Inactivation of p38 MAPK contributes to stem cell-like properties of non-small cell lung cancer

Abstract

Cancer stem cells (CSCs) are recognized as the major source for cancer initiation and recurrence. Yet, the mechanism by which the cancer stem cell properties are acquired and maintained in a cancer cell population is not well understood. In the current study, we observed that the level of active p38 MAPK is downregulated, while the level of the stemness marker SOX2 is upregulated in lung cancer tissues as compared to normal tissues. We further demonstrated that inactivation of p38 is a potential mechanism contributing to acquisition and maintenance of cancer stem cell properties in non-small cell lung cancer (NSCLC) cells. p38, in particular the p38γ and p38δ isoforms, suppresses the cancer stem cell properties and tumor initiating ability of NSCLC cells by promoting the ubiquitylation and degradation of stemness proteins such as SOX2, Oct4, Nanog, Klf4 and c-Myc, through MK2-mediated phosphorylation of Hsp27 that is an essential component of the proteasomal degradation machinery. In contrast, inactivation of p38 in lung cancer cells leads to upregulation of the stemness proteins, thus promoting the cancer stem cell properties of these cells. These findings have demonstrated a novel mechanism by which cancer stem cell properties are acquired and maintained in a cancer cell population, and have revealed a new function of the p38 pathway in suppressing cancer development. These studies have also identified a new pathway that can potentially serve as a target for cancer therapies aimed at eliminating CSCs.

Keywords: Hsp27; MK2; lung cancer stem cells; p38; stemness markers.

Figure 1. Activated p38 negatively regulates the expression of stemness proteins in non-small cell lung cancer (NSCLC)

(A) IHC staining of SOX2 and p-p38 in serial sections of a human lung cancer tissue array containing 153 intact NSCLC tissues and 31 NAT/normal lung tissues. The scatter diagram shows quantification of area of positive staining for SOX2 and p-p38 in normal and tumor tissues, which is calculated by multiplying staining area (scored as 1, 2, 3, and 4, 1: 0–25%, 2: 25%–50%, 3: 50%–75%, 4: 75%–100%, of positive tissue area) with staining intensity (scored as 1, 2, 3, and 4 based on color). *** indicates significant difference with P < 0.001 vs normal tissues in Mann-Whitney test. (B) Negative correlation between the expression levels of p-p38 and SOX2 in three non-small cell lung cancer (NSCLC) cell lines, as detected by Western blot analysis. (C) Western blot analysis of A549 and H460 cells transduced with constitutively active MKK3 or MKK6 (MKK3E/6E) or vector control (BabePuro, BP) or H460 and H1299 cells transduced with dominant negative MKK3 or MKK6 (MKK3A/6A) or vector control (BabeHygro, BH), showing that p38 activation leads to downregulation, while p38 inhibition leads to upregulation, of stemness protein expression. Arrow indicates the Klf4 band.

Figure 2. Activated p38 downregulates the stem cell properties of NSCLC cells

(A) Flow cytometry showing the percentage of the side population detected by Hoechst33342 staining in A549 and H460 cells transduced with vector control (BP), MKK3E or MKK6E and H460 and H1299 cells transduced with vector control (BH), MKK3A or MKK6A. Bar graphs show quantification of the percentage of side population. (B) Sphere formation assay of A549 and H460 cells transduced with vector control (BP), MKK3E or MKK6E and H460 and H1299 cells transduced with vector control (BH), MKK3A or MKK6A. Scar bar, 100 μm. Bar graphs show quantifications of the number and diameter of the spheres, respectively. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 vs BP or BH control in Student's t-test.

Figure 3. Activated p38γ and p38δ suppress the stem cell properties of NSCLC cells

(A) Western blot analysis of the stemness markers in A549 and H460 cells transduced with vector control (BP), p38aWT, p38αD176A, p38bWT, p38βD176A, p38gWT, p38γD179A, p38dWT, or p38δF324S, and in H460 and H1299 cells transduced with shRNA for GFP or indicated p38 isoforms. (B) Quantification of percentage of the side population in A549 and H460 cells transduced with vector control (BP), p38aWT, p38αD176A, p38bWT, p38βD176A, p38gWT, p38γD179A, p38dWT, or p38δF324S, and in H460 and H1299 cells transduced with shRNA for GFP or indicated p38 isoforms, as determined by flow cytometry. (C) Limit dilution xenograft tumor formation assay, showing number of tumors arising after subcutaneous injection of indicated numbers of A549 cells transduced with vector control (BP), p38γD179A or p38δF324S into 8 injection sites in Nod-scid mice. (D, E) The growth curves of xenograft tumors formed by 1 × 10⁶ (D) or 5 × 10⁵ (E) of A549 cells transduced with vector control (BP), p38γD179A or p38δF324S. Tumor volumes = length × width²/2. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 vs BP or shGFP control in Student's t-test.

Figure 4. Activated p38 MAPK reduces protein stability of the stemness proteins and promotes ubiquitylation and proteasome-mediated degradation of SOX2

(A) Relative mRNA levels of the stemness proteins in A549 cells transduced with vector control (BP), MKK3E or MKK6E and H1299 cells transduced with vector control (BH) or MKK6A, as determined by quantitative real time PCR analysis. ns indicates no significant difference with P > 0.05, * indicates significant difference with P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001 vs BP or BH control in Student's t-test. (B) Western blot analysis of the protein stability of the stemness proteins in A549 cells transduced with vector control (BP) or MKK3E and H1299 cells transduced with vector control (BH) or MKK6A after treated with cycloheximide for indicated time. The pan-ERK was as a negative control. (C) Quantification of the results in (B) using ImageJ. (D) Western blot analysis of A549 cells transduced with vector control (MCS) or HA-SOX2 and vector control (BP) or MKK3E, and H1299 cells transduced with vector control (MCS) or HA-SOX2 and vector control (BH) or MKK6A. (E) Western blot analysis of HA-SOX2 immunoprecipitated from A549 cells transduced with vector control (MCS) or HA-SOX2 and vector control (BP) or MKK3E and H1299 cells transduced with vector control (MCS) or HA-SOX2 and vector control (BH) or MKK6A, with (+) or without (−) treatment with MG132 (IP). Ubiquitylated SOX2 was detected by an anti-ubiquitin antibody, while total SOX2 was detected by an anti-SOX2 antibody. Part of the lysate input (IB) was subjected to direct Western blotting using an anti-HA antibody.

Figure 5. Activated p38γ and p38δ reduce the protein stability of the stemness proteins

(A) Western blot analysis of the protein stability of the stemness proteins in H460 cells transduced with vector control (BP), p38gWT, p38γD179A, p38dWT, or p38δF324S (upper panels), and in H460 cells transduced with shRNA for GFP, p38γ or p38δ (lower panels), after treated with cycloheximide for indicated time. (B) Quantification of results in (A) using ImageJ.

Figure 6. Activated p38 suppresses the stem cell-like properties of NSCLC cells through MK2-dependent phosphorylation of Hsp27 at Ser78 and Ser82

(A) Western blot analysis of Hsp27 phosphorylated at Ser15, Ser78 and Ser82 in A549 cells transduced with vector control (BP), p38gWT, p38γD179A, p38dWT, p38δF324S, MKK3E or MKK6E, and H1299 cells transduced with vector control (BH), MKK6A, or shRNA for p38γ or p38δ. (B) Western blot analysis of Hsp27 phosphorylated at Ser15, Ser78 and Ser82 in A549 cells transduced with a scrambled shRNA (SC) or shRNA for PRAK or MK2, and p38γD179A or p38δF324S. (C) Western blot analysis of the stemness proteins in A549 cells transduced with vector control (MCS), wild type Hsp27 (Hsp27-WT) or phosphomimetic mutants of Hsp27 (Hsp27-S15D, Hsp27-DblD, Hsp27-TriD) (left panels), and in A549 cells transduced with vector control (MCS), wild type Hsp27 (Hsp27-WT) or phosphorylation-resistant mutants of Hsp27 (Hsp27-S15A, Hsp27-DblA, Hsp27-TriA) and MKK3E (right panels). (D) Flow cytometry analysis was performed to determine the percentage of the side population in A549 cells transduced with vector control (MCS), wild type Hsp27 (Hsp27-WT) or phosphomimetic mutants of Hsp27 (Hsp27-DblD, Hsp27-TriD), and in A549 cells transduced with vector control (MCS), wild type Hsp27 (Hsp27-WT) or phosphorylation-resistant mutants of Hsp27 (Hsp27-DblA, Hsp27-TriA) and MKK3E. (E) Quantification of the results in (D). *** indicates significant difference with P < 0.001 in Student's t-test.

Figure 7. p38γ and p38δ regulate the protein stability of the stemness proteins through phosphorylation of Hsp27, which enhances the interaction between Hsp27 and the stemness proteins, in NSCLC cells

(A, B) Western blot analysis of the protein stability of the stemness proteins in A549 cells transduced with vector control (MCS), wild type Hsp27 (Hsp27-WT) or phosphomimetic mutants of Hsp27 (Hsp27-TriD) (A), and in A549 cells transduced with vector control (MCS) or phosphorylation-resistant mutants of Hsp27 (Hsp27-TriA) and MKK3E (B), after treated with cycloheximide for indicated time. The bottom plots show quantification of the Western blotting results using ImageJ. (C) Western blot analysis of the stemness proteins present in Hsp27 immunoprecipitates from A549 cells transduced with vector control (MCS), wild type Hsp27 (Hsp27-WT) or phosphomimetic mutant of Hsp27 (Hsp27-TriD) (IP). Part of the lysate input was subjected to direct Western blotting (IB). (D) Western blot analysis of the stemness proteins present in Hsp27 immunoprecipitates from A549 cells transduced with MKK3E and vector control (MCS), wild type Hsp27 (Hsp27-WT) or unphosphorylatable mutant of Hsp27 (Hsp27-TriA) (IP). Part of the lysate input was subjected to direct Western blotting (IB).