CDK 12 drives breast tumor initiation and trastuzumab resistance via WNT and IRS 1‐ErbB‐ PI 3K signaling
2019; Springer Nature; Volume: 20; Issue: 10 Linguagem: Inglês
10.15252/embr.201948058
ISSN1469-3178
AutoresHee‐Joo Choi, Sora Jin, Hani Cho, Hee‐Young Won, Hee Woon An, Ga‐Young Jeong, Young‐Un Park, Hyung‐Yong Kim, Mi Kyung Park, Taekwon Son, Kyueng‐Whan Min, Kiseok Jang, Young‐Ha Oh, Jeong‐Yeon Lee, Gu Kong,
Tópico(s)Ubiquitin and proteasome pathways
ResumoReport30 August 2019free access Source DataTransparent process CDK12 drives breast tumor initiation and trastuzumab resistance via WNT and IRS1-ErbB-PI3K signaling Hee-Joo Choi Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, Korea Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Sora Jin Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hani Cho Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hee-Young Won Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hee Woon An Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Ga-Young Jeong Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Young-Un Park Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hyung-Yong Kim Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Mi Kyung Park National Cancer Center, Goyang, Korea Search for more papers by this author Taekwon Son College of Pharmacy, Seoul National University, Seoul, Korea Search for more papers by this author Kyueng-Whan Min orcid.org/0000-0002-4757-9211 Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Ki-Seok Jang Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Young-Ha Oh Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Jeong-Yeon Lee Corresponding Author [email protected] orcid.org/0000-0003-1298-7466 Department of Medicine, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Gu Kong Corresponding Author [email protected] orcid.org/0000-0001-9206-8210 Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, Korea Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hee-Joo Choi Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, Korea Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Sora Jin Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hani Cho Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hee-Young Won Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hee Woon An Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Ga-Young Jeong Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Young-Un Park Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Hyung-Yong Kim Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Mi Kyung Park National Cancer Center, Goyang, Korea Search for more papers by this author Taekwon Son College of Pharmacy, Seoul National University, Seoul, Korea Search for more papers by this author Kyueng-Whan Min orcid.org/0000-0002-4757-9211 Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Ki-Seok Jang Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Young-Ha Oh Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Jeong-Yeon Lee Corresponding Author [email protected] orcid.org/0000-0003-1298-7466 Department of Medicine, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Gu Kong Corresponding Author [email protected] orcid.org/0000-0001-9206-8210 Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, Korea Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea Search for more papers by this author Author Information Hee-Joo Choi1,2,‡, Sora Jin2,‡, Hani Cho2, Hee-Young Won2, Hee Woon An2, Ga-Young Jeong2, Young-Un Park2, Hyung-Yong Kim2, Mi Kyung Park3, Taekwon Son4, Kyueng-Whan Min2, Ki-Seok Jang2, Young-Ha Oh2, Jeong-Yeon Lee *,5 and Gu Kong *,1,2 1Institute for Bioengineering and Biopharmaceutical Research (IBBR), Hanyang University, Seoul, Korea 2Department of Pathology, College of Medicine, Hanyang University, Seoul, Korea 3National Cancer Center, Goyang, Korea 4College of Pharmacy, Seoul National University, Seoul, Korea 5Department of Medicine, College of Medicine, Hanyang University, Seoul, Korea ‡These authors contributed equally to this work *Corresponding author. Tel: +82 2 2220-0634; Fax: +82 2 2295 1091; E-mail: [email protected] *Corresponding author. Tel: +82 2 2290 8251; Fax: +82 2 2295 1091; E-mail: [email protected] EMBO Rep (2019)20:e48058https://doi.org/10.15252/embr.201948058 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Cyclin-dependent kinase 12 (CDK12) has emerged as an effective therapeutic target due to its ability to regulate DNA damage repair in human cancers, but little is known about the role of CDK12 in driving tumorigenesis. Here, we demonstrate that CDK12 promotes tumor initiation as a novel regulator of cancer stem cells (CSCs) and induces anti-HER2 therapy resistance in human breast cancer. High CDK12 expression caused by concurrent amplification of CDK12 and HER2 in breast cancer patients is associated with disease recurrence and poor survival. CDK12 induces self-renewal of breast CSCs and in vivo tumor-initiating ability, and also reduces susceptibility to trastuzumab. Furthermore, CDK12 kinase activity inhibition facilitates anticancer efficacy of trastuzumab in HER2+ tumors, and mice bearing trastuzumab-resistant HER2+ tumor show sensitivity to an inhibitor of CDK12. Mechanistically, the catalytic activity of CDK12 is required for the expression of genes involved in the activation of ErbB-PI3K-AKT or WNT-signaling cascades. These results suggest that CDK12 is a major oncogenic driver and an actionable target for HER2+ breast cancer to replace or augment current anti-HER2 therapies. Synopsis CDK12, a RNA polymerase II kinase, promotes breast cancer stem cell-like properties mediated by WNT and ErbB-PI3K signaling. Targeting CDK12 improves trastuzumab therapy in breast cancers characterised by HER2/CDK12 co-amplification. CDK12 amplification induces tumor initiation and progression, and mediates trastuzumab resistance. CDK12 is required for transcriptional upregulation of genes involved in ErbB-PI3K and WNT pathway activation. CDK12 inhibition alone or in combination with trastuzumab therapy has anti-tumor activity against HER2+ breast cancers. Introduction Human epidermal growth factor receptor 2 (HER2)-positive (HER2+) breast cancer, which is defined by HER2 amplification or overexpression, accounts for 15–20% of all breast cancers, is clinically defined as a distinct subtype of breast cancer that benefits from anti-HER2 therapies 12. Trastuzumab, the first approved anti-HER2 monoclonal antibody, is the most commonly used drug in the world as a standard regimen for HER2+ breast cancer patients 34. However, accumulating clinical evidence reveals that the response of HER2+ breast cancers to trastuzumab therapy varies widely 4, with > 50% of patients either not responding or acquiring resistance to trastuzumab 567. Recent large-scale whole-genome sequencing and transcriptome analysis of HER2+ breast cancer showed that it comprises several subgroups exhibiting different gene expression and distinct genomic features 8. Furthermore, this genomic heterogeneity causes a variety of responses to HER2-targeted therapies 4910. Although the abnormalities in chromosome 17 (chr17) that cause HER2 amplification are among the most representative characteristics of HER2+ breast cancer 1211, it remains largely unknown whether genes co-amplified with HER2 at chr17 play a key role in driving tumorigenesis and serve as alternative therapeutic targets in HER2+ breast cancer with anti-HER2 therapy. Cyclin-dependent kinase 12 (CDK12) is located at chr17q12 and exhibits high concurrent amplification along with HER2, which accounts for ~90% of HER2+ breast cancer 121314. CDK12 phosphorylates the C-terminal domain (CTD) of RNA polymerase II at serine 2 (PolII CTD-ser2) and regulates multiple biological processes, including transcription elongation, cell cycle progression, and DNA damage repair 15161718. Accumulating studies have proposed the dysregulation of CDK12 in human cancer. Genomic analysis identified frequent alterations of CDK12 through mutation, rearrangements, or amplification in various types of human tumors, including breast, ovarian, and prostate cancers 1219202122. In large-scale screening of phosphoproteins, CDK12 has been nominated as a candidate of highly phosphorylated kinase related to breast cancer 1223. Indeed, CDK12 was associated with aggressive phenotypes of breast cancer in clinical specimens 1824, and its kinase activity promoted increased the migration and invasion ability of breast cancer cells in vitro 18. Particularly, growing evidence has suggested the CDK12 kinase inhibition as a promising therapeutic strategy to improve sensitivity to DNA-damaging anticancer drugs. For instance, in ovarian cancer harboring a functionally inactive CDK12 mutation, CDK12 deficiency enhanced the sensitivity to olaparib, a poly (ADP-ribose) polymerase (PARP)1/2 inhibitor 25. Similarly, resistance to the PARP1/2 inhibitor was reversed by administration of dinaciclib, a pan-CDK inhibitor with potent activity against CDK12 and other CDKs, in triple-negative breast cancer (TNBC) 26. Despite the therapeutic potential of targeting CDK12 in human cancer, little is known about the putative role of CDK12 in driving tumor initiation and progression. In this study, we explored potential actionable targets among chr17q12 genes to improve current anti-HER2 therapy and found that CDK12 regulates cancer stem cell (CSC)-like properties to drive breast tumor initiation and induce trastuzumab resistance in a manner independent on its ability to modulate DNA repair. Furthermore, we propose that CDK12 kinase inhibition represents a broadly effective therapy against different types of HER2+ breast cancers and could be a replacement therapy for trastuzumab in breast cancer treatment. Results and Discussion Chr17q12 encompasses genes with distinct clinical implications Growing evidence suggests that several genes co-amplified with HER2 can influence biological behavior of HER2+ breast cancer, with co-silencing of these genes improving the growth-inhibitory effects of or apoptosis induction in HER2+ breast cancer 1127. Moreover, higher levels of copy number alterations in chr17q12 were associated with non-responsiveness to anti-HER2 therapy 11. Despite the potential importance of 17q12-amplicon genes in breast cancer, the clinical relevance and functional significance of these genes remain largely unknown. To discover possible candidate drivers and druggable target genes, besides HER2, within the region, genetic alterations and expression levels of individual genes at chr17q12 were first explored in breast cancer patients using the METABRIC dataset (n = 1,980). Among the genes with positive correlation between copy number alteration and expression levels (r > 0.4), clinically significant genes that were associated with poor prognosis both in overall survival (OS) and in disease-free survival (DFS) were sorted, and the druggability of sorted genes was further examined (Fig 1A). In addition to HER2, many genes within the 17q12 amplicon, including MIEN1, PGAP3, TCAP, GRB7, STARD3, and CDK12, were associated with high risk for relapse and death in patients with breast cancer (Fig 1A and B). Additionally, several genes, including RPL19, PIP4K2B, and CACNB1, were associated with favorable survival outcome in the cohort (Fig 1B), implying that chr17q12 comprises both putative oncogenes and tumor-suppressor genes. Figure 1. CDK12 amplification is a candidate of druggable target that is associated with poor prognosis in breast cancer The schematic diagram showing the process to determine candidate target genes from chr17q12 amplicons in the METABRIC dataset. Forest plots display the hazard ratios of genes at the 17q12 amplicon according to the DFS (top right) and OS (bottom right) of breast cancer patients in the METABRIC dataset. Genes located at the HER2 amplicon were nominated according to hazard ratio levels (all P-values < 0.01). The HER2 co-amplification percentage of the indicated genes is represented as bar graphs (left). The frequency of CDK12 amplification in HER2-amplified- and HER2-non-amplified breast cancer. The bar graphs indicate the frequency of HER2 and CDK12 co-amplified cases among the patients with HER2 gene amplification (left). Tables show the percentage of CDK12 amplification in the indicated cohorts (right). Amp, amplification; non-amp, non-amplification. Scatter plot showing the correlation between CDK12 expression and its copy number alteration (CNA) in METABRIC (left). The r value was calculated as Pearson's correlation coefficient. Box plots showing the mRNA levels of CDK12 in the indicated subtypes from METABRIC (right). The horizontal bands (red) inside the boxes represent the median; the bottom and top of the boxes correspond to the 25th and 75th percentiles, respectively; and the vertical bars from the boxes (whiskers) are the range of data representing lowest and highest values. P-values were calculated using one-way ANOVA, followed by post hoc LSD test. Survival analysis of breast cancer patients according to the expression of CDK12 in METABRIC (top) and KM plotter (bottom) using the Kaplan–Meier method with the log-rank test. The results of NPI- and AOL-adjusted analysis showing the association between CDK12 expression and the risk of metastatic relapse (MR) in breast cancer datasets generated using bc-GenExMiner 4.0 (http://bcgenex.centregauducheau.fr). Download figure Download PowerPoint Among the genes associated with poor prognosis in both OS and DFS, but whose functions in HER2+ breast cancer remain unclear, CDK12, a kinase associated with phosphorylation of the RNA PolII CTD, was the most prominent candidate gene because of availability on its targeted drugs, such as dinaciclib 26 and THZ-531 28. Consistent with previous reports 813, CDK12 was almost exclusively amplified in 80~90% of HER2-amplified breast cancer (Fig 1C). In HER2 non-amplified cases, only < 0.5% of patients had CDK12 gene amplification (METABRIC, n = 7 of 1,683, 0.14%; TCGA, n = 1 of 711, 0.42%; Fig 1C, right). CDK12 expression was highest in HER2+ breast cancer among breast cancer subtypes, and elevated CDK12 levels were associated with its gene amplification and HER2 expression (Fig 1D; EV1A and B). For these reasons, HER2+ breast cancer patients, most of which harbor high expression levels of CDK12, could not be stratified according to CDK12 expression; thus, the impact of CDK12 on prognostic outcome of breast cancer was examined in all types of breast cancer patients. In the METABRIC and according to the Kaplan–Meier plotter (KM plotter; https://kmplot.com/analysis/), CDK12 expression in breast cancer patients was associated with poor overall survival (OS) and disease-free survival (DFS; OS, P = 0.02; DFS, P < 0.001 in the METABRIC; Fig 1E). Additionally, CDK12 was an independent prognostic factor for high risk of metastatic relapse (MR) relative to the Nottingham Prognostic Index (NPI, P = 0.04) 2930 and Adjuvant Online (AOL, P = 0.01) 31, both well-established breast cancer prognostic indexes (http://bcgenex.centregauducheau.fr) 32 (Figs 1F and EV1C), suggesting CDK12 as a potential prognostic marker in breast cancer. Taken together, these findings revealed that the HER2 amplicon comprises various genes that are significantly associated with distinct prognostic outcomes, supporting a notion that the co-amplified genes are not bystanders of tumor development, but rather cooperate in potentiating the aggressiveness of HER2+ breast cancer. Click here to expand this figure. Figure EV1. Clinical implication of CDK12 amplification in breast cancer Scatter plot (left) showing the correlation between CDK12 mRNA levels and copy numbers in TCGA. The r value was calculated as the Pearson's correlation coefficient. Box plot (right) showing CDK12 mRNA levels in the indicated subtypes of breast cancer from TCGA. The box plots represent the median (red bands inside the boxes), 25th and 75th percentiles (the bottom and top of the boxes), and the range of values (whiskers). P-values were calculated using one-way ANOVA, followed by a post hoc LSD test. The correlation between CDK12 and HER2 expression in METABRIC and TCGA. The r value was calculated as Pearson's correlation coefficient. Forest plot representing the association between CDK12 expression and the risk of metastatic relapse (MR) in different types of breast cancer datasets (bc-GenExMiner 4.0; http://bcgenex.centregauducheau.fr). Download figure Download PowerPoint CDK12 drives breast CSCs and induces trastuzumab resistance To clarify whether the high degree of co-amplifications between HER2 and CDK12 is functional, CDK12 effect on HER2-associated biological features was examined in various subgroups of HER2+ breast cancer. In ZR-75-30 and HCC-1419 cells with low levels of CDK12 expression due to its non-amplification, lentiviral overexpression of CDK12 accelerated cell growth (Fig EV2A and B, left). In contrast, short-hairpin (sh)RNA-mediated CDK12 knockdown in BT474 and HCC-1954 cells harboring CDK12 amplification diminished cell growth (Fig EV2A and B, right). These results were confirmed in a mice xenograft model (Fig EV2C). Click here to expand this figure. Figure EV2. CDK12 accelerates tumor growth in HER2+ breast cancer The amplification status and mRNA levels of CDK12 in HER2+ and HER2− breast cancer cells from the Cancer Cell Line Encyclopedia were displayed by OncoPrint (top). In the indicated cell lines, levels of CDK12 protein were confirmed by Western blot (bottom). Growth curve of the indicated cell lines as measured by SRB assay. Data represent the mean ± SD (n = 3 technical replicates). P-value was calculated using RM ANOVA with a post hoc LSD test. The effect of CDK12 on in vivo tumor growth was analyzed in an orthotopic tumor xenograft model. NOD/SCID mice were injected with the indicated cell lines following implantation with 17-β estradiol pellets. The tumor growth curve was analyzed by measuring tumor size twice weekly (n = 7 mice/group; mean ± SEM). Representative images of tumors were from orthotopic xenograft of CDK12-overexpressing ZR-75-30 or CDK12-knockdown BT474 cells. P-values were calculated using RM ANOVA with a post hoc LSD test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; S, trastuzumab-sensitive. Source data are available online for this figure. Download figure Download PowerPoint BecauseHER2 is a major regulator of CSCs in breast cancer 33, we evaluated the CDK12 effect on the CSC-like properties of HER2+ breast cancer. FACS analyses for the detection of CD44+/CD24−/ESA+ cell population or cells with high aldehyde dehydrogenase (ALDH) activity showed that CDK12 expanded self-renewing CSC-like populations in different types of HER2+ breast cancer cell lines with various response rates to trastuzumab (Figs 2A and B, and EV3A). Likewise, CDK12-overexpressing cells showed an increased ability to form primary and secondary tumorspheres, whereas CDK12 knockdown decreased the tumorsphere formation during serial passage (Figs 2C and EV3B). Consistent with the in vitro results, orthotopically xenografted mice harboring CDK12-overexpressing ZR-75-30 cells exhibited accelerated tumor formation, whereas mice harboring CDK12-knockdown BT474 cells showed impaired tumor-initiating ability (Table 1). To investigate whether the CDK12-induced CSC-like phenotypes were dependent on HER2, the effect of CDK12 on CSC activity was further examined in HER2-negative (HER2−) breast cancer. In MCF7 ER+/HER2− breast cancer cells and MDA-MB-231 ER−/HER2− cells, CDK12 overexpression increased CD44+/CD24−/ESA+ cell population and tumorsphere formation regardless of HER2 (Appendix Fig S1A and B). These data suggested that CDK12 enhanced CSC self-renewal in a HER2-independent manner in breast cancer. Figure 2. CDK12 regulates cancer stemness and response to trastuzumab therapy in HER2+ breast cancer FACS analysis of the percentages of CD44+/CD24−/ESA+ breast CSC-like populations in CDK12-overexpressing ZR-75-30 and HCC-1419 cells and CDK12-siRNA-transfected BT474 and HCC-1954 cells. Top, representative dot plot of CD44-APC versus CD24-PE expression for viable cells (red) and ESA-positive cells (blue) from the viable cells. Bottom, the percentage of CD44+/CD24−/ESA+ cell population (blue dots at lower right quadrant in the plots) was quantified in the indicated cell lines. Mean ± SD of three independent samples. P-values were based on two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) and one-way ANOVA with a post hoc LSD test (BT474 and HCC-1954 cells). Amp, amplification; non-amp, non-amplification; Trz, trastuzumab. ALDEFLUOR assay for analyzing cell population with a high ALDH activity in the indicated cell lines. Cells either treated with ALDH inhibitor DEAB (with DEAB; negative control) or untreated (without DEAB) were incubated with ALDEFLURO substrate (BAAA). In the FACS analysis, the percentage of ALDEFLUOR-positive population was determined based on the shift of fluorescent cells seen in the dot plots in the absence of DEAB. Mean ± SD of three independent samples. P-values were calculated based on a two-tailed Student's t-test The self-renewal of CSCs in the indicated stable cell lines was analyzed using a tumorsphere-formation assay. The number of tumorsphere cells (> 100 μm in diameter) was counted after 3, 5, and 7 days. Data represent the mean ± SD (n = 3 biological replicates). P-value was calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post hoc LSD test (BT474 and HCC-1954 cells). *P < 0.05, **P < 0.01, ***P < 0.001 versus controls (CON or shCON). Cell growth of the indicated stable cell lines treated with trastuzumab (Trz) or vehicle (veh) as measured by SRB assay. Trastuzumab-sensitive HER2+ breast cancer cell lines (S) and trastuzumab-insensitive HER2+ breast cancer cell lines (partial responsive, PR; resistant, R) were treated with 1 or 100 μg/ml trastuzumab, respectively. Data represent the mean ± SD of three technical replicates. P-values were calculated using RM ANOVA with a post hoc LSD test. Analysis of the effect of CDK12 deficiency on the in vivo trastuzumab response of HER2+ breast cancer. Mice were orthotopically xenografted with the indicated HER2+ breast cancer cells and treated with 20 mg/kg trastuzumab. The growth curve of each group was analyzed twice weekly for 5–6 weeks (n = 8 mice/group; mean ± SEM). P-values were calculated using one-way ANOVA with a post hoc LSD test for the last day of tumor measurement. Data information: In (B–E), cell line information was indicated as follows: a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive; S, trastuzumab-sensitive. Source data are available online for this figure. Source Data for Figure 2 [embr201948058-sup-0004-SDataFig2.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. CDK12 impact on CSC-like properties in HER2+ breast cancer Top, representative dot plots showing the cell population with a high ALDH activity in the FACS analysis. Bottom, the quantification of ALDEFLUOR-positive population in the indicated cell lines. DEAB, an inhibitor of ALDH. Data represent the mean ± SD (n = 3 biological replicates). P-values were calculated based on a two-tailed Student's t-test. a/a, CDK12-amplified/HER2-amplified cells; n/a, CDK12 non-amplified/HER2-amplified cells; R, trastuzumab-resistant; PR, trastuzumab-partial responsive. The number of secondary (P2) tumorsphere cells (> 100 μm in diameter) formed from primary tumorspheres (Fig 2C) of indicated stable cell lines was counted after 3, 5, and 7 days. Error bars represent the mean ± SD (n = 3 biological replicates). P-values were calculated based on a two-tailed Student's t-test (ZR-75-30 and HCC-1419 cells) or ANOVA with a post hoc LSD test (BT474 and HCC-1954 cells). *P < 0.05, **P < 0.01, ***P < 0.001 versus controls (CON or shCON). Source data are available online for this figure. Download figure Download PowerPoint Table 1. CDK12 enhances in vivo tumor-initiating ability Cell numbers ZR-75-30 Cell numbers BT474 CON CDK12 shCon shCDK12 5 × 102 0/7 0/7 5 × 103 1/7 0/7 1 × 103 0/7 4/7 1 × 104 3/7 0/7 5 × 103 3/7 7/7 5 × 104 7/7 2/7 1 × 104 7/7 7/7 1 × 105 7/7 7/7 TIC frequency 1/6,315 1/1,619 1/16,559 1/74,865 (1/3,368–1/11,839) (1/834–1/3,145) (1/8,544–1/32,091) (1/39,049–1/43,530) P-value 0.004 0.004 Limiting dilution assay. From 500 to 10,000 cells of ZR-75-30 Con or ZR-75-30 CDK12 and 5,000 to 100,000 cells of BT474 shCon or BT474 shCDK12 were injected into the fat pad of NOD/SCID mice. The tumor-initiating cell (TIC) frequency was calculated using L-Calc software (Stemcell Tech, Vancouver, Canada, http://www.stemcell.com). We further explored whether the oncogenic potential of CDK12 affected response to anti-HER2 therapy in breast cancer. In HER2+ breast cancer cell lines that are sensitive or partially responsive to trastuzumab, CDK12 overexpression induced insensitivity to trastuzumab, whereas its knockdown further sensitized cells to trastuzumab (Figs 2D and EV4A). In trastuzumab-resistant HER2+ breast cancer cells with CDK12 amplification, CDK12 knockdown did not confer trastuzumab sensitivity, but induced growth retardation (Figs 2D and EV4A). These data were confirmed in orthotopic tumor xenograft models (Fig 2E). Taken together, these findings indicated that CDK12 induces tumor growth, cancer stemness, and trastuzumab insusceptibility in HER2+ breast cancer, suggesting CDK12 as a potential oncogenic driver and the cooperative function of CDK12 with HER2 in promoting the initiation and progression of HER2+ breast cancer. Click here to expand this figure. Figure EV4. The effect of CDK12 on trastuzumab response in HER2+ breast cancer Cell viability following treatment with the indicated doses (μg/ml) of trastuzumab (Trz) or vehicle for 5 days as measured by SRB assay. Data represent the mean ± SD (n = 3 technical replicates). P-values were calculated using RM ANOVA with a post hoc LSD test. Cell viability was measured by SRB assay in SKBR3 cells following treatment with 1 μg/ml trastuzumab (Trz) or/and 5 nM dinaciclib (Dina). Data represent the mean ± SD (n = 3 technical replicates). P-value was calculated using RM ANOVA with a post hoc LSD test. Representative images of tumors were from orthotopic xenograft of BT474 or HCC-1954 cells following treatment with trastuzumab and/or dinaciclib. Source data are available online for this figure. Download figure Download PowerPoint CDK12 kinase inhibition alters oncogenic transcriptional programs and has antitumor activity in both trastuzumab-sensitive and trastuzumab-resistant HER2+ breast cancer To elucidate how CDK12 regulates tumorigenesis and trastuzumab response in HER2+ breast cancer, genome-wide CDK12-target genes were investigated by RNA-seq analysis in CDK12-overexpressing ZR-75-30 cells. The differentially expressed genes in the presence of CDK12 overexpression were enriched in several oncogenic signaling cascades, such as EGFR signaling, the PI3K cascade, and the WNT-signaling pathway (Fig 3A and B, Dataset EV1). We also identified CDK12-target genes associated with its enzymatic activity toward PolII CTD phosphorylation, including the ErbB-PI3K-AKT regulator insulin receptor substrate 1 (IRS1) and several WNT ligands, by comparative analysis of RNA-seq data with a gene expression microarray data from breast cancer cells treated with dinaciclib, a potent inhibitor of CDK proteins, including CDK9 and CDK12 26 and PolII chromatin immunoprecipitation (ChIP)-seq data from cells treated with THZ-531, a recently developed selective inhibitor of CDK12/CDK13 28 (Fig 3C). Changes in the expression of genes involved in these pathways were validated by quantitative reverse transcription–polymerase chain reaction (qRT–PCR) (Fig
Referência(s)