TIPRL Regulates Stemness and Survival in Lung Cancer Stem Cells through CaMKK2-CaMK4-CREB Feedback Loop Activation

Abstract

Frequent recurrence and metastasis caused by cancer stem cells (CSCs) are major challenges in lung cancer treatment. Therefore, identifying and characterizing specific CSC targets are crucial for the success of prospective targeted therapies. In this study, it is found that upregulated TOR Signaling Pathway Regulator-Like (TIPRL) in lung CSCs causes sustained activation of the calcium/calmodulin-dependent protein kinase kinase 2 (CaMKK2) signaling pathway by binding to CaMKK2, thereby maintaining stemness and survival. CaMKK2-mediated activation of CaM kinase 4 (CaMK4) leads to phosphorylation of cAMP response element-binding protein (CREB) at Ser129 and Ser133, which is necessary for its maximum activation and the downstream constitutive expression of its target genes (Bcl2 and HMG20A). TIPRL depletion sensitizes lung CSCs to afatinib-induced cell death and reduces distal metastasis of lung cancer in vivo. It is determined that CREB activates the transcription of TIPRL in lung CSCs. The positive feedback loop consisting of CREB and TIPRL induces the sustained activation of the CaMKK2-CaMK4-CREB axis as a driving force and upregulates the expression of stemness- and survival-related genes, promoting tumorigenesis in patients with lung cancer. Thus, TIPRL and the CaMKK2 signaling axis may be promising targets for overcoming drug resistance and reducing metastasis in lung cancer.

Keywords: CaMKK2‐CaMK4‐CREB feedback loop activation; TIPRL; cancer stem cell; drug resistance; lung cancer; metastasis.

Figure 1

TIPRL expression is upregulated in lung cancer tissues and CSCs, correlating with poor prognosis. A) TIPRL levels in the tissues of patients with LUAD and LUSC compared with those in adjacent normal tissues, measured using immunoblotting (Figure S1A,B, Supporting Information); TIPRL band intensity was normalized to that of tubulin for each patient. B) TIPRL transcript levels in the tissues of patients with LUAD and SCLC, measured using qRT‐PCR (Figure S1C,D, Supporting Information); results were calculated as the ratio of the transcript level in the cancer tissue to that in the non‐cancer tissue after normalizing against the B2M level in each patient. C) TIPRL expression in lung cancer cell lines. TIPRL D) mRNA and E) protein levels in CD44⁺CD133⁺ and CD44^–CD133^– A549 cells, analyzed using qRT‐PCR and immunoblotting, respectively. F) Transcription levels of TIPRL, CD133, and CD44 in freshly isolated CSCs from lung cancer tissues, analyzed using qRT‐PCR; the mRNA level of each gene was normalized to that of B2M in each sample. G) TIPRL and CD133 expression in lung cancer and adjacent normal tissues, assessed using immunohistochemistry; red arrows indicate highly positive cells. TIPRL, target of rapamycin signaling pathway regulator; CD, cluster of differentiation; LUAD, lung adenocarcinoma; SCLC, small cell lung cancer; LUSC, lung squamous cell carcinoma; CSCs, cancer stem cells; B2M, β2‐microglobulin; qRT‐PCR, quantitative reverse‐transcription PCR.

Figure 2

TIPRL binds CaMKK2. Immunoblotting for A) TIPRL and B) CaMKK2 immunoprecipitated from A549 cells; normal IgG was used as a negative control. C) IP assay of A549 cells transfected with pTIPRL‐His and pHA‐CaMKK2 plasmids. D) GST pull‐down assay of recombinant TIPRL and CaMKK2. E) IP assay and ICC F) results for ICC HEK293 cells co‐transfected with pTIPRL‐His and pHA‐CaMKK2 or deletion mutants. G) ICC analysis for CD133⁺ A549 cells using antibodies against CaMKK2 and TIPRL. TIPRL, target of rapamycin signaling pathway regulator; IP, immunoprecipitation; ICC, immunocytochemistry; PP2Ac, catalytic subunit of protein phosphatase 2A; WCL, whole cell lysate; DAPI, 4′,6‐Diamidino‐2‐Phenylindole.

Figure 3

TIPRL depletion inactivates CaMKK2, downregulating the CaMKK2‐CaMK4‐CREB signaling pathway. Phosphorylation of A) CaMKK2 and B) CaMK4 immunoprecipitated from A549 cells transfected with the siRNA against TIPRL gene and control siRNA, analyzed using a phospho‐serine antibody. C) Phosphorylation of CaMK4 proteins by CaMKK2, as well as that of CREB by the activated CaMK4, in TIPRL‐depleted A549 cells, analyzed using an in vitro kinase assay with a phospho‐serine antibody. D) Immunoblotting of ionomycin‐ and CaCl₂‐stimulated TIPRL‐depleted A549 cells using phospho‐CREB antibodies. E) Immunoblotting of A549, H1299, H1793, and H460 lung cancer cells transfected with an siRNA against TIPRL gene. F) Protein analysis of cytosolic and nuclear fractions of TIPRL‐depleted A549 cells using antibodies against tubulin (cytosolic marker) and histone H3 (nuclear marker). G) Dual‐Luciferase assay of the indicated lung cancer cells co‐transfected with a CRE‐driven luciferase reporter plasmid and siRNA against TIPRL gene. H) FACSCanto II analysis of CD133‐positive siTIPRL‐transfected A549 cells after staining with phospho‐CREB antibodies. TIPRL, target of rapamycin signaling pathway regulator; CRE, cAMP response element; CREB, cAMP response element‐binding protein; CaMKK2, calcium/calmodulin‐dependent protein kinase kinase 2; CaMK4, calcium/calmodulin‐dependent protein kinase 4.

Figure 4

The CD133⁺ population of CSCs decreased significantly after TIPRL depletion. FACSCanto II analysis of CD133⁺ populations in A) siTIPRL‐transfected A549 cells and B) the stable TIPRL‐depleted A549‐sgTIPRL cell line. C) qRT‐PCR analysis to assess alterations in stemness‐related gene expression caused by TIPRL depletion in lung cancer cells. D) Soft agar and E) sphere‐formation assays of lung cancer cells transfected with an siRNA against TIPRL and control siRNA for 72 h; results show the number of colonies and spheroids generated per 10 000 counted after 3 and 2 weeks, respectively. Measurement of F) the mitochondrial membrane potential (TMRE staining), G) reactive oxygen species (ROS) levels (Mito‐SOX staining), H) Ca²⁺ levels (Rhod2‐AM staining), and I) ATP levels in CD133^– and CD133⁺ siTIPRL‐transfected A549 cells. TIPRL, target of rapamycin signaling pathway regulator; TMRE, tetramethylrhodamine; ROS, reactive oxygen species; CD, cluster of differentiation; siRNA, short interfering RNA; CaMKK2, calcium/calmodulin‐dependent protein kinase kinase 2.

Figure 5

TIPRL promotes the expression of CREB target genes, including Bcl2 and HMG20A, in lung CSCs. A) qRT‐PCR and B) immunoblotting analysis of CREB target gene expression in lung cancer cells transfected with siTIPRL or siCont. C) Immunoblotting analysis of the expression of Bcl2 and HMG20A in CD133⁺ TIPRL‐depleted A549 cells. D) qRT‐PCR analysis of CREB target gene expression in CD133^– and CD133⁺ cells lung cancer cell lines. E) FACSCanto II analysis to identify the CD133⁺ population of CSCs in TIPRL‐depleted A549 cells infected with HMG20A, Bcl2, or mock lentivirus. F) Soft agar and G) sphere‐formation assays; the number of colonies and spheroids were counted after 2 weeks. TIPRL, target of rapamycin signaling pathway regulator; ROS, reactive oxygen species; CREB, cAMP response element‐binding protein; CD, cluster of differentiation; qRT‐PCR, quantitative real‐time reverse‐transcription PCR; CSCs, cancer stem cells.

Figure 6

TIPRL depletion enhances cell death induced by afatinib. A) Cell death of lung CSCs in CD133^– and CD133⁺ TIPRL‐depleted A549 cells treated with the indicated dose of afatinib for 24 h, measured using FACSCanto II after staining with Annexin V‐FITC/PI. Sphere‐formation assay of B) siTIPRL‐transfected A549 cells and C) the A549‐sgTIPRL cell line treated with 20 µm afatinib for 24 h and incubated with 20 ng mL⁻¹ epidermal growth factor and 10 ng mL⁻¹ basic fibroblast growth factor for 3 weeks. A549‐sgTIPRL cells express to the EGFP supported as a selection tool for gene editing using a CRISPR/Cas9. The generated spheroids were analyzed using fluorescence microscopy to detect the GFP‐expressing cells. D) Images of tumors in TIPRL‐depleted mice subjected to the following treatments: sgCont, sgCont+afatinib, sgTIPRL, and sgTIPRL+afatinib; n = 5 each treatment group. E) Mean tumor volumes recorded at 3‐day intervals ± standard error. F) Terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick‐end labeling staining of tumor samples from severe combined immunodeficiency mice intrathoracically injected with CD133⁺ A549‐sgCont and sgTIPRL cells to determine the effects on lung metastasis. G) Analysis of tumor growth and metastasis after 7 weeks post‐injection using a nano positron emission tomography‐magnetic resonance imaging system with ¹⁸F‐FDG. H) Mean SUV ratio in the lungs. I) Survival curves for animals from the two groups. TIPRL, target of rapamycin signaling pathway regulator; CD, cluster of differentiation; CSCs, cancer stem cells; FITC, fluorescein isothiocyanate; PI, propidium iodide; SUV, standard uptake value; PET, positron emission tomography; MRI, magnetic resonance imaging; ¹⁸F‐FDG, ¹⁸F‐fludeoxyglucose.

Figure 7

TIPRL is transcriptionally activated by CREB, leading to sustained activation of the CaMKK2 signaling axis via a positive feedback loop. A) Comparison of TIPRL and CREB1 expression in the TCGA lung cancer cohort. B) TIPRL promoter activity in HEK 293 cells co‐transfected with TIPRL promoter‐driven luciferase reporter plasmids and His‐tagged‐CREB‐overexpressing plasmids. C) qRT‐PCR results showing the association of CREB with the TIPRL promoter in HEK 293 cells co‐transfected with pCaMK4‐flag and wild‐type or mutant forms of CREB‐His (CREB S129A, S133A, and S129/133A), followed by a cut‐and‐run assay using a His antibody. D) Results of a cut‐and‐run assay of TIPRL‐depleted A549 cells using a CREB antibody or normal IgG. qRT‐PCR primer location and CRE‐binding site on the promoter of the TIPRL gene. E) mRNA expression, determined by qRT‐PCR with primers against TIPRL, CD133, and CREB1 genes, and F) protein levels, determined through immunoblotting with antibodies against TIPRL and His, in HEK293 cells transfected with CREB1‐overexpressing plasmids or mock. Analysis of G) alterations in protein levels by immunoblotting with the indicated antibodies and H) cell death using FACSCanto after staining with Annexin V‐FITC/PI in A549 cells treated with the CREB inhibitor 666‐15 and afatinib for 24 h. I) Schematic illustration of the expression and mechanism of action of TIPRL and the CaMKK2‐CaMK4‐CREB axis in lung CSCs. In CSCs, TIPRL, Bcl2, and HMG20A were significantly upregulated by CREB, via activation of the CaMKK2‐CaMK4‐CREB signaling pathway. TIPRL, as a positive feedback regulator, activates the CaMKK2 axis, while elevated Bcl2 maintains the mitochondrial membrane potential and contributes to ATP production by the mitochondrial OXPHOS in lung CSCs. Thereby, the functions of the TIPRL‐CaMKK2 signaling loop seem to be critical for maintaining the self‐renewal and tumorigenic abilities of lung CSCs. CaMKK2, calcium/calmodulin‐dependent protein kinase kinase 2; TIPRL, target of rapamycin signaling pathway regulator; CSCs, cancer stem cells; OXPHOS, oxidation phosphorylation; CREB, cAMP response element‐binding protein; CD, cluster of differentiation; CaMK4, calcium/calmodulin‐dependent protein kinase 4; qRT‐PCR, quantitative real‐time reverse‐transcription PCR; FITC, fluorescein isothiocyanate; PI, propidium iodide; TCGA, The Cancer Genome Atlas.