Interaction of delta-like 1 homolog (Drosophila) with prohibitins and its impact on tumor cell clonogenicity

Abstract

Cancer stem cell characteristics, especially their self-renewal and clonogenic potentials, play an essential role in malignant progression and response to anticancer therapies. Currently, it remains largely unknown what pathways are involved in the regulation of cancer cell stemness and differentiation. Previously, we found that delta-like 1 homolog (Drosophila) or DLK1, a developmentally regulated gene, plays a critical role in the regulation of differentiation, self-renewal, and tumorigenic growth of neuroblastoma cells. Here, we show that DLK1 specifically interacts with the prohibitin 1 (PHB1) and PHB2, two closely related genes with pleiotropic functions, including regulation of mitochondrial function and gene transcription. DLK1 interacts with the PHB1-PHB2 complex via its cytoplasmic domain and regulates mitochondrial functions, including mitochondrial membrane potential and production of reactive oxygen species. We have further found that PHB1 and especially PHB2 regulate cancer cell self-renewal as well as their clonogenic potential. Hence, the DLK1-PHB interaction constitutes a new signaling pathway that maintains clonogenicity and self-renewal potential of cancer cells.

Implications: This study provides a new mechanistic insight into the regulation of the stem cell characteristics of cancer cells.

Figure 1. DLK1 interacts with the PHB1-PHB2 complex

(A) Whole-cell lysates (WCL) were prepared by solubilizing BE(2)C cells in the modified RIPA buffer and incubated with monoclonal anti-DLK1 N-terminus antibody. Naïve mouse IgG2b was used as the isotype control. Immune complexes were separated by 10% SDS-PAGE and visualized by silver staining. PHB2 was identified by mass spectrometry. Asterisks represent heavy and light chains of IgG. (B) Reciprocal immunoprecipitation to confirm the interaction between DLK1 and PHB proteins using specific antibodies against DLK1 N-terminus, PHB2, and PHB1, respectively. Clean-Blot™ IP Detection Reagents (Thermo Scientific) were used to eliminate cross-reaction with IgG heavy and light chains. BE(2)C cells were either maintained under normal culture conditions (normoxia) or under hypoxia (1% O₂, 16 hr) conditions. (C) Intracellular co-localization of DLK1 and PHBs as revealed by confocal microscopy. BE(2)C cells were fixed in cold methanol and then stained with anti-DLK1 + anti-PHB1 or anti-DLK1+anti-PHB2 as described in Materials and Methods. Nuclei were counterstained with To-PRO3. Images were acquired using a Zeiss LSM 510 Meta confocal microscope.

Figure 2. DLK1 cytoplasmic domain interacts with the PHB1-PHB2 complex

Whole-cell lysates (WCL) were prepared from ER cells overexpressing full-length DLK1 (DLK1-FL), DLK1 extracellular domain (DLK1-EC), DLK1 Y339F/S355A mutant (DLK1-DM), or DLK1 without cytoplasmic domain (DLK1-ΔIC). Expression of the different DLK1 constructs (indicated by arrows) was validated by Western blots (A). Interactions between each DLK1 construct and PHBs were examined by immunoprecipitation with anti-DLK1 N-terminus antibody (B) and anti-PHB2 antibody (C). DLK1 cytoplasmic domain interacts with PHB2 (D). HEK293 cells were transiently transfected with Flag-tagged full-length DLK1 (Flag-DLK1 FL), Flag-tagged DLK1 cytoplasmic domain (Flag-DLK1-Cyto), or the empty vector. WCL was subjected to immunoprecipitation using anti-Flag antibodies. DLK1 and PHB2 were then detected by Western blots.

Figure 3. Secreted DLK1 extracellular domain enhances DLK1-PHB interaction

(A) ER cells expressing DLK1 extracellular domain (ER-EC), ER cells with empty vector (ER-V), or BE(2)C cells were cultured for 48 hours to approximately 80% confluence and maintained in the serum-free medium for 24 hr. Secreted proteins in conditioned media (CM) were precipitated by trichloroacetic acid and then subjected to Western blot analysis. DLK1-EC was detected by anti-DLK1 N-terminus antibodies, and the WCL from BE(2)C was used as a positive control for full-length DLK1. (B) Secreted DLK1 extracellular domain enhances DLK1-PHB interaction. BE(2)C or 3T3-L1 cells were incubated overnight with a 1:1 mixture of the ER cell-conditioned medium (ER-EC or ER-V) and the fresh culture medium containing 10% FBS. WCLs from these CM-treated cells were subjected to immunoprecipitation using anti-DLK1 N-terminus antibodies. The presence of PHB2 was examined by Western blots. (C) Secreted DLK1 extracellular domain enhances clonogenicity. BE(2)C cells were plated at a clonal density (300 cells/well in 6-well plates) in a 1:1 mixture of the ER cell-conditioned medium (ER-EC or ER-V) and the fresh culture medium containing 10% FBS. After incubation for 10 days, tumor colonies were fixed and stained with Crystal Violet. (D) Colonies were counted and Clonogenic Efficiency or Plating Efficiency was calculated as # colonies/# input cells x 100 (mean ± sem, *p<0.05).

Figure 4. DLK1 modulates mitochondrial functions

(A–C) DLK1 regulates mitochondrial membrane potential. BE(2)C cells (DLK1-high) stably expressing DLK1-DM or DLK1-ΔIC (A) as well as those cells transduced with lentivirus expressing DLK1-targeting shRNA shDLK1-2 or shDLK1-6 (B) were stained with JC-1. Formation of J-aggregates (red fluorescence) indicates high mitochondrial membrane potential. The percentage of cells with J-aggregates (JC-1⁺) was calculated. Data shown are mean ± sem from three independent experiments. ***p<0.0001 versus vector control. Other fluorescence: green = GFP from viral vectors and blue = Hoechst 33342. (C) ER cells (DLK1-low) stably expression DLK1-FL or DLK1-EC were stained with JC-1, and fluorescence intensity of the J-aggregates was measured by FACS. CCCP treatment reduces mitochondrial membrane potential. (D) DLK1 regulates production of reactive oxygen species (ROS). BE(2)C cells were transduced with shDLK1-2, shDLK1-4, shDLK1-6 or vector and stained with mitochondrial ROS indicator CMH₂XROS. Cells were then fixed and analyzed by FACS for CMH2ROS fluorescence. One of the three independent experiments was shown. (E) Validation of DLK1 knockdown by Western blots.

Figure 5. The DLK1-PHB pathway regulates cancer cell stemness

BE(2)C cells were treated with pooled siRNA oligos specific for DLK1, PHB1, PHB2, and control (SMARTpool, Dharmacon). Efficiency of knockdown was examined by Western blots (A) and by quantitative RT-PCR (B), respectively. The qRT-PCR data were analyzed by one-way ANOVA (p<0.001). For pairwise comparison, *p<0.01 versus their respective siCtrl. (C) The DLK1-PHB pathway is required for maintaining self-renewal. BE(2)C cells were treated with siRNAs discussed in (A). Tumor sphere formation assay was performed by plating 5000 cells/well in polyHEMA-coated 12-well plates. Tumor cell spheres were allowed to grow for three days in the serum-free sphere medium. Data shown were mean numbers of spheres per well ± sem. Data in the left panel were first analyzed by one-way ANOVA (p<0.0001, n=6), followed by pairwise comparison (*p<0.0001 versus siCtrl and #p<0.01 versus siPHB1 or siDLK1). Data in the right panel were analyzed by unpaired, two-tailed Student’s t-test of siDLK1 versus siCtrl in each PHB group, *p<0.003, n=3. (D) The DLK1-PHB pathway is required for clonogenicity. siRNA-treated BE(2)C cells were plated at 300 cells/well in 6-well plats and were cultured for 10 days. Colonies were fixed and stained with Crystal Violet and counted. Clonogenic Efficiency = % colonies per input ± sem (n=6). Data in the left panel were first analyzed by one-way ANOVA (p<0.0001), followed by pairwise comparison (*p<0.0001 versus siCtrl and #p<0.01 versus siPHB1 or siDLK1). Data in the right panel were also analyzed by one-way ANOVA (p<0.03 for the siDLK1 group). For comparison with Vector/siCtrl, *p<0.04 (Student’s t-test).