-1 and CK-5 mRNA and lower levels of breast cancer resistance protein 1 (BCRP-1) and CD133 mRNA, 2 antigens that have been previously associated with (cancer) stem cell phenotype (refs. 31–33; Figure 1C). The higher level of CD133 expression in N-MS compared with T-MS was also evident in IHC analysis (Supplemental Figure 4A).
We then assessed IL-6 mRNA in a set of archival breast tumor samples (Table 2), including ductal (n = 10) and basal-like (n = 6) breast carcinomas, a subtype of cancer showing stem cell features (34–37). This tumor type, similar to MS, was characterized by the expression of CK-5, CK-14, and EGFR protein as well as Bmi-1 and CD133 mRNA (Supplemental Figure 4, B and C), thereby reinforcing the notion of a tight similarity between MS and basal-like breast carcinoma cells (37).
Clinical-pathologic parameters of 16 archival breast carcinoma tissues assessed by RT-PCR
In keeping with this reasoning, we detected IL-6 mRNA in basal-like breast carcinoma tissues, but not in ductal breast carcinoma (Figure 1D). These data indicate that IL-6 expression occurs in MS obtained from aggressive ductal breast carcinoma and in basal-like breast carcinoma tissues, wherein stem cell–like phenotypes are particularly apparent.
IL-6 promotes MS self renewal and MCF-7–derived spheroid formation. To assess the functional role of IL-6 expression in MS, we exposed secondary T-MS to a mAb that blocks the IL-6 receptor/ligand interaction (anti–IL-6; 1.5 μg/ml). Exposure of T-MS to anti–IL-6 substantially blunted their secondary regeneration capacity, a functional property that has been referred to as MS self-renewal capability (refs. 21, 22, 25; Figure 2A). Accordingly, we observed that administration of IL-6 (10 ng/ml) to N-MS and T-MS from the same patient yielded an increase in secondary MS formation compared with MS not exposed to the cytokine, a phenomenon that was hampered by the simultaneous addition of anti–IL-6 (1.5 μg/ml; Figure 2B). We further investigated this phenomenon in the context of MCF-7–derived spheroids [MCF-7(S)], which have been recently shown to contain a substantial proportion of CD44+CD24– cells (38). MCF-7(S) expressed high levels of IL-6 mRNA, whereas the mRNA of the cytokine was absent in MCF-7 cells cultured in standard conditions (Figure 2C). Moreover, the administration of anti–IL-6 (1.5 μg/ml) caused a substantial reduction in MCF-7(S) size (Figure 2C). These data indicate that IL-6 mRNA expression promotes growth in suspension and that both autocrine and exogenous IL-6 promotes MS self renewal.
IL-6 sustains MS self renewal and MCF-7 spheroid formation. (A) Day 7 secondary T-MS, generated from primary T-MS in the presence or absence of the mAb anti–IL-6, which blocks the IL-6 receptor/ligand interaction (1.5 μg/ml). Phase-contrast microscopy analysis and number of MS per well (n = 3). *P = 0.029, **P = 0.042, ANOVA. (B) Phase-contrast microscopy analysis and number of MS per well in day 7 secondary T-MS and N-MS generated from primary MS in the presence or absence of IL-6 (10 ng/ml) and anti–IL-6 (1.5 μg/ml), respectively (n = 3). Χ_P_ = 0.027, ΧΧ_P_ = 0.020, #P = 0.048; ##P = 0.035, ANOVA plus post-hoc tests adjusted for multiple comparisons. (C) RT-PCR analysis of IL-6 mRNA in MCF-7 and day 2 MCF-7–derived spheroids and MCF-7(S) generated in the presence or absence of anti–IL-6 (1.5 μg/ml). Also shown are phase-contrast microscopy analysis and MCF-7(S) size distribution. n denotes the number of spheroids counted for each sample. °P = 0.02, Monte Carlo χ2 test. β2μ was assessed as quantitative control for RT-PCR analysis. Scale bars: 100 μm.
The MCF-7(S) growth-promoting activity of IL-6 requires Notch-3 gene. Notch genes play a pivotal role in MS self renewal (24–26). In particular, Notch-3 is highly expressed in N-MS and T-MS (21, 26), and its blockage induces a marked reduction in MS self renewal and survival (26). On these bases, we tested the hypothesis that the effect of IL-6 on MS self renewal and MCF-7(S) formation may depend upon Notch-3 gene expression. We found that administration of anti–IL-6 (1.5 μg/ml) to T-MS for 24 hours yielded downregulation in the level of Notch-3 mRNA and that administration of IL-6 (10 ng/ml) to N-MS for 24 hours elicited upregulation of Notch-3 mRNA (Figure 3A). A similar regulation was observed in MCF-7 cells and MCF-7(S) exposed to IL-6 (10 ng/ml for 24 hours) and in MCF-7(S) exposed to anti–IL-6 (1.5 μg/ml for 24 hours; Figure 3B). To better characterize the role of IL-6/Notch-3 interplay in substrate-independent growth, we generated MCF-7(S) using MCF-7 cells stably transduced with a retroviral vector expressing Notch-3–specific (shNotch-3) or control short hairpin RNA (shRNA). We found that MCF-7(S) obtained from control shRNA–transduced MCF-7 cells and generated in the presence of IL-6 (10 ng/ml) showed an increase in size, whereas shNotch-3 MCF-7 cells did not produce MCF-7(S), even in presence of exogenous IL-6 (10 ng/ml; Figure 3C). These data indicate that Notch-3 signaling is of pivotal importance to sustain the IL-6–dependent growth of breast cancer cells in suspension culture.
IL-6 induces Notch-3 gene upregulation and Notch-3–dependent MCF-7(S) formation. (A) RT-PCR analysis of Notch-3 mRNA in day 10 primary N-MS in the presence or absence of IL-6 (10 ng/ml) and in T-MS in the presence or absence of anti–IL-6 (1.5 μg/ml) for 24 hours. (B) RT-PCR analysis of Notch-3 mRNA in MCF-7 cells cultured in the presence or absence of IL-6 (10 ng/ml) and in MCF-7(S) in the presence or absence of anti–IL-6 (1.5 μg/ml) or IL-6 (10 ng/ml) for 24 hours. (C) Day 7 MCF-7(S) generated from MCF-7 cells infected with a pSuperPuro retroviral vector encoding a Notch-3-specific (N3) or control (CT) shRNA (sh) in the presence or absence of IL-6 (10 ng/ml). Phase-contrast microscopy analysis, MCF-7(S) size distribution (n denotes number of spheroids counted per sample), and Western blot analysis of Notch-3 and β-actin protein levels. *P = 0.034, Monte Carlo χ2 test. β2μ was assessed as quantitative control for RT-PCR analysis. Scale bars: 100 μm.
IL-6 elicits a Notch-3–dependent upregulation of Jagged-1 mRNA expression, which sustains MCF-7(S) formation and promotes MS self renewal. We recently reported that Notch-3 promotes MS survival by interacting with its ligand Jagged-1 (26). Therefore we next evaluated whether Jagged-1 was involved in Notch-3–dependent MS growth. Indeed, either exposing N-MS to IL-6 (10 ng/ml) or adding anti–IL-6 (1.5 μg/ml) to T-MS modulated the expression of Jagged-1 mRNA (Figure 4A). Moreover, we found that in MCF-7 cells, IL-6 elicited upregulation of Jagged-1 mRNA, which was blocked by the coadministration of IL-6 with the MEK/ERK inhibitor UO-126 (Figure 4B). Furthermore, we found that the upregulation of Jagged-1 induced by IL-6 was negligible in shNotch-3 MCF-7 cells and that the transfection of pCDNA3.1 vector encoding Notch-3 intracellular active cleaved fragment (pNICD3) into MCF-7 cells triggered an upregulation of Jagged-1 mRNA, which was prevented by the concurrent administration of UO-126 (Figure 4B). In addition, we observed that MCF-7(S) formation was extremely reduced when MCF-7 cells were transfected with a Jagged-1 specific siRNA compared with scrambled control siRNA (Figure 4C). Finally, we observed that an antibody blocking Jagged-1/Notch-3 interaction reduced MS regeneration capacity (Figure 4D), indicating that the Notch-3/Jagged-1 pathway is functionally relevant for IL-6–induced MS formation. Notably, we also found that basal-like breast carcinoma tissues expressed higher Jagged-1 and Notch-3 mRNA levels than did ductal breast carcinoma tissues (Figure 4E). These data suggest that upregulation of Jagged-1 via Notch-3 signaling is crucial for the growth in suspension of breast cancer cells and MS and that this phenomenon may also occur in basal-like breast cancer tissues.
Notch-3/Jagged-1 interplay sustains MCF-7(S) formation and MS self-renewal. (A) Day 10 primary N-MS and T-MS cultured in the presence or absence of IL-6 (10 ng/ml) or anti–IL-6 (1.5 μg/ml) for 24 hours. RT-PCR analysis of Jagged-1 mRNA. (B) RT-PCR analysis of Jagged-1 mRNA and Western blot analysis of phosphorylated ERK and total ERK protein in MCF-7 cells exposed to IL-6 (10 ng/ml) in the presence or absence of the MEK1 inhibitor UO-126 (20 μM) or DMSO for 24 hours, in shNotch-3 and control MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours), in MCF-7 cells transfected with 1 μg pNICD3 or empty control vector (pEMPTY) for 24 hours, and in MCF-7 cells transfected with pNICD3 in the presence or absence of UO-126 (20 μM) or DMSO. (C) Day 7 MCF-7(S) generated from MCF-7 cells transfected with Jagged-1–specific or scrambled (JAG1 and SCR, respectively) siRNA (1 μg, 72 hours’ pre-exposure). RT-PCR analysis of Jagged-1 mRNA, phase-contrast microscopy analysis, and MCF-7(S) size distribution. #P = 0.001, Monte Carlo χ2 test. (D) Day 7 secondary N-MS generated in the presence of IL-6 (10 ng/ml) and in the presence or absence of anti-N3 mAb, which blocks Notch-3 activity (1.5 μg/ml). Shown are phase-contrast microscopy and N-MS size distribution (n denotes number of spheroids counted per sample). Χ_P_ = 0.039, **P = 0.009, Monte Carlo χ2 plus post-hoc tests adjusted for multiple comparisons. (E) RT-PCR analysis of Jagged-1 and Notch-3 mRNA (ratio to β2μ) in basal-like or ductal carcinoma tissues. ##P = 0.005, ΧΧ_P_ = 0.042, Mann-Whitney test. β2μ was assessed as quantitative control for RT-PCR; β-actin was assessed as quantitative control for Western blot. Scale bars: 100 μm.
IL-6 induces a Notch-3–dependent upregulation of carbonic anhydrase IX. ERK upregulation has recently been found to enhance the expression of the hypoxia survival gene carbonic anhydrase IX (CA-IX; refs. 26, 39). Thus given our above observations, we next evaluated whether IL-6 signaling modulates CA-IX gene expression. Indeed, adding IL-6 (10 ng/ml) to N-MS induced upregulation of CA-IX mRNA (Figure 5A). Increased CA-IX expression was also observed in MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours), whereas CA-IX gene expression was markedly reduced by the administration of UO-126 (Figure 5B). Similar to what we observed for Jagged-1 (Figure 4B), CA-IX gene expression was inhibited in shNotch-3 MCF-7 cells, but not control MCF-7 cells, exposed to IL-6, while it was enhanced by transfection of the pNICD3 vector but not in the presence of UO-126 (Figure 5B). Because CA-IX is a hypoxia response gene (39), we investigated whether IL-6 plays a role in the hypoxia response. Exposure of MCF-7 cells to hypoxic stimuli (100 μM desferoxamine [DFX] or low oxygen tension, <0.1% O2, for 48 hours), as well as the exposure of N-MS and T-MS to 50 μM DFX (48 hours), enhanced the expression of IL-6, Notch-3, and CA-IX mRNAs (Figure 5C). Importantly, upon blocking the upregulation of hypoxia-responsive genes with 100 μM DFX, the administration of anti–IL-6 (1.5 μg/ml) to MCF-7 cells caused downregulation of Notch-3 and CA-IX mRNA. In addition, CA-IX mRNA was also downregulated in shNotch-3 MCF-7 cells exposed to 100 μM DFX compared with control MCF-7 cells (Figure 5D). Taken together, these results indicate the CA-IX gene expression is regulated by the IL-6/Notch-3 pathway in MCF-7 cells and MS.
IL-6/Notch-3 cross-talk promotes the upregulation of CA-IX mRNA and protein. (A) RT-PCR analysis of CA-IX mRNA in day 10 primary N-MS cultured in the presence or absence of IL-6 (10 ng/ml) for 24 hours. (B) RT-PCR analysis of CA-IX mRNA and Western blot analysis of CA-IX (phosphorylated ERK, total ERK, and β-actin protein levels shown in Figure 4B) in MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours) in the presence or absence of UO-126 (20 μM) or DMSO, in shNotch-3 and control MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours), in MCF-7 cells transiently transfected with pNICD3/pEMPTY vector (1 μg), and in MCF-7 cells transfected with pNICD3 and coadministered with UO-126 (20 μM) or DMSO for 24 hours. (C) RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA in MCF-7 cells exposed to low oxygen (<0.1% O2) or 100 μM DFX and in N-MS and T-MS exposed to 50 μM DFX for 48 hours. (D) RT-PCR analysis of Notch-3 and CA-IX mRNA in MCF-7 cells in the presence or absence of anti–IL-6 (1.5 μg/ml) and in shNotch-3 and control-infected MCF-7 cells exposed to DFX (100 μM for 24 hours), Western blot analysis of Notch-3 and β-actin protein. β2μ was assessed as quantitative control for RT-PCR analysis.
IL-6/Notch-3/CA-IX axis promotes hypoxia survival in MCF-7 and MS. CA-IX has been found to play a crucial role in hypoxia survival of breast cancer cells and MS (26). In keeping with these data, we observed a substantial increase compared with matched controls in cell death of MCF-7 cells exposed to 100 μM DFX in the presence of anti–IL-6 (1.5 μg/ml) or transfected with a CA-IX–specific siRNA (Figure 6A). Furthermore, a higher degree of hypoxia-induced cell death accompanied by downregulation of CA-IX mRNA was observed in shNotch-3 MCF-7 cells compared with control MCF-7 cells (Figure 6A). In line with these results, we found that exposure of T-MS to anti–IL-6 or anti-N3 (1.5 and 1 μg/ml, respectively) or transfection with CA-IX siRNA, in the presence of 50 μM DFX, increased cell death compared with a matched scrambled siRNA control (Figure 6B). Interestingly, detectable levels of CA-IX mRNA were found only in tissues from basal-like breast carcinoma (Figure 6C). These data indicate that IL-6/Notch-3–induced CA-IX gene expression promotes hypoxia survival in MS and support the similarity between the gene expression profiles of MS and basal-like breast carcinoma tissues.
The IL-6/Notch-3/CA-IX axis promotes hypoxia survival. (A) MCF-7 cells in the presence or absence of DFX (100 μM for 48 hours) and in the presence or absence of anti–IL-6 (1.5 μg/ml for 24 hours), with transient transfection with the CA-IX–specific or scrambled siRNA (1 μg for 72 hours), and shNotch-3 and control MCF-7 cells. Shown are Western blot analysis of Notch-3 and β-actin protein and cell death analysis and RT-PCR analysis of Notch-3 and CA-IX mRNA (n = 3). *P = 0.017, **P = 0.008, ***P = 0.002, ANOVA. (B) Cell death analysis and RT-PCR analysis of Notch-3 and CA-IX mRNA in day 7 secondary T-MS exposed to 50 μM DFX for 48 hours in the presence or absence of anti–IL-6 (1.5 μg/ml for 48 hours) or anti-N3 (1.5 μg/ml for 48 hours) or transfected with CA-IX or scrambled siRNA (1 μg for 72 hours). n = 3 per group. #P = 0.022, ##P = 0.025, ###P = 0.044, ANOVA. (C) RT-PCR analysis and representative IHC analysis of CA-IX protein expression of breast carcinoma tissues from patients affected by basal-like or ductal breast carcinoma (see Table 2). Data are shown as CA-IX/β2μ mRNA ratio. Χ_P_ = 0.002, Mann-Whitney test. β2μ was assessed as quantitative control for RT-PCR analysis. Scale bar: 100 μm.
IL-6 triggers a Notch-3/CA-IX–dependent increase in the invasiveness of MS and MCF-7 cells. The results illustrated in Figure 5B pointed out that IL-6 induces a Notch-3/ERK–mediated upregulation of CA-IX expression in absence of hypoxia. We then investigated the activity of the IL-6/Notch-3/CA-IX axis in normoxic conditions. We found that exposure to IL-6 (10 ng/ml) enhanced the capacity of MCF-7 cells to invade the extracellular matrix. This increase was negligible in shNotch-3 MCF-7 cells, and it was also substantially reduced when CA-IX, but not scrambled, siRNA was administered to IL-6–exposed MCF-7 cells (Figure 7A). In keeping with these observations, we found that administration of anti–IL-6 (1.5 μg/ml) or transfection of an IL-6–specific siRNA or CA-IX siRNA caused a substantial decrease in the invasive potential of T-MS compared with scrambled siRNA (Figure 7B). Further, the administration of IL-6 (10 ng/ml) enhanced the invasive potential of N-MS, yet this phenomenon was blocked by coadministration of anti-N3 (1.5 μg/ml) or transfection of CA-IX, but not scrambled, siRNA (Figure 7C). Parallel to these findings, we observed that IL-6 enhanced the activity of the extracellular matrix–degrading enzyme MMP-2 in control MCF-7 cells and in scrambled siRNA–transfected MCF-7 cells, but not in shNotch-3 MCF-7 cells or in CA-IX siRNA–transfected ones (Figure 7D). These data suggest that the IL-6/Notch-3–dependent upregulation of the CA-IX gene enhances the invasive behavior of MCF-7 cells and MS.
IL-6/Notch-3 cross-talk enhances the invasive potential of MS and MCF-7 cells by means of CA-IX mRNA upregulation. (A) Boyden invasion chamber assay in MCF-7 cells, in shNotch-3 and control MCF-7 cells, and in MCF-7 cells transiently transfected with scrambled or CA-IX siRNA (1 μg, 72 hours’ pre-exposure), in the presence or absence of IL-6 (10 ng/ml for 24 hours). n = 5 per group. *P = 0.0001, **P = 0.0001, #P = 0.0001, ANOVA. Inset: RT-PCR analysis of CA-IX mRNA in cells administered scrambled or CA-IX siRNA. (B) Boyden chamber invasion assay of day 7 secondary T-MS in the presence or absence of anti-IL6 (1.5 μg/ml for 24 h) or transfected with IL-6 or CA-IX or scrambled siRNA (1 μg, 72 hours’ pre-exposure). n = 3 per group. *P = 0.003, **P = 0.042, Χ_P_ = 0.0001, ANOVA. RT-PCR analysis of IL-6 and CA-IX mRNA is shown. (C) Boyden chamber invasion assay of day 7 secondary N-MS exposed to IL-6 (10 ng/ml for 24 hours) in the presence or absence of anti-N3 (1.5 μg/ml for 24 hours) or scrambled or CA-IX siRNA (1 μg, 72 hours’ pre-exposure). n = 3 per group. #P = 0.036, ##P = 0.037, Χ_P_ = 0.0001, ANOVA. RT-PCR analysis of IL-6, CA-IX, and β2μ mRNA is shown. (D) Zymographic analysis of MMP-2 activity in shNotch-3 and control MCF-7 cells in the presence or absence of IL-6 (10 ng/ml for 24 hours) and in MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours) transfected with CA-IX or scrambled siRNA (1 μg, 72 hours’ pre-exposure). n = 3 per group. *P = 0.032, #P = 0.025, ##P = 0.027, ANOVA.
Autocrine IL-6 sustains a CA-IX–dependent aggressive phenotype in MCF-7–derived, hypoxia-selected cells. Taken together, these results suggest that the establishment of an autocrine IL-6 loop may engender cancer cells with a substantial growth advantage over their normal counterparts. To explore this idea, we next examined a MCF-7–derived cell population, HYPO-7, which was obtained by selecting parental MCF-7 cells in the presence of 100 μM DFX (see Methods). Such cells, cultured for an extensive time period (up to 1 year) in the absence of DFX were found to constitutively express high levels of IL-6, Notch-3, and CA-IX mRNA (Figure 8A). We found that, compared with scrambled siRNA, administration of IL-6 siRNA to HYPO-7 yielded a decrease in Notch-3 and CA-IX mRNA expression, an increase in the susceptibility to DFX-induced cell death, and a reduction in their invasive potential and MMP-2 activity (Figure 8A). In agreement with the data obtained in MCF-7 cells and MS, we found that the administration of CA-IX, but not scrambled, siRNA to HYPO-7 cells recapitulated the phenotypic changes induced by IL-6 siRNA in HYPO-7 cells (Figure 8B). Interestingly, the effects elicited by IL-6 siRNA were also observed when HYPO-7 cells were exposed to anti–IL-6 (1.5 μg/ml for 24 hours; data not shown). Of particular importance, however, was the observation that administration of anti–IL-6 (1.5 μg/ml for 24 hours) caused downregulation of IL-6 mRNA in HYPO-7 cells as well as in MCF-7(S) and T-MS (Figure 8C). These data suggest that autocrine IL-6 production could promote the aggressiveness of breast cancer cells.
Autocrine IL-6 loop sustains a CA-IX–dependent malignant phenotype in HYPO-7 cells. (A) HYPO-7, a MCF-7–derived cell population, in the presence of IL-6 or scrambled siRNA (1 μg, 48 hours’ pre-exposure). RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA; cell death analysis in the presence of DFX (600 μM for 48 hours); and Boyden chamber invasion assay (n = 5) and zymographic analysis (n = 3) of MMP-2 activity (24 hours). *P = 0.042, **P = 0.0001, ***P = 0.015, ANOVA. (B) HYPO-7 cells in the presence of CA-IX or scrambled siRNA (1 μg, 48 hours’ pre-exposure). RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA; cell death analysis in the presence of DFX (600 μM for 48 hours); and Boyden chamber invasion assay (n = 5) and zymographic analysis (n = 3) of MMP-2 activity (24 hours). #P = 0.034, ##P = 0.0001, ###P = 0.018, ANOVA. (C) HYPO-7 cells, MCF-7(S), and T-MS exposed to anti–IL-6 (1.5 μg/ml) for 24 hours. RT-PCR analysis of IL-6 mRNA level. β2μ was assessed as quantitative control for RT-PCR analysis.
IL-6 induces an autocrine IL-6 loop that triggers Notch-3–dependent aggressive behavior in MCF-7 cells. Prompted by these observations, we reasoned that IL-6 might regulate the production of its own mRNA. Accordingly, we found that administration of IL-6 (10 ng/ml) upregulated IL-6 mRNA in MCF-7 cells and N-MS (Figure 9A). Furthermore, once exposed to IL-6 (10 ng/ml for 24 hours), MCF-7 cells expressed IL-6 mRNA, even 2 weeks after the withdrawal of IL-6 from the medium (Figure 9B), suggesting that IL-6 autoregulation might perpetuate phenotypic changes caused by exposing breast cancer cells to IL-6. Compared with untreated MCF-7 cells, the cells described above displayed upregulation of Notch-3 and CA-IX mRNA levels, paralleled by an enhancement in their invasive potential and an increase in MMP-2 activity (Figure 9B). The gene upregulation and the increase in invasive behavior of MCF-7 cells 2 weeks after withdrawal of IL-6 was abolished by administration of anti–IL-6 (1.5 μg/ml), indicating that such features were dependent upon an autocrine IL-6 loop (Figure 9C). Notch-3 signaling was also required for this effect, because shNotch-3 MCF-7 cells did not show upregulation in CA-IX mRNA nor an enhancement of invasive potential, which were both observed in control MCF-7 cells 2 weeks after exposure to IL-6 (Figure 9D). As expected, the enhanced invasive capacity of IL-6–treated control MCF-7 cells was reduced by the transfection of CA-IX siRNA, but not scrambled siRNA (Figure 9E). These data support the argument that an IL-6 autocrine loop could induce long-term enhancement in the aggressive features of breast cancer cells by sustaining upregulation of the Notch-3/CA-IX axis.
Autocrine IL-6 loop sustains a Notch-3/CA-IX–dependent aggressive phenotype in MCF-7 cells. (A) RT-PCR analysis of IL-6 mRNA in MCF-7 cells and N-MS exposed to IL-6 (10 ng/ml) for 24 hours. (B) MCF-7 cells exposed to IL-6 (10 ng/ml for 24 hours) and assessed at various times (1 or 2 weeks) after the withdrawal of the cytokine. RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA and Boyden chamber invasion assay (n = 5) and zymographic analysis (n = 3) of MMP-2 activity (24 hours). *P = 0.010, #P = 0.012, ##P = 0.002, ANOVA with post-hoc test for multiple comparisons. (C) MCF-7 cells exposed to IL-6 (10 ng/ml) for 24 hours and assessed 2 weeks after cytokine withdrawal in the presence or absence of anti–IL-6 (1.5 μg/ml) for 24 hours. RT-PCR analysis of IL-6, Notch-3, and CA-IX mRNA and Boyden chamber invasion assay (24 hours). n = 5 per group. **P = 0.004, ANOVA with post-hoc test for multiple comparisons. (D) RT-PCR analysis of IL-6 and CA-IX mRNA, Western blot analysis of Notch-3 and β-actin protein level, and Boyden chamber invasion assay (24 hours) in shNotch-3 and control MCF-7 cells either untreated or exposed to IL-6 for 24 hours and assessed 2 weeks after cytokine withdrawal (n = 5). Χ_P_ = 0.001, ANOVA with post-hoc test for multiple comparisons. (E) Boyden chamber invasion assay (24 hours) and RT-PCR analysis of CA-IX mRNA in cells as in C and D transfected with CA-IX or scrambled siRNA (1 μg, 48 hours’ pre-exposure). n = 5 per group. ΧΧ_P_ = 0.002, ANOVA. β2μ was assessed as quantitative control for RT-PCR analysis.