Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 1. CDK4 and CDK6 CRISPR-KO impair primary tumor growth and metastatic colonization in TNBC cells.A, Three specificgRNAs targeting distinct genomic sites on each gene were generated and delivered through lentiviral infection. B, Gene modification of the CDK4 and CDK6 gRNAs were determined using genomic cleavageassays. C, CDK4 and CDK6 KO efficiency from three different gRNAs in breast cancer cells (SUM159) was assessed at the protein level by immunoblotting. D, The NT ctrl, CDK4 KO1, and CDK6 KO1 SUM159 cells were xenografted in NSG mice (n ¼ 8 per group) through orthotopic mammary fat pad transplantation and primary tumor growth was assessed by measuring tumor volume. E, SUM159 cells were xenografted in NSG mice through mammary fat pad transplantation and allowed to develop for 26 days. Mice were then separated into two groups (n ¼ 7 per group) with similar average tumor volume and treated daily with vehicle or palbociclib (30 mg/kg) by intraperitoneal injection. Primary mammary tumor growth was assessed by measuring tumor volume. F, MDA-MB-231 KO cells from gRNA1 (n ¼ 10 mice per group) were xenografted through orthotopic mammary fat pad transplantation in NSG mice and primary tumor growth was assessed by measuring tumor volume. (Continued on the following page.)

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: CRISPR, Generated, Infection, Western Blot, Transplantation Assay, Injection

Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 2. CDK4 and CDK6 CRISPR-KO impair primary tumor growth and distant metastasis in prostate and pancreatic cancer cells. A and B, Knockout efficiencies of the CDK4 and CDK6 KOs were assessed by immunoblotting in PC3 and HPAFII cells. C, Primary tumor growth was assessed following subcutaneous transplantation of PC3 KO cells in NSG mice (n ¼ 5). D–F, NT, CDK4, and CDK6 KO PC3 cells were injected intravenously in NSG mice (n ¼ 6–10 mice per group) and the number of metastatic nodules were quantified in lung (D), liver (E), and kidney (F) tissues. The midlines show median values. G, CDK4 and CDK6 KO HPAF-II cells were xenografted through orthotopic pancreatic transplantation in NSG mice (n ¼ 4 mice per group). After 22 days, primary pancreatic tumors were collected for tumor volume measurement. H, Liver metastasis was monitored following intrasplenic injections of CDK4 and CDK6 KO HPAF-II pancreatic cancer cells. Metastatic nodules in liver were counted and are represented as dot plots for individual mice. The midlines show median value. All data are mean SEM from three biological replicates unless otherwise stated. , P < 0.05 by Student t test.

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: CRISPR, Knock-Out, Western Blot, Transplantation Assay, Injection

Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 3. CDK4 and CDK6 regulate distinct transcriptomes. RNA-seq was performed in NT, CDK4, and CDK6 KO SUM159 cells as well as in palbociclib (pal)-treated conditions. A and B, Visualization of principal component analyses plot (A) and unsupervised clustering (B) analyses is presented for two biological replicates for each condition. C, Differential RNA-seq analyses comparing palbociclib-treated versusvehicle-treated NT SUM159, CDK4-KO versus NT, and CDK6-KO versus NT SUM159. Number of significantly up-/downregulated genes (FC >1.5, < 1.5; FDR < 0.05) are shown. D, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis using downregulated genes from palbociclib-treated versus vehicle-treated condition in NT SUM159 shows the number of genes in all significantly ranked pathways. E, KEGG pathway enrichment analysis of downregulated genes from CDK4-KO versus NT condition shows the number of genes in all significantly ranked pathways. F, RNA-seq Log2 (FC) value for TNF signaling pathway–enriched genes in palbociclib and CDK4-KO targets. G, KEGG pathway enrichment analysis using downregulated genes from CDK6-KO versus NT condition shows the number of genes in all significantly ranked pathways. H, Venn diagram of overlapping differentially expressed genes between the CDK4 and CDK6 KOs and palbociclib treatment conditions.

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: RNA Sequencing

Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 4. CDK6 regulates expression of the HR DNA repair pathway in TNBC cells. A, Tumorsphere cell numbers were quantified from NT, CDK4 KO, CDK6 KO SUM159 cells. B–G, mRNA expression of DNA replication (TK1, DTL, POLD3) and HR repair genes (Rad51, BRCA1, Rad54B) was measured by RT-PCR in NT, CDK4 KO, and CDK6 KO SUM159 cells. H, Immunoblotting of the indicated antibodies in NT, CDK4/6 KO SUM159 cells. I, Immunoblotting of the indicated antibodies in SUM159 treated with vehicle and 100 nmol/L palbociclib for 24 hours. J, Immunoblotting of the indicated antibodies in NT and CDK6 KO SUM159 cells as well as CDK6 KO cells overexpressed HA-CDK6 cDNA. K, Immunoblotting of the indicated antibodies in NT, CDK4/6 KO SUM159 cells as well as cells treated with palbociclib. L, Immunoblotting of the indicated antibodies in NT, CDK4/6KO MDAMB468 cells. M, Immunofluorescent intensity of gH2AX in NT, CDK4/6 KO SUM159 cells. N, Cell viability was quantified by Prestoblue staining in NT, CDK4/6 KO SUM159 cells. O, Apoptotic cells were quantified by Annexin V/PI staining in NT, CDK4/6 KO SUM159 cells. All data are mean SEM from three biological replicates unless otherwise stated. , P < 0.05 by Student t test.

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining

Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 5. CDK6-regulated DNA replicative genes facilitate mammary tumor growth and metastatic colonization in vivo. A, RNA-seq Log2 (FC) value for CDK6-targeted genes in response to palbociclib. B, STRING network visualization of 12 CDK6 target genes. C, Gene modification of specific gRNAs targeting distinct genomic sites on each gene were assessed by using genomic cleavage assays. D–G, gRNAs targeting the 12 CDK6 target genes in SUM159 cells were xenografted in NSG mice (n ¼ 5 per group) through orthotopic mammary fat pad transplantation and primary tumor growth was assessed by measuring tumor volume. H-J, SUM159 cells knocked out individually for each of the 12 genes were injected intravenously in NSG mice to assess for metastasis. Lungs were collected and fixed in Bouin solution and metastatic nodules counted. Data are represented as dot plot for individual mice. The midlines show median value. All data are mean SEM from three biological replicates unless otherwise stated. , P < 0.05 by Student t test.

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: In Vivo, RNA Sequencing, Transplantation Assay, Injection

Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 6. Clinical outcomes of CDK6-regulated DNA repletion/repair genes. A-C, mRNA expression of CDK6 and its five DNA replication targets (TK1, DTL, POLD3, POLE2, CENPI) in 2,509 primary breast tumors using METABRIC dataset. The heatmaps of these patients categorized into TNBCs and non-TNBCs (A); different breast cancer subtypes (B) including basal-like, HER2þ, luminal A, luminal B, normal-like, and claudin-low tumor tissues; tumor grades (C). D, Kaplan–Meier survival analysis of five CDK6 targets in distant metastasis-free survival (DMFS) using GOBO gene set analysis. Number of patientswith breast cancer at risk with high (blueline), median (red line), low expression (gray line) of five genes.

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: Expressing

Journal: Cancer Research

Article Title: Differential Regulation of Cancer Progression by CDK4/6 Plays a Central Role in DNA Replication and Repair Pathways

doi: 10.1158/0008-5472.can-20-2121

Figure Lengend Snippet: Figure 7. In vivo delivery of a CRISPR/Cas9/anti-CDK6 gRNA efficiently prevents mammary tumor growth and metastatic colonization in a cell- and patient-derived xenograft model. A, Experimental procedure for CDK6 genomic deletion on a preclinical model of preestablished mammary tumors and metastatic lesions using a CRISPR/ Cas9-based in vivo gene therapy delivery system. B, SUM159 breast cancer cells were transplanted into the mammary fat pad of NSG mice (n ¼ 5 per group) and tumors allowed to develop for 35 days. (Continued on the following page.)

Article Snippet: For rescue CDK6, 2 mg HA-tagged CDK6 cDNA (Addgene, 1868; ref. 23) was transfected with Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol for 3 days.

Techniques: In Vivo, CRISPR, Derivative Assay