Thr 163 Phosphorylation Causes Mcl-1 Stabilization when Degradation Is Independent of the Adjacent GSK3-Targeted Phosphodegron, Promoting Drug Resistance in Cancer

<div><p>The antiapoptotic Bcl-2 family member Mcl-1 is a PEST protein (containing sequences enriched in proline, glutamic acid, serine, and threonine) and is subject to rapid degradation via multiple pathways. Impaired degradation leading to the maintenance of Mcl-1 expression is an important determinant of drug resistance in cancer. Phosphorylation at Thr 163 in the PEST region, stimulated by 12-O-tetradecanoylphorbol acetic acid (TPA)-induced activation of extracellular signal-regulated kinase (ERK), is associated with Mcl-1 stabilization in BL41-3 Burkitt lymphoma cells. This contrasts with the observation that Thr 163 phosphorylation in normal fibroblasts primes glycogen synthase kinase (GSK3)-induced phosphorylation at Ser 159, producing a phosphodegron that targets Mcl-1 for degradation. In the present follow-up studies in BL41-3 cells, Mcl-1 degradation was found to be independent of the GSK3-mediated pathway, providing a parallel to emerging findings showing that Mcl-1 degradation through this pathway is lost in many different types of cancer. Findings in Mcl-1-transfected CHO cells corroborated those in BL41-3 cells in that the GSK3-targeted phosphodegron did not play a major role in Mcl-1 degradation, and a phosphomimetic T163E mutation resulted in marked Mcl-1 stabilization. TPA-treated BL41-3 cells, in addition to exhibiting Thr 163 phosphorylation and Mcl-1 stabilization, exhibited an ∼10-fold increase in resistance to multiple chemotherapeutic agents, including Ara-C, etoposide, vinblastine, or cisplatin. In these cancer cells in which Mcl-1 degradation is not dependent on the GSK3/phosphodegron-targeted pathway, ERK activation and Thr 163 phosphorylation are associated with pronounced Mcl-1 stabilization and drug resistance – effects that can be suppressed by inhibition of ERK activation.</p> </div

TPA-induced Mcl-1 stabilization is associated with increased resistance to various chemotherapeutic agents in BL41-3 cells.

<p><b>A:</b> BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours, followed by the addition of the indicated concentrations of etoposide. After 6 hours, PARP cleavage, Mcl-1 expression, and Thr 163 phosphorylation were assayed (ChemiDoc), where the values for PARP Cleavage (%) and the Decrease in Mcl-1 Expression (%) are indicated below the respective blots. <b>B:</b> BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours prior to the addition of the indicated concentrations of vinblastine. After 6 hours, PARP cleavage and Mcl-1 expression were assayed as in Panel A. <b>C:</b> BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours prior to the addition of the indicated concentrations of cisplatin. After 24 hours, PARP cleavage and Mcl-1 expression were assayed as in Panel A. The blot shown is representative of 3 independent experiments. The SE of the values shown in successive lanes for PARP Cleavage was 2, 2, 5, 11, 17, 0.2, 4, 2, 3, 4, 6, and 13, respectively. The Decrease in Mcl-1 Expression was calculated relative to untreated control cells; the SE of the other values shown was 10, 12, 18, 12, 7, 9, and 11.</p

Ara-C-induced Mcl-1 degradation and cell death are inhibited upon TPA-induced ERK activation in BL41-3 cells.

<p><b>A:</b> BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours, followed by the addition of the indicated concentrations of Ara-C. After 24 hours, PARP cleavage and Mcl-1 expression were assayed (ChemiDoc). A representative experiment is included (upper photograph) along with the average (± SE) of 5 independent experiments. Also shown for comparative purposes is the percentage of cells exhibiting morphological apoptosis, assayed with a concentration of 1 nM TPA applied 1 hour before the addition of Ara-C (mean of 3 independent experiments ± SE run in parallel with previous studies; some of the BL41-3 cell samples not treated with TPA were included in a previous publication comparing these to BL41 parental cells (Fig. 4 in reference <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047060#pone.0047060-Vrana2" target="_blank">[23]</a>). *significant difference in the presence of Ara-C as compared to its absence (p<0.05; ANOVA, post-hoc Holm-Sidak test). <b>B:</b> BL41-3 cells were incubated in the absence or presence of TPA (5 nM) for 0.5 hours, followed by the addition of the indicated concentrations of Ara-C. After 6 hours, PARP cleavage and Mcl-1 expression were assayed as in Panel A. The average of 4 experiments is shown below representative blots, where the values for Ara-C concentrations that had similar effects are shown together and overlined. For PARP Cleavage, the SE of the values shown in successive lanes was 1, 8, 7, 3, and 3, respectively. The Decrease in Mcl-1 Expression was calculated relative to untreated control cells; the SE of the values shown (other than the untreated control value) was 11, 8, 8, and 7. Linear regression analysis of the Decrease in Mcl-1 Expression versus PARP Cleavage for 0–10 micromolar Ara-C in the absence of TPA demonstrated an average slope of 1.2 [±0.49 (SE) for the 4 experiments)], with an average r² value of 0.87.</p

TPA-induced ERK activation, Thr 163 phosphorylation, and Mcl-1 stabilization occur rapidly and are subsequently downregulated. A:

<p>The phosphorylation sites at Thr 163 and Ser 159 in the human Mcl-1 protein are diagrammed. Thr 163 is subject to phosphorylation by MAP kinases, as seen upon TPA-induced ERK activation in BL41-3 cells. Ser 159 is subject to phosphorylation by GSK3. Mcl-1 is a PEST protein subject to rapid turnover, the half-life of decay being ∼3 hours in BL41-3 cells (stippling of lesser versus greater density indicates poor PEST and potential PEST sequences; PESTFIND). However, Mcl-1 exhibits striking stabilization upon TPA-induced ERK activation and Thr 163 phosphorylation in these cells. Mcl-1 is also subject to another posttranslational modification involving truncation of the extreme N-terminus (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047060#pone.0047060-Ding2" target="_blank">[28]</a> indicated with an arrowhead). This results in a closely spaced 42/40 kd doublet, where the lower 40 kd band lacks ∼16 amino acid residues and is the most abundant band present in BL41-3 cells. The doublet is best visualized on large format electrophoresis gels (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047060#pone.0047060.s001" target="_blank">Fig. S1B</a>) but can sometimes be detected on standard gels (Fig. 1C). As Thr 163 phosphorylation does not prevent N-terminal truncation, Mcl-1 stabilization in the presence of this phosphorylation is seen as slowed decay of the 40 kd band. <b>B:</b> BL41-3 cells were either left untreated or exposed to TPA (5 nM) for the indicated times, and monitored for expression of Thr 163 phosphorylated Mcl-1 (Mcl-1 pT163), total Mcl-1, phosphorylated ERK (pERK), and GAPDH (ChemiDoc). All samples were run at the same time and subjected to the same autoradiographic exposure, where the black vertical line in this and subsequent figures indicates that lanes have been rearranged to facilitate comparison. Thr 163 phosphorylation was also induced by lower concentrations of TPA (e.g., 1 nM; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047060#pone.0047060.s001" target="_blank">Fig. S1C</a>), and was inhibited by U0126 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0047060#pone.0047060.s001" target="_blank">Fig. S1D</a>). m <b>C:</b> BL41-3 cells were either left untreated or exposed to TPA (1 nM). Some cells were immediately exposed to CHX to monitor Mcl-1 protein decay on Day 0. Additional TPA-treated cells were incubated for 24 hours (Day 1 after TPA Addition) and then exposed to CHX, where a portion of these cells was retreated with TPA at this time. The untreated cell sample (Time 0) is identical for each pair of Western blots. The blot at the bottom confirmed that pERK was reduced at 24 hours.</p

Mcl-1 degradation is slowed by a T163E mutation (but not T163A) in transfected CHO cells.

<p><b>A:</b> CHO cells were transfected with WT-Mcl-1 and incubated in the presence of LiCl (+LiCl; 20 mM) or in its absence (−LiCl) where 20 mM NaCl was added in the latter case. After 10 hours, CHX was applied and expression of the introduced WT-Mcl-1 gene product, and endogenous beta-catenin and GAPDH, was assayed after the indicated times (ChemiDoc). The half-life of Mcl-1 decay was estimated to be ∼4 hours in cells exposed to LiCl, and 3.7 hours in the controls exposed to NaCl. No substantial difference was seen in parallel cells not exposed to NaCl or LiCl. The blot shown is representative of three independent experiments. <b>B–C:</b> CHO cells were transfected with the indicated constructs, replated on the following day, and incubated for a 24-hour period to allow expression. CHX was then added and expression of the introduced Mcl-1 gene product and endogenous beta-tubulin was assayed after the indicated times by Western blotting. In Panel B, the decline in Mcl-1 expression at 3 hours in 2 independent experiments ranged from 53–66% with WT-Mcl-1, 25–54% with Mcl-1-S162A, and 25–50% with Mcl-1-T163A. While the decline in expression with WT-Mcl-1 appeared be slightly greater than that seen with Mcl-1-T163A, this was not a consistent finding. A short autoradiographic exposure is also shown because additional bands were detected at the high levels of expression obtained with Mcl-1-T163E. At the end of the 12-hour observation period, expression of Mcl-1-T163E was decreased by ∼15% in Panel B and ∼27% in Panel C, as estimated from short autoradiographic exposures. <b>D:</b> AKR-2B cells transfected with the indicated constructs were replated on the following day, at which time cell viability (trypan blue dye exclusion) was 88–92% in untransfected as well as transfected cultures. One day later, cells were exposed to 25 micrograms/ml CHX and assayed after the indicated times for expression of the introduced Mcl-1 gene product or endogenous actin by Western blotting. Duplicate plates are shown in adjacent lanes.</p

Mcl-1 degradation is insensitive to the GSK3 inhibitor LiCl in BL41-3 cells.

<p><b>A:</b> BL41-3 cells were either left untreated or exposed to TPA (20 nM) and/or LiCl (20 mM, or NaCl as a control). After 18 hours, expression of Mcl-1, beta-catenin, and GAPDH and was assayed by Western blotting. Comparable results were observed when the cells were exposed to TPA for 30 minutes instead of 18 hours (not shown). <b>B:</b> BL41-3 cells were exposed to the indicated concentrations of LiCl (or KCl or NaCl as controls), and assayed for expression of Mcl-1 and beta-catenin after 24 hours. A large format gel that separates the Mcl-1 doublet bands was used. Slight inhibition of cell growth was seen at 10–20 mM LiCl (13 and 27%, respectively as compared to 5% with 20 mM NaCl). Results similar to those shown were obtained with an exposure time of 12 hours or a LiCl concentration of 40 mM (not shown). <b>C:</b> BL41-3 cells were incubated in the absence or presence of LiCl (20 mM) for 0.5 hours, at which time CHX was applied. Expression of Mcl-1, beta-catenin, and GAPDH was assayed after the indicated times. No substantial difference was seen in parallel cells exposed to NaCl (20 mM, not shown) instead of LiCl.</p

Table_2_Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.docx

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.</p

DataSheet_1_Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.docx

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.</p

Table_1_Intratumoral CXCR4hi neutrophils display ferroptotic and immunosuppressive signatures in hepatoblastoma.docx

Pediatric hepatoblastoma (HB) is the most common primary liver malignancy in infants and children. With great diversity and plasticity, tumor-infiltrating neutrophils were one of the most determining factors for poor prognosis in many malignant tumors. In this study, through bulk RNA sequencing for sorted blood and tumor-infiltrated neutrophils and comparison of neutrophils in tumor and para-tumor tissue by single-cell sequencing, we found that intratumoral neutrophils were composed of heterogenous functional populations at different development stages. Our study showed that terminally differentiated neutrophils with active ferroptosis prevailed in tumor tissue, whereas, in para-tumor, pre-fate naïve neutrophils were dominant and ferroptotic neutrophils dispersed in a broad spectrum of cell maturation. Gene profiling and in vitro T-cell coculture experiment confirmed that one of main functional intratumoral neutrophils was mainly immunosuppressive, which relied on the activation of ferroptosis. Combining the bulk RNA-seq, scRNA-seq data, and immunochemistry staining of tumor samples, CXCL12/CXCR4 chemotaxis pathway was suggested to mediate the migration of neutrophils in tumors as CXCR4 highly expressed by intratumoral neutrophils and its ligand CXCL12 expressed much higher level in tumor than that in para-tumor. Moreover, our study pinpointed that infiltrated CXCR4hi neutrophils, regardless of their differential distribution of cell maturation status in HB tumor and para-tumor regions, were the genuine perpetrators for immune suppression. Our data characterized the ferroptosis-dependent immunosuppression energized by intratumoral CXCR4 expression neutrophils and suggest a potential cell target for cancer immunotherapies.</p