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Cancer cells have an efficient antioxidant system to counteract their increased generation of ROS. However, whether this ability to survive high levels of ROS has an important role in the growth and metastasis of tumors is not well understood. Here, we demonstrate that the redox protein thioredoxin-like 2 (TXNL2) regulates the growth and metastasis of human breast cancer cells through a redox signaling mechanism. TXNL2 was found to be overexpressed in human cancers, including breast cancers. Knockdown of TXNL2 in human breast cancer cell lines increased ROS levels and reduced NF-κB activity, resulting in inhibition of in vitro proliferation, survival, and invasion. In addition, TXNL2 knockdown inhibited tumorigenesis and metastasis of these cells upon transplantation into immunodeficient mice. Furthermore, analysis of primary breast cancer samples demonstrated that enhanced TXNL2 expression correlated with metastasis to the lung and brain and with decreased overall patient survival. Our studies provided insight into redox-based mechanisms underlying tumor growth and metastasis and suggest that TXNL2 could be a target for treatment of breast cancer.
ROS, including superoxide O–2, hydroxyl radical OH, and H2O2, are constantly generated during intracellular metabolism and in response to environmental stimuli (1). Generally, ROS are regarded as host-defending molecules that destroy exogenous pathogens (2) and act as secondary messengers in signal transduction (1, 3). However, increased production of ROS is involved in committing cells to apoptosis (3, 4). Although ROS are involved in tumorigenesis and progression, as reflected by ROS activation of tumor-promoting signaling pathways (5), excess oxidative stress, due to further elevated ROS levels beyond a threshold or weakened antioxidative defense, can damage macromolecules vital for cellular functions (6, 7). This in turn results in pathophysiological changes, such as apoptosis, cell cycle disruption, and necrosis (8). As such, induction of ROS-mediated damage in cancer cells by proper pharmacological agents that either promote ROS generation or disable the cellular antioxidant system has been considered as a “radical” therapeutic strategy to preferentially kill cancer cells (9).
The redox state in the normal cell is balanced by the cellular antioxidant capacity to maintain a viable steady-state environment that is predominantly reducing (10). A key mechanism by which cells regulate redox processes is the reversible formation of disulfides through the oxidation of thiol groups in cysteine residues (11). To maintain the cellular thiol-disulfide redox balance, living cells possess 2 major regulatory systems: the thioredoxin/thioredoxin (Trx/Trx) reductase system and the glutaredoxin/glutathione/glutathione (Grx/GSH/GSH) reductase system (12). Trx-1 (12 kDa) is a well-documented member of the Trx regulatory system that reduces disulfide bonds and thus regulates the activity of transcriptional factors like AP-1, NF-κB, and p53 (13, 14). Overexpression of Trx-1 inhibits apoptosis (15). Grxs can readily reduce S-glutathionylated protein (protein-SSG) mixed disulfide and can be regenerated by the reduced form of GSH (16). Grxs protect cells against oxidative stress by catalyzing protein de-glutathionylation and has therefore been implicated in various cellular processes, including regulation of transcription factor binding activities and redox regulation (17–19). For example, Grx-1 regulates intracellular and extracellular homeostasis of protein glutathionylation (20–22).
Elevated oxidative status has been observed in many types of cancer cells, due in part to their high metabolic rate. On the other hand, many tumor cells possess stronger antioxidative defense mechanisms to counterbalance excessive ROS, maintain their redox status, and thus suppress apoptosis (23). This phenomenon may be a consequence of cellular adaption to ROS stress and may play an important role in the development of highly malignant behaviors and drug resistance (9). Overexpression of Trx-1 in MCF-7 human breast cancer cells enhances cell growth (24). Increased Trx-1 protein levels are found in several human cancers (25, 26). Interestingly, Grx-1 expression can be induced by oxidative stress in breast cancer cells and thus inhibits apoptosis (27).
During a recent microarray analysis of the IGF-regulated genes in breast cancer cells, we found that a novel Trx-related protein, Trx-like 2 (TXNL2; also known as Grx3 and PICOT), is significantly induced by IGFs (28). The 38-kDa TXNL2 protein is much larger than typical Trx proteins and has a unique protein structure consisting of an N-terminal Trx homology region, followed by 2 tandem repeats of Grx domains (22, 29, 30). Grx3/4, the yeast homolog of TXNL2, was implicated in the regulation of the oxidative stress response (31). Although TXNL2 is conserved in eukaryotes, the physiological function in mammalian cells is still poorly understood (30). Recent reports showed that it can inhibit cardiac hypertrophy through enhancing ventricular function and cardiomyocyte contractility and can regulate FcεRI-mediated mast cell activation (32, 33). Deletion of TXNL2 in mice causes embryonic lethality (34), indicating its role in protecting cells against oxidative stress during embryogenesis.
We hypothesized that TXNL2 may play an important role in antagonizing oxidative stress in cancer cells. This study was designed to determine how TXNL2 contributes to the regulation of the cellular redox state in cancer cells and redox-mediated signaling pathways. We characterized the molecular actions of TXNL2 and its involvement in tumor development and metastasis and also examined the clinical significance of its expression in primary human breast cancers. Our studies demonstrate that TXNL2 is essential for cancer cell functions by regulating ROS levels and NF-κB activity.
We first compared the expression levels of TXNL2 in normal breast tissues and breast cancer tissues using the Oncomine database, which includes publicly available cDNA microarray data on cancer. Analysis of a representative data set (35) indicated TXNL2 mRNA levels were higher in breast cancer tissue than in normal breast tissues (Figure (Figure1A).1A). As different cancers commonly possess strong antioxidant capacity to counteract ROS, we also analyzed gene expression profiles of other cancers. TXNL2 mRNA levels were also elevated in lung cancer (36), colon cancer (ref. 37 and Figure Figure1A),1A), gastric cancer (38), bladder cancer (39), and cervical cancer (40) (Supplemental Figure 1, A–C; supplemental material available online with this article; doi: 10.1172/JCI43144DS1). Thus, increased TXNL2 expression is associated with multiple human cancers.
We then examined TXNL2 expression in human breast cancer cell lines and normal human mammary epithelial cells (HMECs). Immunoblotting showed higher TXNL2 levels in breast cancer cell lines compared with HMECs (Figure (Figure1B).1B). Next, we performed immunohistochemistry of TXNL2 on tissue microarrays containing 190 samples of human invasive breast carcinomas of various subtypes and 15 samples of normal human breast tissues. Tissue staining was scored on the basis of the intensity of TXNL2 labeling and the percentage of TXNL2-positive tumor cells. As illustrated in Figure Figure1C1C and Table Table1,1, TXNL2 protein was not detected or was weakly detected in most normal breast tissues but was readily detectable in most invasive breast carcinomas. Normal mouse IgG was used as a negative control (Supplemental Figure 1D). Breast cancer tissues showed significantly higher TXNL2 levels than normal breast tissues (P < 0.0001).
Since TXNL2 levels were consistently higher in breast cancer cell lines, we used shRNA to generate TXNL2-knockdown (TXNL2-KD) cell models to study the function of TXNL2. As shown in Supplemental Figure 2A, 2 TXNL2 shRNAs caused a similar degree of TXNL2 knockdown, without affecting Trx-1 and Grx-1 levels. For simplicity of interpretation, the effects of a representative shRNA are described here. We first explored the effect of TXNL2 downregulation on cell growth using MDA-MB-231 and BT549 human breast cancer cells. Inhibition of TXNL2 suppressed the growth of breast cancer cells but not normal HMECs (Figure (Figure2A),2A), suggesting that TXNL2 is essential for the growth of breast cancer cells. Next, we performed soft agar assays and Matrigel 3D culture. As shown in Figure Figure2B,2B, depletion of TXNL2 prevented the colony formation of MDA-MB-231 and BT549 cells in soft agar, indicating that anchorage-independent growth was suppressed. Similarly, TXNL2 knockdown disrupted the growth of these cells in Matrigel (Figure (Figure2C).2C). Interestingly, in contrast to that of control cells, which formed stretching-out stellate structures, TXNL2-KD MDA-MB-231 and BT549 cells showed a spherical structure in Matrigel culture (Figure (Figure2C).2C). Similar effects of TXNL2 depletion on growth of 4T1 and MCF-7 breast cancer cells were observed (Supplemental Figure 2, B and C). These results indicate that deficiency of TXNL2 specifically inhibits breast cancer cell growth in monolayer and 3D culture.
Notably, knockdown of TXNL2 resulted in a change from mesenchymal-to-epithelial morphology in MDA-MB-231 and BT549 cells (Supplemental Figure 3A). Using immunofluorescence microscopy, we found that TXNL2-KD MDA-MB-231 cells displayed a flattened and cubical morphology, with non-polarized lamellar extensions around the entire cell periphery but no stress fiber formation when plated on different extracellular matrices, such as collagen IV, fibronectin, and laminin (Figure (Figure3A).3A). In contrast, control cells exhibited spindle-like fibroblast morphology, with bundles of stress fibers paralleling the long axis of cells. Similar results were observed in TXNL2-KD BT549 and 4T1 cells (data not shown).
We then explored whether these morphological changes were associated with altered cell motility and invasiveness. To this end, Boyden chamber assays were conducted. TXNL2 knockdown in MDA-MB-231 cells inhibited cell migration and invasion in vitro (Figure (Figure3B).3B). TXNL2-KD cells also showed reduced migration capabilities in wound-healing assays (Supplemental Figure 3B). Furthermore, TXNL2-KD cells at the edges of the wound did not form lamellipodia and moved collectively toward the wound center, as shown by F-actin staining, while control cells displayed polarization toward the wound site and migrated individually (Supplemental Figure 3B). TXNL2 depletion in 4T1 (Supplemental Figure 3, C and D) and BT549 cells (data not shown) also led to impaired migration and invasion.
As small GTPase proteins are well known to be involved in stress fiber formation and cell movement (41), we measured Ras, RhoA, and Rac1 activity in control and TXNL2-KD cells using kinase activity assays. Activation of the focal adhesion kinase (FAK), which relays extracellular matrix signaling, was examined using Western blotting. As expected, activity/activation of Ras, RhoA, Rac1, and FAK was lower in TXNL2-KD cells than in control cells (Figure (Figure4A). 4A).
We next compared the transcriptional profiles of control and TXNL2-KD MDA-MB-231 cells using cDNA microarray analysis. As illustrated in Figure Figure4B,4B, genes with the highest fold changes are involved in cell survival and metastasis. Semiquantitative RT-PCR results corroborated that the invasion/metastasis inhibitors KISS-1 and TIMP3 were upregulated, while the invasion/metastasis promoters MMP-1 (42) and IL-1β (43) were downregulated in TXNL2-KD cells (Figure (Figure4C4C and Supplemental Table 1). Since TXNL2 knockdown induced a change from a mesenchymal to an epithelial shape, we then measured epithelial-mesenchymal transition (EMT) markers in control and TXNL2-KD cells. Although the expression of EMT transcriptional factors Snail, Slug, and Twist did not change in TXNL2-KD cells (Figure (Figure4C),4C), epithelial markers (E-cadherin and α-, β-, and γ-catenin) were upregulated, while mesenchymal markers (vimentin and α-SMA) were downregulated in TXNL2-KD cells (Figure (Figure4D).4D). Similar results were also observed in BT549 TXNL2-KD cells (Supplemental Figure 3E). In summary, our data suggest that TXNL2 knockdown in breast cancer cells results in reversal of EMT and inhibition of cell migration and invasion.
TXNL2 has Trx-like and Grx domains and can rescue the yeast grx3/4 mutant under oxidative stress (data not shown). We thus hypothesized that TXNL2 may play an important role in maintaining cell redox homeostasis in response to stress. To address this question, we first measured ROS levels using flow cytometry after dichlorofluorescein diacetate (DCFH-DA) staining in control and TXNL2-KD cells. ROS levels in TXNL2-KD MDA-MB-231 and BT549 cells were higher than those in control cells (Figure (Figure5A).5A). Apoptosis was also increased by TXNL2 depletion, as detected by Annexin V/propidium iodide (Annexin V/PI) staining, followed by flow cytometry (Figure (Figure5A)5A) and cleaved caspase-3 staining (Supplemental Figure 4A). Similar results were found in cells treated with the ROS inducers H2O2 and diamide (Figure (Figure5B). 5B).
GSH is the most abundant ROS scavenger in mammalian cells, and GSH/GSH disulfide (GSH/GSSG) equilibrium is a major indicator for oxidative stress (44). The GSH/GSSG ratio, commonly used to define oxidative stress in culture cells (45), was markedly lowered in TXNL2-KD cells (Supplemental Figure 4B). The GSH biosynthesis inhibitor buthionine sulfoximine (BSO), like TXNL2 shRNA, increased cell apoptosis and elicited an epithelial shape in MDA-MB-231 cells (Figure (Figure5C).5C). In contrast, N-acetyl-cysteine (NAC, a metabolic precursor of GSH) (46) partially inhibited apoptosis and restored fibroblast morphology in TXNL2-KD cells (Figure (Figure5D).5D). The same effects were shown in control and TXNL2-KD BT549 cells (Supplemental Figure 4C) treated with BSO and NAC. Taken together, these results suggest that TXNL2 modulates intracellular oxidative stress in cancer cells.
Both Trx-1 and Grx-1 are well-known ROS scavengers and involved in cellular redox regulation (30). PX-12, a small-molecule inhibitor of Trx-1 (24), did not inhibit growth or increase ROS levels in MDA-MB-231 cells, although it had a moderate effect on MCF-7 cells (Supplemental Figure 4, D and E). These results suggest that the effects of TXNL2 depletion were not primarily mediated by Trx-1 or Grx-1 inhibition. Of note, TXNL2 knockdown did not induce a significant increase of ROS levels in HMECs (data not shown), which may explain why TXNL2 knockdown did not affect the growth of HMECs.
Because the expression of NF-κB–regulated genes such as RELB, IL1B, IL8, BCL2A1, A20, and IKBA (47–49) was altered in TXNL2-KD cells (Figure (Figure6A)6A) and because NF-κB is regulated by ROS (50), we speculated that NF-κB activity might be downregulated by TXNL2 depletion. Thus, we examined NF-κB components by Western blotting. As illustrated in Figure Figure6B,6B, levels of total RelB and phosphorylated p65 (p-p65; Ser536) were reduced by TXNL2 knockdown, implicating lower NF-κB activity. Immunofluorescence staining also showed less p65 nuclear localization in TXNL2-KD cells (Figure (Figure6C6C and Supplemental Figure 5A), which was confirmed by immunoblotting with nuclear and cytosolic extracts (Figure (Figure6D).6D). Using the TransAM DNA-binding ELISA, we found that p65 and RelB DNA binding activity was suppressed in TXNL2-KD MDA-MB-231 and BT549 cells, while c-Rel activity was unaltered (Figure (Figure6E6E and Supplemental Figure 5, A and B). These results suggest TXNL2 depletion inhibits NF-κB activity. To determine the clinical relevance of this association between TXNL2 and p65, we further examined the nuclear localization of p65, which has been used to recognize the active state of NF-κB (51, 52). In 190 human breast cancer specimens, immunohistochemical analysis demonstrated that TXNL2 positivity was significantly associated with the nuclear expression of p65 (Table (Table2). 2).
In line with the inactivation of p65 by TXNL2 deficiency, immunoblotting showed that levels of IκBα, an NF-κB inhibitor causing p65 cytoplasmic retention, were increased, whereas IκBα phosphorylation at Ser32 (a key signal for IκBα degradation) was decreased in TXNL2-KD MDA-MB-231 cells (Figure (Figure6F).6F). This suggests stabilization of IκBα protein. IKK activity is reportedly inhibited by oxidative stress (53). Not surprisingly, lower IKK activity was found in TXNL2-KD cells (Figure (Figure6F),6F), as shown by less IκBα phosphorylation in IKK kinase assays (54, 55). Although AP-1 transcriptional factors can also be regulated by redox state (56), their activity was not consistently and significantly inhibited in TXNL2-KD cells (data not shown).
Given that high levels of GSSG can convert the NF-κB heterodimers into an oxidized form with little or no DNA binding activity (57) and that TXNL2-KD cells contain low GSH/GSSG ratio, we investigated whether NF-κB was oxidized in these cells. GSH immunoprecipitation followed by immunoblotting indicated increased glutathionylation of p65 and RelB (Figure (Figure6G),6G), which would decrease their activity. Of note, actin displayed increased glutathionylation in TXNL2-KD cells, as indicated by the increased amount of immunoprecipitated actin by GSH antibody. In line with these results, the nuclear localization of p65 and RelB as well as the expression of NF-κB–regulated genes (see Figure Figure6A)6A) in TXNL2-KD cells was rescued by NAC treatment (Figure (Figure6H6H and Supplemental Figure 5C).
To further rule out potential off-target effects of shRNA, we suppressed TXNL2 expression by siRNA that had TXNL2-targeting sequences distinct from those of shRNA (see Methods and Supplemental Table 2). As expected, NF-κB activity decreased and ROS levels increased (Supplementary Figure Figure5D).5D). Furthermore, ectopic expression of the mouse Txnl2 gene in TXNL2-KD MDA-MB-231 cells restored levels of nuclear p65, p-p65, IκBα, and EMT markers and reversed fibroblast morphology (Supplemental Figure 5E). Collectively, these observations demonstrate that TXNL2 depletion inhibits NF-κB activity by increasing ROS levels.
Because NF-κB regulates cell functions, including EMT (58), we asked whether NF-κB mediates the effects of TXNL2 knockdown. When the IKK inhibitor BMS-345541 was used to block NF-κB activity in MDA-MD-231 and BT549 cells, the fibroblast morphology changed to a cuboidal shape (Figure (Figure7A)7A) and was accompanied by changes in the expression of NF-κB–regulated genes (Figure (Figure7B).7B). These BMS-induced phenotypes were reminiscent of those in TXNL2-KD cells. Similar results were found in MDA-MB-231 cells treated by other NF-κB inhibitors, such as BAY-117082 and JSH-23 (data not shown). As expected, pharmacologic inhibition of NF-κB inhibited cell growth, migration, and invasion (Supplemental Figure 6A).
We next examined Akt activation by immunoblotting p-Akt, since Akt is reportedly involved in the stress response (59). Akt activation was lower in TXNL2-KD MDA-MB-231 (Figure (Figure7C)7C) and BT549 (Supplemental Figure 6B) cells. Blocking Akt activity using Akt inhibitor IV (AI-IV), unlike NF-κB blockade, did not induce mesenchymal-epithelial transition (MET) in both cell lines (Supplemental Figure 6C) but elicited apoptosis and growth inhibition (Figure (Figure7D).7D). This suggests that Akt may be involved in mediating the effect of TXNL2 on cell survival but not the EMT process in breast cancer cells.
Akt can be downstream (60) or upstream of NF-κB (61). To address this, we generated IκBα S32A/S36A super-repressor–overexpressing (IκBα-SR-overexpressing) MDA-MD-231 cells. As shown in Figure Figure7E,7E, p-Akt was downregulated in these cells, which also displayed MET. In agreement with this, p65 nuclear localization and p-Akt were markedly reduced by the IKK inhibitor BMS. In contrast, p65 nuclear localization was not blocked by AI-IV (Figure (Figure7F).7F). To further investigate the mechanism of p65 in TXNL2-regulated signaling, we overexpressed p65 in TXNL2-KD MDA-MB-231 cells. RelB expression, which is a known target of p65, was dramatically increased, whereas TXNL2 expression was not upregulated by NF-κB (Supplemental Figure 6D). Notably, the p-Akt level was induced by p65 overexpression, further indicating that NF-κB mediates the effect of TXNL2 on Akt activation. Fibroblast morphology and invasion ability were also restored in p65-overexpressing TXNL2-KD MDA-MB-231 cells. Together with our findings that Snail, Slug, and Twist were not inhibited by TXNL2 depletion, these data suggest that NF-κB mediates the effects of TXNL2 on breast cancer cells and that Akt acts downstream of NF-κB to regulate cell survival and proliferation (Figure (Figure7G). 7G).
We next tested whether TXNL2 knockdown inhibits tumor growth in vivo. Control and TXNL2-KD MDA-MB-231 cells were injected orthotopically into the mammary fat pads of SCID mice and tumor growth was examined. Mice injected with control cells formed large tumors within 37 days, whereas mice injected with TXNL2-KD cells showed greatly reduced tumor growth (Figure (Figure8A).8A). These results indicate that TXNL2 expression is necessary for optimal growth of breast cancer cells in the mammary fat pads of mice. Because MDA-MB-231 cells have a high metastatic potential in vivo (62), we examined the lungs from mice with control and TXNL2-KD mammary tumors. As shown in Figure Figure8B,8B, TXNL2 depletion substantially suppressed the formation of lung metastases after injection of MDA-MB-231 cells in the mammary fat pads.
Because the reduced lung metastasis might be due to slower primary tumor growth, we further evaluated the in vivo effects of TXNL2 on breast cancer lung metastasis, using an experimental metastasis assay. Control or TXNL2-KD cells were injected into the lateral tail veins of SCID mice. After 4 weeks, the average number of metastatic nodules per lung was 120 in mice injected with control cells but only 30 in mice injected with TXNL2-KD cells (Figure (Figure8C).8C). The lungs from control mice were not extensively stained by black India ink, a commonly used method to visualize pulmonary macrometastases, as the lung tissue was replaced by tumor cells. In contrast, the lungs from mice injected with TXNL2-KD cells were stained by the dye. Our data indicate that TXNL2 controls the highly metastatic behavior of MDA-MB-231 cells.
Given the above results, we further examined the clinical significance of TXNL2 overexpression in primary human breast cancer, using gene expression microarray analysis of a 192-sample data set with known metastasis profiles (63). Elevated TXNL2 expression was positively associated with lung metastasis (P = 0.0097) and brain metastasis (P = 0.0267) and significantly correlated with shorter lung metastasis-free survival (P < 0.0001; Figure Figure9A)9A) and brain metastasis-free survival (P < 0.011; Figure Figure9B).9B). This statistically significant association was consistently observed at multiple arbitrary percentile cutoff points (25th, 50th, 75th, and 90th) used for purposes of creating dichotomous groups to facilitate data analysis. The risk of developing lung metastasis was 44.1% for each unit increase relative to the TXNL2 mRNA level (hazard ratio 1.441, 95% CI, 1.217–1.707). Thus, TXNL2 may serve as a prognostic marker for breast cancer metastasis. Analysis of 2 additional data sets (64, 65) lent further support to the clinical impact of enhanced TXNL2 mRNA expression. Elevated TXNL2 expression was found to correlate with decreased recurrence-free survival (P < 0.003; Figure Figure10A),10A), decreased distant-metastasis free survival (P < 0.012; Figure Figure10A),10A), decreased disease-specific survival (P < 0.005; Figure Figure10B),10B), and decreased overall survival (P < 0.030; Figure Figure10B).10B). Thus, TXNL2 may serve as a prognostic marker for breast cancer metastasis and survival.
Although increased TXNL2 expression in various solid tumors has been reported in another study (66), our study is the first to our knowledge to investigate TXNL2 function in cancer cells. We found that TXNL2 knockdown increased intracellular ROS levels and decreased the GSH/GSSG ratio, thereby inhibiting cell growth, survival, and invasion. These TXNL2 effects were mediated by NF-κB signaling (see Figure Figure7G).7G). Xenograft models showed that TXNL2 deficiency inhibited tumor growth and lung metastasis. The correlation between elevated TXNL2 expression and metastasis to the lung or brain was demonstrated using gene expression microarray analysis. Our data suggest that TXNL2 may be a critical factor for redox homeostasis and breast cancer tumorigenesis and metastasis and may serve as a therapeutic target.
Cancer cells generate higher ROS levels, partially due to a higher metabolic rate, but cancer cells have strong ROS scavenging systems (67). Upsetting this balance by further increasing ROS generation or inhibiting antioxidative capacity can cause cell death. As mentioned above, TXNL2 is consistently overexpressed in many breast cancer cell lines. Further increasing its levels in noninvasive MCF-7 cells only mildly enhanced cell growth and invasion (our unpublished observations). We postulate that TXNL2 is essential but may not be sufficient to induce aggressive behavior associated with tumor cells.
TXNL2, although lacking the conserved Cys-Gly-Pro-Cys Trx motif that is essential for catalytic activity (29), may be able to modulate ROS levels via its Grx domains. A recent structural analysis of the TXNL2 yeast homolog confirmed that the cysteine in Grx domain may have catalytic activity (68). This is also supported by a recent study, indicating that TXNL2 binds 2 bridging [2Fe-2S] clusters in a homodimeric complex with the active site Cys residues of its 2 Grx domains and GSH bound non-covalently to the Grx domains (69).
Recent studies indicate that TXNL2 is able to protect yeast cells against oxidative stress and that TXNL2 deficiency causes embryonic lethality (34) (our unpublished observations). By using a yeast 2-hybrid technique, we identified several TXNL2-interacting proteins, including oxidation-regulating proteins such as metallothionein 2A, malate dehydrogenase 1, and cystatin C (our unpublished observations). These results implicate TXNL2 in cellular responses to stress signals, particularly ROS. Mechanically, how TXNL2 controls ROS levels is not understood. The increase of ROS by TXNL2 depletion may lead to a lower GSH/GSSG ratio. Alternatively, TXNL2 may regulate ROS levels through first modulating the GSH/GSSG pool. Both scenarios can be supported by our findings that the GSH precursor NAC restored TXNL2-impaired cell functions, while the GSH synthesis inhibitor BSO mimicked the effect of TXNL2 knockdown.
Constitutive NF-κB DNA binding activity has been reported in 65% of primary breast cancers (70, 71) and is also associated with other malignancies (72). Our studies showed that TXNL2-KD unregulated proapoptotic genes, such as CRADD (73), downregulated antiapoptotic genes, such as BCL2A1 (74), and indirectly decreased Akt activity via reduced NF-κB activity. These results further support that NF-κB plays a central role in mediating the effects of TXNL2 on cell survival.
Notably, MMP1 emerges as a strongly downregulated gene, while KISS1 is a strongly upregulated gene in TXNL2-KD cells. MMP-1 is reportedly induced by NF-κB (48, 75) and is a potential prognostic marker of breast cancer (76). Recently, MMP-1 was found to promote breast cancer cell metastasis to bone (42, 62, 77) and lung (78). Consistent with this notion, our experimental metastasis assays showed that TXNL2 knockdown inhibited lung metastasis. KISS-1 is a well-established cancer metastasis inhibitor (79). The MET process, which is also associated with less aggressive cancer cell activity, is induced by inhibition of NF-κB, as shown in our and other studies (58, 80). In addition to the classical NF-κB pathway, RelB also inhibits breast cancer cell apoptosis and maintains the invasive phenotype and mesenchymal characteristics (81). Thus, suppression of RelB activity may also be involved in TXNL2 knockdown-induced effects.
One critical regulator of NF-κB is the IKKα/IKKβ/IKKγ complex, which phosphorylates IκBα and thereby releases NF-κB. Our data showed that IKK activity was significantly downregulated in TXNL2-KD cells, consistent with the previous finding that IKKβ activity is regulated by redox state (53). Interestingly, the DNA binding activity and expression level of RelB were suppressed in TXNL2-KD cells. This may be due to reduced NF-κB activity, which controls RelB transcription (48), and reduced IKK activity (82), which governs RelB activation. This mechanism might explain why TXNL2 knockdown has a dramatic effect on NF-κB.
A high ratio of GSH/GSSG provides cells with a reducing environment and maintains proteins in a reduced state. An oxidative environment with high GSSG can promote protein-SSG formation (83). NF-κB activity can be suppressed by oxidation (84). A decrease in the GSH/GSSG ratio may create a more oxidative environment, which subsequently oxidizes NF-κB and diminishes its DNA binding activity (85). S-glutathionylation of the cysteine residues close to the DNA-binding loops inhibits p65 DNA binding activity (86, 87). A redox balance is required for optimal activation of NF-κB (88). It is known that ROS can activate transcription factors, including NF-κB (85). However, ROS accumulation beyond threshold levels can also inhibit NF-κB activity (89–91). We recently also showed that elesclomol, a novel small-molecule oxidative stress inducer, dramatically suppressed NF-κB activity in breast cancer cells by increasing ROS (92). We found that TXNL2 depletion increased ROS and oxidation of p65 and RelB (indicated by their glutathionylation) and inhibited NF-κB. This effect is similar to the effects of ROS inducers BSO, H2O2, and diamide and can be reversed by NAC. Although glutathionylation is reversible, an oxidizing environment due to low GSH/GSSG ratio in TXNL2-KD cells may prevent protein-SSG reduction and thus suppress NF-κB activity.
Additionally, IL-1β and IL-8 are inhibited in TXNL2-KD cells. Thus, feedback regulatory loops between cytokines and NF-κB may also play a role in regulation of NF-κB activity (93–95). Taken together, our results suggest that the regulation of NF-κB by TXNL2 may involve multiple mechanisms.
In summary, the data presented here demonstrate what we believe to be a novel and important role of TXNL2 in regulating cancer cell growth and metastasis through a redox signaling mechanism. The findings that TXNL2 depletion preferentially impacts cancer cells but does not affect normal mammary epithelial cells and that TXNL2 is a critical regulator of NF-κB in cancer cells may have significant clinical implications. Further investigation is warranted to determine whether targeting TXNL2 may be an effective approach to induce ROS in cancer cells.
Details regarding cell culture, immunofluorescence staining, in vitro migration/invasion, Western blotting, kinase assays, and RT-PCR are provided in the Supplemental Methods.
Immunohistochemistry was performed by using a highly sensitive streptavidin-biotin-peroxidase detection system with breast cancer tissue microarrays and an in-house generated TXNL2 monoclonal antibody (1:200 dilution). Immunohistochemistry of p65 was conducted using a monoclonal anti-p65 antibody (Santa Cruz Biotechnology Inc.). Positive or negative nuclear staining of p65 was assessed. The tissue microarray slide included 190 cases of breast tumors and 15 cases of normal breast tissues, with duplicate cores for each case (Shanghai Biochip Co.). Methyl green was used as a counterstain. The slides were evaluated microscopically, and the signal was scored using the Allred scoring system (96). The χ2 test was used to determine the association of TXNL2 staining with normal and tumor tissues.
shRNA constructs in pLKO.1-puro specifically targeting human and mouse TXNL2 sequences were purchased from Sigma-Aldrich. The human TXNL2 shRNA1 sequence is 5′-CCGGGAACGAAGTTATGGCAGAGTTCTCGAGAACTCTGCCATAACTTCGTTCTTTTTG-3′. The human TXNL2 shRNA2 sequence is 5′-CCGGGCTCTTTATGAAAGGAAACAACTCGAGTTGTTTCCTTTCATAAAGAGCTTTTTG-3′. The mouse TXNL2 shRNA1 sequence is 5′-CCGGCCGAAGCTGTTCCTGAAGTATCTCGAGATACTTCAGGAACAGCTTCGGTTTTTG-3′. The mouse TXNL2 shRNA2 is 5′-CCGGGCTAAAGAACACCCTCATGTTCTCGAGAACATGAGGGTGTTCTTTAGCTTTTTG-3′. Control cells were transfected with a control shRNA that does not match any known human or mouse coding cDNA. Stable knockdown clones were pooled and used for experiments.
Gene expression analysis of MDA-MB-231 cells and TXNL2-KD cells was performed using the Human OneArray (Phalanx Biotech Group). Detailed methods are described in the Supplemental Methods. The log2-transformed expression ratio (TXNL2 shRNA clones versus control clones) of each gene was calculated.
ROS generation was measured with DCFH-DA (Invitrogen). Cells were incubated with 5 μM DCFH-DA for 30 minutes, followed by flow cytometry using a FACSCalibur (BD Biosciences). The data were analyzed using Cell Quest software (BD Biosciences).
ROS generation was measured using DCFH-DA (Invitrogen) staining. Apoptotic cells were analyzed by flow cytometry using Annexin V–FITC and PI staining (BD Biosciences). FACSCalibur was used for flow cytometry, and the data were analyzed using Cell Quest software.
The NF-κB–responsive luciferase reporter construct in pGL4 was from Promega. The construct was cotransfected with the TXNL2 vector or the control vector. After 24 hours of incubation, the luciferase activity was measured and normalized by β-galactosidase activity (Promega). The data presented are the mean value of 3 independent experiments.
The transcription factor NF-κB family activation assay was measured using the TransAM NF-κB Family Kit (Active Motif), according to the manufacturer’s instructions.
All animal experiments were performed with the approval of Baylor College of Medicine and Tianjin Medical University animal care and use committees. Details are provided in the Supplemental Methods.
The expression of TXNL2 and its association with metastasis to the lung or brain were examined in breast cancer patients in a 192-patient data set (63). The Wilcoxon rank-sum test was used to assess statistical significance for this comparison. Brain-specific and bone-specific metastasis-free survival was also examined in this data set using the log-rank test, and survival plots were created using Kaplan-Meier methods. The prognostic significance of TXNL2 in predicting recurrence-free survival and distant metastasis-free survival in breast cancer patients was examined in the 189-sample (65) microarray data set. The prognostic significance of TXNL2 in predicting disease-specific survival and overall survival in breast cancer patients was examined in the 159-sample (64) microarray data set. Details regarding the gene expression microarray analysis for the above data sets are provided in the Supplemental Methods.
Values represented mean ± SD of samples measured in triplicate. Each experiment was repeated 3 times, unless otherwise indicated. The significance of differences between experimental groups was analyzed using the Student’s t test and 2-tailed distribution. The χ2 test was performed to determine the association between TXNL2 levels and p65 nuclear staining in human breast cancer samples.
We thank Dave Hoon and Myles Cabot for technical help. We thank Oncothyreon Inc. for providing PX-12. We thank Gwen Berry and Yukun Cui for helpful suggestions and critical reading of the manuscript. This work was supported by Susan G. Komen Breast Cancer Foundation (BCTR0601346 to X. Cui), Avon Foundation (02-2008-081 to X. Cui), Del Webb Foundation (to X. Cui), and the United States Department of Agriculture/Agricultural Research Service under Cooperation Agreement (6250-51000-055 to N.-H. Cheng).
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article: J Clin Invest. 2011;121(1):212–225. doi:10.1172/JCI43144.