Dependency of human and murine LKB1-inactivated lung cancer on aberrant CRTC-CREB activation

  1. Xin Zhou
  2. Jennifer W Li
  3. Zirong Chen
  4. Wei Ni
  5. Xuehui Li
  6. Rongqiang Yang
  7. Huangxuan Shen
  8. Jian Liu
  9. Francesco J DeMayo
  10. Jianrong Lu
  11. Frederic J Kaye
  12. Lizi Wu  Is a corresponding author
  1. Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, United States
  2. UF Health Cancer Center, United States
  3. Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, United States
  4. UF Genetics Institute, United States
  5. State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, China
  6. Zhejiang University-University of Edinburgh Institute (ZJU-UoE Institute), Zhejiang University School of Medicine, International Campus, Zhejiang University, China
  7. Reproductive & Developmental Biology Laboratory, National Institute of Environmental Health Sciences (NIEHS), United States
  8. Department of Medicine, University of Florida College of Medicine, United States
7 figures, 1 table and 2 additional files

Figures

Figure 1 with 3 supplements
Three members of the CRTC co-activator family, CRTC1, CRTC2, and CRTC3, are expressed at varying levels in human lung epithelial and cancer cell lines.

(A) The transcript levels of the three CRTC genes were determined by RT-qPCR assays. All three CRTC transcript levels were normalized against the level of the housekeeping gene GAPDH individually. …

Figure 1—figure supplement 1
CRTC1, CRTC2, and CRTC3 showed predominantly de-phosphorylated, nuclear forms in LKB1-null cells (A549), and phosphorylated, cytoplasmic forms in LKB1-expressing cells (H322).

(A) H322 cell lysates were treated with or without alkaline calf intestinal phosphatase (CIP, one unit per ug protein) for 60 min at 37°C. Samples were loaded together with untreated A549 and H322 …

Figure 1—figure supplement 2
LKB1 re-introduction to LKB1-null lung cancer cells (A549) induced the phosphorylation and cytoplasmic retention of CRTCs.

A549 cells were transduced with retroviruses generated from a pBabe-LKB1 construct or pBabe vector and then selected with puromycin (1.5 µg/ml) for 48 hr. (A) A549-LKB1 cell lysates were treated …

Figure 1—figure supplement 3
CRISPR/Cas9-mediated LKB1 knockout in LKB1-expressing lung cancer cells (H322) resulted in the dephosphorylation and nuclear translocation of CRTCs.

(A) Cas9-expressing H322 cells (H322-Cas9) were transduced with lentiviruses containing non-targeting gRNA (gNT) or two gRNAs targeting LKB1 (gLKB1-1, gLKB1-2). H322 gNT cell lysates were treated …

Figure 2 with 1 supplement
Individual knockouts of the CRTC family members in human LKB1-null lung cancer cells inhibit the CREB-mediated target gene expression and moderately affect cell viability and anchorage-independent growth.

(A) Western blot analysis of endogenous CRTC proteins in parental A549 cells, A549 cells stably transduced with non-targeting sgRNA, and two independent single knockout clones for each CRTC1, CRTC2, …

Figure 2—figure supplement 1
CRISPR/Cas9-edited alleles in two independent single knockout clones for each CRTC gene were validated by genomic DNA sequencing.

The altered sequences of CRTC1 (a), CRTC2 (b), and CRTC3 (c) genes in two independent single knockout clones were shown.

A dominant negative CRTC mutant (dnCRTC) interacted with CREB on the target gene promoters and blocked CRTC co-activation of CREB transcription.

(A) A diagram of CRTC co-activator and dnCRTC was shown. The dnCRTC consists of CRTC1 (1-55aa) followed by a nuclear localization signal (nls) and GFP, cloned into the retroviral pMSCV vector. (B) …

Figure 4 with 3 supplements
Gene expression profiling revealed dnCRTC repressed CRTC-CREB target gene expression.

(A) Western blotting confirmed dnCRTC-expressing and GFP-expressing control cells. (B, C) The heatmap and volcano plots showed gene expression changes in dnCRTC-expressing and GFP-expressing cells. …

Figure 4—figure supplement 1
Effects of dnCRTC expression on gene expression in LKB1- expressing and LKB1-null lung cancer cells.

Two LKB1-null (A549 and H157) and two LKB1-positive (H322 and H522) NSCLC cells were transduced with dnCRTC or control GFP retroviruses. Expression of the top dnCRTC-downregulated gene candidates …

Figure 4—figure supplement 2
CRISPR/Cas9-mediated LKB1 knockout in in LKB1-expressing lung cancer cells led to enhanced expression of multiple dnCRTC-regulated targets.

The Cas9-expressing H322 cells (H322-Cas9) were first generated and then transduced with lentiviruses containing non-targeting gRNA (gNT) or two gRNAs targeting LKB1 (gLKB1-1, gLKB1-2). The …

Figure 4—figure supplement 3
The Box and whisker plots show gene expression levels in LKB1 mutant (Mut) and wildtype (Wt) groups of lung adenocarcinoma (TCGA-LUAD, PanCancer Atlas) and lung squamous cell carcinoma (TCGA-LSCC, PanCancer Atlas).

The RNA-seq gene expression data from the TCGA-LUAD cohort (n = 503 cases with 428 WT, 75 mut) and the TCGA-LSCC cohort (n = 466 cases with 461 WT, five mut) were downloaded from cBioportal. The top …

Figure 5 with 1 supplement
dnCRTC expression suppressed the growth of LKB1-null but not LKB1-positive lung cancer cells.

(A) Two LKB1-null (A549 and H157) and two LKB1-positive (H322 and H522) NSCLC cells were transduced with dnCRTC or control GFP retroviruses. The MOI was optimized to obtain an infection rate of …

Figure 5—figure supplement 1
Effects of dnCRTC expression on the growth of human immortalized lung bronchial epithelial BEAS-2B cells and mouse LKB1-null NSCLC mLSCCLP cells.

(a) BEAS-2B cells were transduced with dnCRTC or GFP retroviruses and the dnCRTC and GFP expression were confirmed by western blotting. (b) The transduced dnCRTC- or GFP BEAS-2B cells were …

Figure 6 with 1 supplement
Expression of dnCRTC significantly inhibited the growth of LKB1-null NSCLC xenograft tumors.

(A–E) A549-luc were transduced with GFP control or dnCRTC for 72 hr and the transduced cells (1 × 106 per mouse) were injected subcutaneously to the right flanks of NOD/SCID mice. Tumor volumes of …

Figure 6—figure supplement 1
Expression of GFP and dnCRTC in the transduced human LKB1-null lung cancer cells at the time of tumor cell injection and in the resulting xenograft tumors.

(A) Western blot analysis of GFP and dnCRTC proteins in luciferase-expressing A549 (A549-luc) cells and xenograft tumors. Lane 1: A549-luc (parental); Lane 2: A549-luc transduced with GFP control …

Expression of dnCRTC reduced lung colonization of LKB1-null lung cancer cells.

(A–C) Luciferase-expressing LKB1-null A549 cells (A549-luc) were transduced with retroviruses expressing GFP control or dnCRTC for 72 hr, and transduced cells (2 × 106 cells per mouse) were …

Tables

Key resources table
Reagent type
(species) or
resource
DesignationSource or
reference
IdentifiersAdditional
information
Gene (Homo sapiens) STK11GenBankGene ID: 6794This gene is commonly known as LKB1 in the field
Gene (Homo sapiens)CRTC1GenBankGene ID: 23373
Gene (Homo sapiens)CRTC2GenBankGene ID: 200186
Gene (Homo sapiens)CRTC3GenBankGene ID: 64784
Gene (Homo sapiens)NR4A2GenBankGene ID: 4929
Gene (Homo sapiens)INSL4GenBankGene ID: 3641
Gene (Homo sapiens)LINC00473GenBankGene ID: 90632
Gene (Homo sapiens)PDE4DGenBankGene ID: 5144
Cell line (Homo-sapiens)A549ATCCCCL-185
Cell line (Homo-sapiens)H157ATCCCRL-5802
Cell line (Homo-sapiens)H322ATCCCRL-5806
Cell line (Homo-sapiens)H522ATCCCRL-5810
Cell line (Homo-sapiens)H2126ATCCCCL-256
Cell line (Homo-sapiens)H1819ATCCCRL-5897
Cell line (Homo-sapiens)H2087ATCCCRL-5922
Cell line (Homo-sapiens)H2009ATCCCRL-5911
Cell line (Homo-sapiens)H3123Frederic J Kaye labCVCL_Y295
PMID:11030152
Cell line (Homo-sapiens)H23ATCCCRL-5800
Cell line (Homo-sapiens)H460ATCCHTB-177
Cell line (Homo-sapiens)H2122ATCCCRL-5985
Cell line (Homo-sapiens)H358ATCCCRL-5807
Cell line (Homo-sapiens)BEAS-2BATCCCRL-9609
Cell line (M. musculus)mLSCCLPFrancesco J DeMayo labPMID:31089135
Antibodyanti-CRTC1 (Rabbit Polyclonal)Rockland Immunochemicals IncCat: #600-401-936WB 1:1000
Antibodyanti-CRTC1 (Rabbit Polyclonal)Bethyl LaboratoriesCat: #A300-769AChIP 3 ug/ml
Antibodyanti-CRTC2 (Rabbit Polyclonal)Bethyl LaboratoriesCat: #A300-637AWB 1:1000 ChIP 3 ug/ml
Antibodyanti-CRTC3 (Rabbit Polyclonal)Bethyl LaboratoriesCat: #A302-703A,ChIP 3 ug/ml
Antibodyanti-CRTC3 (Rabbit monoclonal)Cell Signaling TechnologyCat: #2720WB 1:1000
Antibodyanti-LKB1 (Rabbit monoclonal)Cell Signaling TechnologyCat: #3050WB 1:1000
Antibodyanti-β-TUBULIN (Rabbit monoclonal)EpitomicsCat: #1878WB 1:2000
Antibodyanti-HDCA1 (Rabbit Polyclonal)Santa Cruz BiotechnologyCat: #sc7872WB 1:2000
Antibodyanti-β-ACTIN (Mouse monoclonal)Sigma-AldrichCat: #A5316WB 1:2000
Recombinant DNA reagentlentiCRISPR v2 (plasmid)AddgenePlasmid #52961
Recombinant DNA reagentsgCtr- LentiCRISPRv2 (plasmid)AddgenePlasmid #107402
Recombinant DNA reagentsgCRTC1- lentiCRISPR v2 (plasmid)This papersgRNA sequence cloned into lentiCRISPR v2
Recombinant DNA reagentsgCRTC2- lentiCRISPR v2 (plasmid)This papersgRNA sequence clone into lentiCRISPR v2
Recombinant DNA reagentsgCRTC3- lentiCRISPR v2 (plasmid)This papersgRNA sequence clone into lentiCRISPR v2
Recombinant DNA reagentpMSCV-GFP (plasmid)AddgenePlasmid #86537
Recombinant DNA reagentpMSCV-dnCRTC (plasmid)This paperdnCRTC sequence cloned into pMSCV-GFP
Recombinant DNA reagentpcDNA FLAG TORC1 (plasmid)AddgenePlasmid #25718
Recombinant DNA reagentpcDNA FLAG TORC2 (plasmid)AddgenePlasmid #22975
Recombinant DNA reagentpcDNA FLAG TORC3 (plasmid)AddgenePlasmid #22976
Recombinant DNA reagentlentiCas9-Blast (plasmid)AddgenePlasmid #52962
Recombinant DNA reagentnon-targeting control gRNA (plasmid)AddgenePlasmid #80180
Recombinant DNA reagentSTK11 gRNA-1 (plasmid)AddgenePlasmid #75912
Recombinant DNA reagentSTK11 gRNA-2 (plasmid)AddgenePlasmid #75913
Recombinant DNA reagentpMD2.G (plasmid)AddgenePlasmid #12259Lentiviral Envelope
Recombinant DNA reagentpsPAX2 (plasmid)AddgenePlasmid #12260Lentiviral Packaging
Sequence-based reagentCRTC1-qRT-FThis paperqPCR primersTGTCTCTCTGACCCCCTTCCAATCC
Sequence-based reagentCRTC1-qRT-RThis paperqPCR primersGTCCGCGGGTGGTGAGAGGTA
Sequence-based reagentCRTC2-qRT-FThis paperqPCR primersAGCCCCCTGAGTTTGCTCGC
Sequence-based reagentCRTC2-qRT-RThis paperqPCR primersTGGGGGTAACCGCTGGTCAGT
Sequence-based reagentCRTC3-qRT-FThis paperqPCR primersTGACCAGCAGTCCATGAGGCCA
Sequence-based reagentCRTC3-qRT-RThis paperqPCR primersGGTCTTTGAACAGGCTGGTGCTGG
Sequence-based reagentLINC00473-qRT-FThis paperqPCR primersAAACGCGAACGTGAGCCCCG
Sequence-based reagentLINC00473-qRT-RThis paperqPCR primersCGCCATGCTCTGGCGCAGTT
Sequence-based reagentFOS-qRT-FThis paperqPCR primersCACTCCAAGCGGAGACAG
Sequence-based reagentFOS-qRT-RThis paperqPCR primersAGGTCATCAGGGATCTTGCAG
Sequence-based reagentNR4A2-qRT-FThis paperqPCR primersGCCGGAGAGGTCGTTTGCCC
Sequence-based reagentNR4A2-qRT-RThis paperqPCR primersAGGGTTCGCCTGGAACCTGGAA
Sequence-based reagentINSL4-qRT-FThis paperqPCR primersGATGTGGTCCCCGATTTGGA
Sequence-based reagentINSL4-qRT-RThis paperqPCR primersAGGTTGACACCATTTCTTTGGG
Sequence-based reagentCPS1-qRT-FThis paperqPCR primersCTGATGCTGCCCACACAAAC
Sequence-based reagentCPS1-qRT-RThis paperqPCR primersAGGGGAAGGATCGAGAAGCT
Sequence-based reagentPDK4-qRT-FThis paperqPCR primersACAGACAGGAAACCCAAGCC
Sequence-based reagentPDK4-qRT-RThis paperqPCR primersGTTCAACTGTTGCCCGCATT
Sequence-based reagentNR4A1-qRT-FThis paperqPCR primersGAGTCCCAGTGGCGGAGGCT
Sequence-based reagentNR4A1-qRT-RThis paperqPCR primersCAGGCTGCACCCTACCCGGC
Sequence-based reagentTM4SF20-qRT-FThis paperqPCR primersTCCAGGCTCTCTTAAAAGGTCC
Sequence-based reagentTM4SF20-qRT-RThis paperqPCR primersATGGTGTCGTTACTGGTGGG
Sequence-based reagentNR4A3-qRT-FThis paperqPCR primersGAAGAGGGCAGCCCGGCAAG
Sequence-based reagentNR4A3-qRT-RThis paperqPCR primersACGCAGGGCATATCTGGAGGGT
Sequence-based reagentPTGS2-qRT-FThis paperqPCR primersGTTCCCACCCATGTCAAAAC
Sequence-based reagentPTGS2-qRT-RThis paperqPCR primersCCGGTGTTGAGCAGTTTTCT
Sequence-based reagentSIK1-qRT-FThis paperqPCR primersAGCTTCTGAACCATCCACACA
Sequence-based reagentSIK1-qRT-RThis paperqPCR primersTTTGCCAGAACTTCTTCCGC
Sequence-based reagentPDE4B-qRT-FThis paperqPCR primersCCGATCGCATTCAGGTCCTTCGC
Sequence-based reagentPDE4B-qRT-RThis paperqPCR primersTGCGGTCTGTCCATTGCCGA
Sequence-based reagentPDE4D-qRT-FThis paperqPCR primersAACACATGAATCTACTGGCTGA
Sequence-based reagentPDE4D-qRT-RThis paperqPCR primersTCACACATGGGGCTTATCTCC
Sequence-based reagentGAPDH-qRT-FThis paperqPCR primersCAATGACCCCTTCATTGACC
Sequence-based reagentGAPDH-qRT-RThis paperqPCR primersGACAAGCTTCCCGTTCTCAG
Sequence-based reagentID1-qRT-FThis paperqPCR primersTTCTCCAGCACGTCATCGAC
Sequence-based reagentID1-qRT-RThis paperqPCR primersCTTCAGCGACACAAGATGCG
Sequence-based reagentLINC00473 promotor-qRT-FThis paperqPCR primersCTACAGACGTCATCGCCTCC
Sequence-based reagentLINC00473 promotor-qRT-RThis paperqPCR primersCACATTTGGGGGTGCTTGTG
Sequence-based reagentNR4A2 promoter-qRT-FThis paperqPCR primersGGGGAAAGTGAAGTGTCG
Sequence-based reagentNR4A2 promoter-qRT-RThis paperqPCR primersCCGCGCTCGCTTTGGTAT
Sequence-based reagentsgCRTC1-AThis papergRNA targetsTGGCGACTTCGAACAATCCG
Sequence-based reagentsgCRTC1-BThis papergRNA targetsTTACCCGCGCGGCCCGCGTC
Sequence-based reagentsgCRTC1-CThis papergRNA targetsCCCAGCCGAGGCCAGTACTA
Sequence-based reagentsgCRTC2-AThis papergRNA targetsGCAGCGAGATCCTCGAAGAA
Sequence-based reagentsgCRTC2-BThis papergRNA targetsAGGATATGTGGCGGGTGTAT
Sequence-based reagentsgCRTC2-CThis papergRNA targetsACAGGCCCAAAAACTGCGAC
Sequence-based reagentsgCRTC3-AThis papergRNA targetsCTGACGCACTGCTCCGCAGC
Sequence-based reagentsgCRTC3-BThis papergRNA targetsAAAAAGGATATTTGTCGCCC
Sequence-based reagentsgCRTC3-CThis papergRNA targetsAACCCGCCATCACGGGCTGG
Sequence-based reagentsg-CtrThis papergRNA targetsCTTCCGCGGCCCGTTCAA
Commercial assay or kitBronchial Epithelial Cell Growth Medium kitLonzaCat: # CC-4175BEAS-2B cell culture
Commercial assay or kitEffectene Transfection ReagentQIAGENCat: #301425Transfection
Commercial assay or kitRNeasy Mini KitQIAGENCat: #74106RNA extraction
Commercial assay or kitcDNA Reverse Transcription KitApplied BiosystemsCat: #4368814
Commercial assay or kitSYBR Green SupermixBio-RadCat: #1725120
Commercial assay or kitAlkaline Phosphatase, Calf IntestinalNew England BioLabsCat: #M0290
Commercial assay or kitNuclear and Cytoplasmic Extraction ReagentsThermo ScientificCat: #78833
Commercial assay or kitWest Dura Extended Duration SubstrateThermo ScientificCat: # 34076
Commercial assay or kitGFP-Trap Magnetic AgaroseChromoTekCat:# #gtma-10
Commercial assay or kitVeriBlot for IP Detection ReagentabcamCat:# ab131366
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaCat:# E1910
Commercial assay or kitFITC Annexin V Apoptosis Detection KitBD BioscienceCat: #556547
Chemical compound, drugHexadimethrine bromideSigma-AldrichCat: # H9268polybrene
Chemical compound, drugPuromycin DihydrochlorideGibcoCat: #A1113803
Chemical compound, drugMatrigelCorningCat: #356231
Chemical compound, drugD-LuciferinPerkinElmerCat: #122799
Software, algorithmGraphPad Prism 7GraphPad Prism
Software, algorithmImageJ softwareImageJ

Additional files

Supplementary file 1

dnCRTC-reguated gene analysis and oligo sequences used in this study.

(a) Differentially expressed genes in dnCRTC-expressing A549 lung cancer cells in comparison with GFP-expressing control cells were shown. (b) GSEA analysis revealed that multiple oncogenic signatures were negatively associated with the dnCRTC-regulated target genes. (c) Primer and sgRNA sequences used in this study.

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