HOXC10 Expression Supports the Development of Chemotherapy Resistance by Fine Tuning DNA Repair in Breast Cancer Cells Helen Sadik1, Preethi Korangath1, Nguyen K

Published OnlineFirst June 14, 2016; DOI: 10.1158/0008-5472.CAN-16-0774 Cancer Molecular and Cellular Pathobiology Research

HOXC10 Expression Supports the Development of Chemotherapy Resistance by Fine Tuning DNA Repair in Breast Cancer Cells Helen Sadik1, Preethi Korangath1, Nguyen K. Nguyen1, Balazs Gyorffy2,3, Rakesh Kumar4, Mohammad Hedayati5, Wei Wen Teo1, Sunju Park1, Hardik Panday1, Teresa Gonzalez Munoz1, Otilia Menyhart2,3, Nilay Shah1, Raj K. Pandita6, Jenny C. Chang7, Theodore DeWeese5, Howard Y. Chang8, Tej K. Pandita6, and Saraswati Sukumar1

Abstract

Development of drug resistance is a major factor limiting tant phases. For double-strand break repair, HOXC10 recruits the continued success of cancer chemotherapy. To overcome HR proteins at sites of DNA damage. It enhances resection and drug resistance, understanding the underlying mechanism(s) lastly, it resolves stalled replication forks, leading to initiation is essential. We found that HOXC10 is overexpressed in of DNA replication following DNA damage. We show that primary carcinomas of the breast, and even more significantly HOXC10 facilitates, but is not directly involved in DNA in distant metastasis arising after failed chemotherapy. High damage repair mediated by HR. HOXC10 achieves integration HOXC10 expression correlates with shorter recurrence-free of these functions by binding to, and activating cyclin-depen- and overall survival in patients with estrogen receptor– dent kinase, CDK7, which regulates transcription by phos- negative breast cancer undergoing chemotherapy. We found phorylating the carboxy-terminal domain of RNA polymerase that HOXC10 promotes survival in cells treated with doxoru- II. Consistent with these findings, inhibitors of CDK7 reverse bicin, paclitaxel, or carboplatin by suppressing apoptosis and HOXC10-mediated drug resistance in cultured cells. Blocking upregulating NF-kB. Overexpressed HOXC10 increases S- HOXC10 function, therefore, presents a promising new strat- phase–specific DNA damage repair by homologous recombi- egy to overcome chemotherapy resistance in breast cancer. nation (HR) and checkpoint recovery in cells at three impor- Cancer Res; 76(15); 1–14. 2016 AACR.

Introduction Large scale interaction screens as well as reciprocal afﬁnity puriﬁcations have been instrumental in identifying several HOX Many chemotherapeutic agents induce DNA damage, trigger- proteins that interact with proteins critical for DNA damage repair ing cells to activate their DNA damage repair machinery. Nucle- processes (reviewed in ref. 1). HOXB7 associates with Ku70, otide excision repair (NER) is a major DNA repair pathway that Ku80, and DNA-Pkcs (2), and with PARP1 (3), whose poly- mediates repair of DNA adducts that cause inter- or intrastrand ADP-ribosylation activity enhances the kinase activity of DNA- cross links (ICL) and the formation of pyrimidine dimers. Alky- PK at the initiation of non-homologous end joining (NHEJ). lating- and platinum-based agents often induce bulky DNA Recent evidence supports HOX protein participation in the cell damage that is repaired via NER. replication machinery by associations at DNA replication origins (4). HOXC10 is one such example (5, 6); HOXC10 also has important functions in tissue regeneration (7). Recently, loss of 1 Department of Oncology, Johns Hopkins University School of Med- HOXC10 expression was implicated in the development of resis- icine, Baltimore, Maryland. 2MTA TTK Lendulet€ Cancer Biomarker Research Group, Budapest, Hungary. 32nd Department of Pediatrics, tance to estrogen response modulators in estrogen receptor (ER)– Semmelweis University, Budapest, Hungary. 4Department of Radia- positive breast cancer (8). At the present time, the function of tion Oncology, University of Texas Southwestern Medical Center, HOXC10 in breast cancer remains poorly understood. Dallas, Texas. 5Department of Radiation Oncology, Johns Hopkins University School of Medicine, Baltimore, Maryland. 6Department of Here, we report that HOXC10 expression is frequently higher in Radiation Oncology,The Houston Methodist Research Institute, Hous- ER-negative breast carcinomas that are also chemotherapy resis- 7 ton, Texas. Methodist Cancer Center, The Houston Methodist tant. We determined that HOXC10 contributes to chemoresis- Research Institute, Houston,Texas. 8Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California. tance through suppression of apoptosis and enhanced DNA damage repair, mediated through direct interaction with, and Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). activation of CDK7. We also show that CDK7 inhibition can reverse chemotherapy resistance. Corresponding Authors: Saraswati Sukumar, Johns Hopkins University School of Medicine, 1650 Orleans Street, CRB1/Rm 143, Baltimore, MD 21287. E-mail: [email protected]; and Tej K. Pandita, 6550 Fannin Street, SM8.026, Houston Materials and Methods Methodist Research Institute, Houston, TX 77030. E-mail: Cell lines, constructs, and reagents [email protected] All cell lines were purchased from the ATCC and passages doi: 10.1158/0008-5472.CAN-16-0774 used were within 6 months of purchase. Stable MCF10A-Ras 2016 American Association for Cancer Research. cells were established by transfection of LXSN-K-Ras vector into

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MCF10A cells; MCF7 wt and drug-resistant sublines were from Luciferase assay Dr.A.M.Parissenti,LaurentianUniversity,Sudbury,ON, A total of 1 105 cells were seeded in 12-well cell culture plates, Canada (9). DR-95 is a human fibroblast cell line stably and transfected with a total of 1.6 mg of plasmids, including expressing a pDR-GFP plasmid containing a mutated GFP gene reporter, expression, and pCMV-b-galactosidase plasmids using with an 18 bp I-SceI endonuclease cleavage site and in-frame Lipofectamine 2000 (14). NF-kB-luc (Igk2-IFN-luc wt or mut) termination codon (10). Retrovirus and lentivirus were pro- was a kind gift from Dr. Joel L. Pomerantz, Johns Hopkins duced in HEK 293T cells. Human HOXC10 cDNA (Thermo University School of Medicine, Baltimore, MD (15). The TOP- Scientific) was cloned into the EcoRI and ClaI sites of pLPCX Flash plasmid (16) was purchased from Addgene. for retroviral production for creating HOXC10-expressing cell lines. Mutated HOXC10 constructs were generated with Clonogenic cell survival assay full-length myc-tagged HOXC10 cloned in pCDNA3.1-Neo Exponentially growing cells were exposed to drugs or ultravi- at EcoRI and XbaI sites. The following reagents were used: olet light (9). Twenty-four hours later, cells were reseeded doxorubicin (Sigma), paclitaxel, and gemcitabine (Tocris Bio- (1–2 103/well) in triplicate in 6-well plates. Viable colonies science) and carboplatin, BS-181 and SNS-032 (gifted by Sell- were fixed, stained with crystal violet, and counted 1 to 2 weeks eckchem; ref. 11), TRC lentiviral Human HOXC10 shRNA later. (set of 4; Thermo Scientific); FlexiTube siRNA for HOXC10 CDK7 (SI04296621), E2F1 (SI00300083), or (SI02664795) MTT assay (Qiagen). All other reagents were from Sigma. A total of 2.5 103 cells per well were plated in 96-well plates in triplicate and treated with drugs alone or in combination, and Human tissue samples MTT assay was performed (17). Values are expressed as percent Fresh frozen primary human tissues were used following survival of the vehicle-treated control. approval of the Johns Hopkins Institutional Review Board. Caspase-3/7 activity Survival analysis in patients with breast cancer Caspase-3 and -7 activities were measured with the Caspase- Kaplan–Meier analyses were performed using 4,117 breast Glo Assay Kit (Promega) according to the manufacturer's instruc- cancer patients. http://kmplot.com/analysis (12). tions. Values were expressed as the percentage of activity of the vehicle-treated control. Real-time quantitative PCR analysis qRT-PCR was conducted using the MaximaSYBR Green/ Western blot analysis ROX Master Mix (Fermentas) per manufacturer protocol. The Cells were lysed with RIPA buffer and processed as described DDCt method was used, with GAPDH expression for normal- previously (17). The following antibodies were used: anti-CDK7 ization (13). and anti-XPD (Santa Cruz Biotechnology) and BCL-xL and BIRC3 (Abcam).All other antibodies were from CellSignaling Technology. Soft agar colony formation assay and Matrigel invasion assay Six-well plates containing a 0.6% agar layer were overlaid with Host-cell reactivation assay 3 103 cells i n 0.3% agar layer. Colonies were counted after 7 The assay was performed (18) with some modifications. pGL3- days. Cell invasion assay was performed using The BD BioCoat basic (Promega) was exposed to UV light [Stratalinker UV-Cross- Matrigel Invasion Chamber assay system (13). linker 1800 (Stratagene)] or treated with 100 to 1,000 nmol/L cisplatin. To measure HR-mediated repair, 1 mg of vector digested Tumor xenograft studies with HindIII was transfected into cells with pCMV-b-galactosi- Approved by Johns Hopkins Institutional Animal Care and Use dase. Luciferase and b-galactosidase activities were measured after Committee (IACUC), BALB/c nu/nu athymic mice (Sprague– 48 hours. Dawley–Harlan) received subcutaneous injections of 3 million cells/100 mL PBS/Matrigel (1:1), and were treated with doxoru- Comet assay bicin (4 mg/kg BW/i.v./weekly) or paclitaxel (10 mg/kg BW/i.p./ The alkaline Comet assay to measure DNA strand breaks in weekly). single cells was performed according to the manufacturer's protocol (CometAssay Kit; Trevigen). Comet tail moments of 100 Growth assay cells were measured and quantified using the CometScore Cells were grown in 12-well plates (2,000 cells/well), fixed with software. formalin, and stained with crystal violet. To quantitate growth, the dye was solubilized by 10% acetic acid, and absorbance was Chromosomal aberration analysis at metaphase measured at 560 nmol/L. Chromosomal aberration analysis was performed as described previously (18, 19). Exponentially growing cells were gamma Flow cytometry analysis irradiated (3 G) and analyzed for metaphase aberrations after 12 Cells at 70% to 80% confluence or after double thymidine hours (20). Cisplatin-induced chromosome aberrations were synchronization were collected, permeabilized, pelleted, and analyzed as described previously (19). resuspended in an isotonic-buffered propidium iodide (PI) stain- ing solution containing RNase A (0.1 mg/mL) and PI (20 mg/mL). Detection of g-H2AX Foci Samples were run on the BD FACSCalibur system (Becton Dick- Cells growing in chamber slides (Nunc Lab-Tek II) were treated inson), and data analyzed using WinMDI 2.9 software. with Dox (200 nmol/L) or gemcitabine (50 nmol/L) for 24 hours.

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After fixation and permeabilization, cells were probed with Results an antibody against phosphorylated H2AX-Ser139 (Upstate HOXC10 overexpression is linked to chemotherapy Biotechnology); H2AX foci were visualized using a Zeiss Axio resistance Scope fluorescent microscope and scored with the ImageJ By HOX tiling mRNA expression array, compared with nor- software (v1.47, NIH, Bethesda, MD). One-hundred cells or mal breast, HOXC10 was among the top five overexpressed more were evaluated. genes in breast cancer (23), and higher than all 39 HOX genes in distant metastasis (Fig. 1A). In Oncomine (24), HOXC10 was I-Sce1 assay for homologous recombination repair activity among the top 1% transcripts in breast compared with other DR-95 cells were transfected with pI-Sce1, pEGFP, or pCMV carcinomas(Fig.1B),andinTCGA(https://tcga-data.nci.nih. (21, 22), harvested after 72 hours, and %GFP-expressing cells was gov/), was equally prevalent in all breast carcinomas (Fig. 1C). measured by flow cytometry. Frequency of recombination events qRT-PCR analysis of our primary tissue panel validated this ¼ mean %GFP-positive cells transfected with pI-Sce1/Mean finding; median HOXC10 expression was 10-fold higher in %GFP-positive cells transfected with pEGFP. primary invasive carcinomas and 30- fold higher in distant metastatic tissues (Fig. 1D). No correlation of HOXC10 expres- Recruitment of HR protein at double-strand break sites sion with relapse-free survival (RFS) or overall survival (OS) DR95 cells were electroporated with I-pCBASce. ChIP was done was observed in breast cancer patients in the GEO (http://www. using antibodies to Rad51 (Abcam); BRCA1 (Cell Signaling ncbi.nlm.nih.gov/geo) and the METABRIC cohorts (Fig. 1E, a Technology); KU80 (Cell Signaling Technology; ref. 20). Quan- and F, a; ref. 25). In chemotherapy treated patients, high tification of ChIP DNA was carried out by real-time PCR in HOXC10 expression correlated with short RFS (Fig. 1E, b and triplicate using the LightCycler Fast Start DNA Master SYBR Green Supplementary Fig. S1A and S1B) and short OS (Fig. 1F, b and I (Roche Applied Sciences). Supplementary Fig. S1C), and even more consistently in the subset of ER-negative patients treated with chemotherapy DNA fiber assay (Fig. 1E, c and F, c). Cox multivariate regression analysis, taking fi fi DNA ber spreads were prepared (19) with minor modi ca- all clinical parameters into account, revealed a highly signifi- tions. Cells in exponential phase were labeled for sites of ongoing cant (P ¼ 0.00013) inverse correlation between HOXC10 m replication with 5-iododeoxyuridine (IdUrd; 50 mol/L) fol- expression and RFS. lowed by exposure to hydroxyurea (4 mmol/L), washed, and labeled with 5-chlorodeoxyuridine (CldUrd; 50 mmol/L). Fibers HOXC10 were analyzed using ImageJ software. is induced during development of chemotherapy resistance Coimmunoprecipitation to detect HOXC10/CDK7 As a first step to investigating the contribution of HOXC10 to One milligram of protein lysate was subjected to immunopre- drug resistance, we analyzed HOXC10 expression in chemother- cipitation overnight at 4C with CDK7 antibody (C-19; Santa apy-resistant MCF7-sublines (9) that displayed resistance (>250- Cruz Biotechnology) or normal rabbit IgG control (sc-2027; Santa fold) to epirubicin, paclitaxel and docetaxel. HOXC10 was Cruz Biotechnology); immunoblotting was performed using: expressed at 2- to 8-fold higher levels in MCF-7- R sublines (Fig. Myc-Tag (9B11; Cell Signaling Technology), RNA Polymerase II 2A) and in newly developed MDA-MB-231-R sublines that (CTD4H8; Millipore), and XPD [sc-101174, Santa Cruz Biotech- emerged (>30 days) following exposure to drugs (Supplementary nology; ref. 14). Fig. S2A). Depleting HOXC10 levels in MCF-7-Tax-R subline restored its response to paclitaxel (Fig. 2B). Further studies were CDK7 kinase activity carried out in eight breast cancer cell lines with varying levels of Three hundred micrograms of protein extract from drug-treated HOXC10 mRNA (Supplementary Fig. S2B). On exposure of cells cells was used for immunoprecipitation with CDK7 antibody. The in culture to drugs, induction of HOXC10 expression was rapid, immunoprecipitate was resuspended in 40 mL of kinase buffer (50 starting at day 1 in MDA-MB-231 (Fig. 2C), SUM159 (Fig. 2D), HOXC10 mmol/L HEPES, pH 7.5, 10 mmol/L MgCl2, 250 mmol/L EGTA, 10 and SUM149 (Supplementary Fig. S2C). Basal levels of mmol/L b- glycerophosphate, 1 mmol/L DTT), 10 mLof50nmol/L expression determined response to Dox. Mouse xenografts of ATP, and 20 ng GST-CDT peptide (P4016,Proteinone) as substrate, low HOXC10-expressing MDA-MB-231 (Fig. 2E) responded to incubated for 1 hour at 30C, added to an equal volume of kinase- Dox significantly better than high HOXC10-expressing SUM159 GLO reagent (Promega), and incubated for 15 minutes at room (Fig. 2F) or HCC1954 (Supplementary Fig. S2D). By qRT-PCR, temperature. Control reactions lacked the CTD peptide substrate. MDA-MB-231 (Fig. 2G) and SUM149 (Supplementary Fig. S2E) Luminescence was recorded and expressed as relative RLU to the tumors that resumed growth during treatment showed signifi- untreated control cells. cantly higher HOXC10 expression compared with SUM159 (Fig. 2H) and HCC1954 (Supplementary Fig. S2F) supporting the Statistical analysis argument that upregulated HOXC10 expression correlated with Results were expressed as mean SEMofatleastthree resistance to chemotherapy. independent experiments. Paired Student t test or ANOVA tests were performed for data analysis. All statistical analyses were HOXC10 overexpression decreases susceptibility to performed using GraphPad Prism version 4.03 (GraphPad chemotherapy Software, Inc.). In all figures, , P < 0.05; , P < 0.01; and , To characterize the effects of HOXC10 overexpression, we P < 0.001. derived stable clones of MCF10A-Ras [stably expressing K-ras Details of constructs, materials, and experimental procedures (Gly12-Val)] and MDA-MB-231–overexpressing myc-tagged are provided in Supplementary Materials and Methods online. HOXC10, and SUM159 and SUM149 cells depleted of HOXC10

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A B Oncomine HOX tiling array Mets Normal 1.5 4 1.0 3

0.5 2

0.0 1 –0.5 0 –1.0 –1 –1.5 –2 –2.0 log2 Median-centered intensity

Lung Liver –2.5 Breast Bladder Ovarian Kidney Prostate CervicalSarcoma Colorectal Pancreatic HOXC10 Head&Neck Lymphoma

C TCGA D Johns Hopkins tumor panel P < 0.0001 3 P < 0.0001 P = 0.0208 2 750 500 1 250 0 200 –1 150 –2 log2 Median intensity –3 100 Normal Invasive breast Invasive ductal Invasive lobular carcinoma carcinoma carcinoma Relative expression 50 n = 61 n = 76 n = 392 n = 36 P P P = 1.7e-17 = 1.1e-25 = 1.87e-14 0 Fold = 3.779 Fold = 3.402 Fold = 4.239 Organoids IDC Distant mets N = 12 N = 31 N = 49 E GEO a All patients b Chemotherapy treated patients c ER negative and chemotherapy (n = 3,005, HR = 1.1, P = 0.11) (n = 548, HR=1.7, P = 0.009) (n = 285, HR=2.0, P = 0.0039)

1 1 1 Low 0.8 0.8 0.8 High 0.6 0.6 0.6 0.4 0.4 0.4 Probability Probability Probability 0.2 0.2 0.2 0 0 0 051005100510 RFS (10 years) RFS (10 years) RFS (10 years) F

METABRIC a All patients b Chemotherapy treated patients c ER negative and chemotherapy (n = 1,112, HR=1.19, P = 0.18) (n = 528, HR=1.4, P = 0.067) (n = 122, HR=2.8, P = 0.021)

1 1 1 0.8 0.8 0.8 Low High 0.6 0.6 0.6 0.4 0.4 0.4 Probability Probability 0.2 0.2 Probability 0.2 0 0 0 051005100510 OS (years) OS (years) OS (years)

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by 50% to 70% (immunoblot in Fig. 3A) using specific shRNAs. HOXA5 in S. cerevisiae upregulates components of the mismatch Properties of anchorage-independent growth (Supplementary repair (MMR) system, important for the detection and repair of Fig. S3A and S3B), colony formation (Supplementary Fig. DNA damage (32). Hence, we investigated whether HOXC10 S3C), invasion through Matrigel (Supplementary Fig. S3D), pro- participates directly in DNA repair and if so, the type of damage liferation (Supplementary Fig. SE), quantified in (Supplementary and cell-cycle stage of repair. Fig. S3F), and tumor growth in mice (Fig. 3B and Supplementary Ahost–cell reactivation assay was used to assess cellular Fig. S3G) were all significantly decreased by depleting HOXC10 ability to repair exogenously damaged DNA. A pGL3 (lucif- and enhanced by overexpressing HOXC10 in tumor cells. By erase-reporter) plasmid was exposed to UV or cisplatin, or colony survival assays, Dox, gemcitabine, carboplatin, and Tax digested with Hind III (to generate double-strand breaks, DSB) were more cytotoxic in SUM159-shC10 (Fig. 3C and Supplemen- followed by transfection into SUM159-shC10 cells or MCF- tary Fig. S3H), MCF7-shC10 and SUM149-shC10 (Supplemen- 10A-Ras-C10 cells and their respective controls. SUM159- tary Fig. S3H) than in MCF10A-Ras-C10 cells (Supplementary Fig. shC10 cells displayed a significantly decreased ability to repair S3I). This body of data, supporting an increased oncogenicity and DNAdamage(Fig.4A),whereasthereversewasshownforUV- aggressiveness of HOXC10-expressing breast cancer cells, was induced DNA damage in MCF-10A-Ras-C10 cells (Fig. 4B). further corroborated by MTT assays in SUM159-shC10 (Supple- Clonogenic cell survival assays in both MCF10A-Ras-C10 and mentary Fig. S3J) and in MCF10A-Ras-10 cells (Supplementary SUM159-shC10 cells exposed to UV (Fig. 4C, quantified Fig. S3K). in S4A) supported these findings.However,repairofDNA The activation of caspase-3/7 is a reliable marker of cells DSBs caused by Hind III digestion (Fig. 4A) remained undergoing apoptosis (26). Susceptibility to the drugs was unchanged in SUM159-shC10 cells, suggesting no involve- reflected by an increase in caspase 3/7 activity in SUM159-shC10 ment of the NHEJ pathway. Furthermore, ionizing radiation cells (Fig. 3D) and a decrease in MCF10A-Ras-C10 cells (Fig. 3E). (IR) induced G1-chromosome damage repair was not affected Apoptotic cells were identified on the basis of DNA content in SUM159-shC10 cells (Supplementary Fig. S4B), wherein frequency histograms as cells with fractional "sub-G1" DNA DSB repair by NHEJ is predominant. On the other hand, content (27). In response to Dox, there was a decrease in sub- SUM159-shC10 cells had a higher frequency of S-phase–spe- G1 population in SUM159shC10 cells (Fig. 3F) and an increase in cific IR-induced chromosomal aberrations (Supplementary MCF10A-Ras-C10 cells (Fig. 3G). Also, several pro- and antia- Fig. S4C), suggesting that HOXC10 expression enhances poptotic mRNAs were modulated in SUM159-shC10 and repair, possibly by the homologous recombination (HR) MCF10A-Ras (Supplementary Fig. S3L and S3M), and further pathway. MCF10A-Ras-C10 cells treated with cisplatin showed intensified by drug treatment (Fig. 3H and Supplementary Fig. significantly reduced number of cells with chromosomal aber- S3N). Western blot analysis of SUM159-shC10 cells showed that rations at metaphase (Supplementary Fig. S4D) compared antiapoptotic genes, hsp27, Birc 3, PI3K, and BCL2 levels were with the vector control cells. Collectively, the data strongly decreased, whereas proapoptotic genes, p21, and BID levels were support the hypothesis that HOXC10 is an important partic- increased by depletion of HOXC10 (Fig. 3I). Dox-treated ipant in DNA DSB repair by HR. MCF10A-Ras-vec xenografts regressed by week 4, whereas We had noted that HOXC10 expression had a dramatic effect MCF10A-Ras-C10 tumors grew after week 2 (Fig. 3J). Consistent on survival following exposure to UV light or the platinum with this observation, MCF10A-Ras-C10 tumors showed high drugs (Fig. 4A–C and Supplementary S4A). DNA damage mRNA expression of many antiapoptotic genes (Fig. 3K). induced by such agents is repaired by nucleotide excision repair Given that the antiapoptotic genes examined are known direct (NER) and HR. Therefore, we measured the activity of the NER targets of NF-kB (28), we measured NF-kB activity using the NF- pathwaybythealkalinecometassay(Fig.4D),asingle-cellgel kB–responsive reporter Igk2-IFN-LUC, with and without Dox electrophoresis method in which the intensity of the comet tail treatment. The basal activity of NF-kB was lower in SUM159- of the migrating cells relative to the head reflects the number of shC10 (Supplementary Fig. S3O) and high in MCF10A-Ras-C10 DNA breaks (33). Cells depleted of HOXC10 had significantly (Supplementary Fig. S3P) compared with their respective controls, more residual UV-, Dox- and gemcitabine-induced DNA dam- and further enhanced by Dox treatment. These data are consistent age 24 hours after treatment (Fig. 4E). Furthermore, time- with findings that the basal activity of NF-kB is much higher, and course analysis after UV treatment showed that DNA damage maintained as such, in Dox-resistant MCF7 cells (29). Thus, in SUM159-scr cells had returned to baseline levels in 24 hours, HOXC10 upregulates NF-kB to achieve both activation of anti- but was still 4-fold higher in SUM159-shC10 cells (Fig. 4E), apoptotic pathways, and increases cell survival following stress. revealing inefficient DNA repair in the absence of HOXC10. Improved response to DNA damage was evident in MCF10A- HOXC10 enhances DNA damage repair and checkpoint Ras-C10 cells compared with the vector-control cells (Fig. 4B, C, recovery and F). The reduced survival of SUM159-shC10 cells in colony Several homeodomain-related proteins have been functionally formation assays (Fig. 4C) also correlated with higher levels of related to DNA damage repair (1, 2, 30, 31). Overexpression of residual DSBs.

Figure 1. HOXC10 overexpression has prognostic signiﬁcance in breast cancer. A, HOX-tiling array analysis of mRNA expression in normal breast and distant metastasis. B, HOXC10 expression in cancers in the Bittner multicancer dataset. , P ¼ 3.18 e40. C, invasive ductal and lobular breast carcinoma. D, qRT-PCR of HOXC10 expression in normal breast organoids (n ¼ 12), invasive ductal carcinoma (n ¼ 31), and distant metastasis (n ¼ 49). Kaplan-M plots of correlation of HOXC10 expression with RFS (E) and OS (F) in all patients with breast cancer (n ¼ 4117; a), chemotherapy-treated (b), chemotherapy-treated ER-negative (c) breast cancer.

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A B MCF-7-R Sublines MCF7-Pacli-R Subline *** scr 6 shC10-1 ** 95 5 shC10-2

3 * * 76 2 % Survival 1

0 60

Relative Fold HOXC10 expression 0 2 4 6 8 10 -1 R h i- p ac-R Ve E Veh-2P μ Doce-R Paclitaxel, mol/L

C MDAMB231 D SUM159 * * * 8 4 * 6 * * 3 * * * * * * 4 * 2 * expression expression 1 2 * Relative fold HOXC10 Relative fold HOXC10 0 0 Days 0 1 2 3 1 2 3 1 2 3 Days 0 1 2 3 1 2 3 1 2 3 Dox Gemc Carbo Dox Gemc Carbo

SUM159 E MDA-MB-231 F 400 2,000 ) ) 3 3 Control Control 1,500 300 Dox Dox n.s 200 1,000 **

500 100 Tumor volume (mm Tumor volume (mm 0 0 0 5 10 15 20 0 5 10 15 Days Days

G 2.0 H ** 1.5 n.s 1.5 1.0 1.0

0.5 0.5

0.0 0.0 Cont Dox Cont Dox Relative fold HOXC10 expression Relative fold HOXC10 expression MDA-MB-231 Tumors SUM159 Tumors

Figure 2. Chemotherapy-resistant cell lines upregulate HOXC10. A, qRT-PCR of HOXC10 expression in drug-resistant MCF7 sublines. Epirubicin (Epi-R), paclitaxel (Pac-R), and docetaxel (Doc-R) resistant. B, MTT assay in HOXC10-depleted MCF7-Pac-R clones. qRT-PCR of HOXC10 expression in MDA-MB-231 cells (C)and SUM159 cells (D) treated with Dox (200 nmol/L), gemcitabine (Gemc; 200 nmol/L), or carboplatin (50 mmol/L). Growth in vivo of xenografts of MDA-MB-231 (n ¼ 12; E) and SUM159 (n ¼ 12; F) treated with doxorubicin. qRT-PCR of HOXC10 expression in Dox-treated xenografts of MDA-MB-231 (G) and SUM159 (H). Mean values SEM are plotted. , P < 0.05; , P < 0.01; , P < 0.001.

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2,000 A B ) 3 P = 0.002 SUM 149 10A-Ras 231 SUM159 SUM149 1,500 v C10 v C10 scr sh-1 sh-2 scr sh-1 sh-2 Scr 1,000 HOXC10

500 shC10 GAPDH Tumor volume (mm 0 scr shC10 SUM149 C D SUM159 E 1,350 MCF10A-RAS 450 v Dox, Dox, Gemc, Gemc, Carbo, Carbo, Tax, scr * 1,050 μ μ * C10 None 50 nmol/L 100 nmol/L 50 nmol/L 100 nmol/L 10 mol/L 25 mol/L 10 nmol/L 360 shC10-1 *** shC10-2 750 SUM159 270 450 scr * * * * * 300 activity 180 SUM159 200 ** * shC10 90 100 % Increase in caspase-3/7 0 0 Dox Gemc Carbo Tax Dox Gemc Carbo Tax F )

1 50 SUM159 159-SCr 159-shC10 scr ** 40 shC10

30 ***

20 * H SUM159: Dox treatment 10 6 scr % Apoptotic cells (sub-G 0 5 * 0DoxGemcUV shC10 * 48h Dox treatment 4 *

G ) 1 30 MCF10A-Ras 3 Ras-V Ras-C10 V * ** 25 ** 2 * C10 * * ** 20 1 * Relative expression ** 15 0 α 10 P - P IM ID K ** B A B A IRC2FLI Κ XI P27 P21 B B B I BIRC3 5 BCL-XL

% Apoptotic cells (sub-G 0 0 Dox Tax UV

I J K 159-SCr 159-shC10 MCF10A-Ras 20 Dox (nmol/L) 0 250 500 1,000 0 250 500 1,000 16 * 10A-Ras * Hsp27 300 V v 12 * ) C10 Birc3 3 C10 8 225 4 Pl3K * 3 BCL2 150 ** 2 * * 1 Bid 75

Fold induction in expression induction Fold 0 p21 Tumor volume (mm 3 P -a 1 0 -XL A B VIN RC XI K I P21 pH2Ax 012345 CL I V MCL RCC3 BI R BIRC2E ERCC2 HSP27 B GADD34 Weeks SU β-Actin

Figure 3. HOXC10 overexpression decreases susceptibility to chemotherapy treatment. A, Western blot analysis of MCF10A-Ras-C10 and MDA-MB-231-C10, and HOXC10 shRNAs expressing clones of SUM159 and SUM149 cells. Loading control, GAPDH. B, growth of xenografts of SUM149-Scr and SUM149-shC10 cells in immunodeficient mice. C, colony survival assay of SUM159-shC10-1 cells treated with Dox, gemcitabine (Gemc), carboplatin (Carbo), and Tax. D and E, caspase-3/7 activity in SUM159-shC10 cells (D) and MCF10A-Ras-C10 cells (E) following treatment with treated with Dox (1 mmol/L), gemcitabine (1 mmol/L), taxol (0.5 mmol/L), docetaxel (0.5 mmol/L), or carboplatin (100 mmol/L) for 24 hours. F and G, flow cytometry analysis of SUM159-shC10 cells (F) and MCF10A-Ras-C10 cells (G) treated with Dox (50 nmol/L), gemcitabine (50 nmol/L), or UV; bar graphs show quantification of cells in subG1. H, qRT-PCR analysis of pro- and antiapoptotic mRNAs with Dox (200 nmol/L) for 72 hours. I, Western blot analysis of select proteins in SUM159-shC10 cells treated with Dox (250–1,000 nmol/L). J, growth of established xenografts of MCF10A-Ras-v (n ¼ 6) and MCF10A-Ras-C10 (n ¼ 9) cells in nude mice, treated three times at weekly intervals (arrows) with Dox (4 mg/kg). K, qRT-PCR of antiapoptotic genes in tumors from I at 5 weeks. Mean values SEM are plotted. , P < 0.05; , P < 0.01; , P < 0.001.

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A Host cell reactivation assay B C MCF10A-Ras UV 120 SUM159 100 0 scr v 100 shC10 80 C10 SUM159-scr 80 60 60 SUM 159-shC10 40 40 MCF10A-Ras-V % Luciferase activity 20 % Luciferase activity 20 0 0 MCF10A-Ras-C10 0UV 0UV Cispl Hindlll

DEFAlkaline comet assay-SUM 159 P < 0.0001 SUM159 0 UV-12 h UV-24 h Dox 50 30 MCF10A-Ras P < 0.0001 scr v shC10 25 159-scr 40 C10 P = 0.0004 20 P = 0.021 30 P = 0.0134 P = 0.0002 15 159-shC10 20 10 Tail moment P < 0.0001 10 5

Key: 0 0 0 Dox Gemc UV UV 0 Dox Gemc UV UV No Increase in pre- 24 h 12 h 24 h 24h h 24 h12 damage DNA damage Apoptotic

G γ-H2AX fociH l-Scel endonuclease–induced I ChIP analysis of HR proteins DSB repair * 100 *** * scr 0.0015 80 shC10 *** 60 0.001 H2AX foci H2AX γ 40

Sce-1-Induced HR Sce-1-Induced 0.0005 Total 20 Relative protein associations 0 0 None Dox Gemc

J K DNA fiber analysis MCF10A-Ras+C10 MCF10A-Ras+vector * SUM159-scr SUM159-shC10 *

SUM159-shC10 MCF10A-Ras-C10

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DNA damage that results in DSBs is always followed by the We therefore considered the possibility that HOXC10 has a role phosphorylation of the histone, H2AX (34). Newly formed phos- in the initiation of new origins of DNA replication. Single-mol- phorylated protein g-H2AX is the initial step of damage detection ecule examination of replication dynamics by DNA fiber analysis to recruit repair proteins (34). We quantified the number of (19) after hydroxyurea (HU) treatment in SUM159-shC10 and residual g-H2AX foci 24 hours after treatment of SUM159-shC10 MCF10A-Ras-C10 cells showed that HOXC10 is required for the cells with Dox or gemcitabine. On average, 20% to 30% more initiation of new origins, but not for the accumulation of stalled g-H2AX foci accumulated in SUM159-shC10 cells after drug forks (Fig. 4J and K). treatment (Fig. 4G) with almost 50% to 60% more cells displaying Collectively, the data suggestthatHOXC10,asatranscrip- 100 foci/nucleus (Supplementary Fig. S4E). A time-course anal- tional factor, affects the chromatin status and sets the stage for ysis of g-H2AX protein after UV or Dox treatment showed no HR by, (i) decreasing S-phase chromosome aberrations, (ii) differences in the initial induction of g-H2AX (10 minutes to 8 enhancing resection as shown by g-H2AX foci, (iii) increasing hours; Supplementary Fig. S4F and S4G). Residual g-H2AX mea- HR based on l-Scel endonuclease–induced DSB repair, and (iv) sured 24 hours after treatment, and not the initial kinetics of resolving stalled replication forks as shown by DNA rfibe g-H2AX formation, is a better predictor of cell viability (35, 36). analysis, possibly leading to initiation of DNA replication These results indicated that HOXC10 might be more involved in following DNA damage. DNA damage processing rather than in damage sensing. No major difference was observed in the phosphorylation status of known HOXC10 binds to CDK7 in vivo after chemotherapy treatment DNA damage sensors of the ATR/ATM pathways after Dox or We had thus far observed that HOXC10 enhances cell gemictabine treatment of SUM159-shC10 (Supplementary Fig. survival by affecting apoptosis, NF-kB activity, and damage S4H), thus confirming that HOXC10 is not involved in DNA repair, mainly by the HR pathways. One key protein that links damage sensing. all three pathways is cyclin-dependent kinase 7 (CDK7). In These findings were further substantiated by the HR-DSB repair metazoans, CDK7 has essential roles in transcription as a assay of lesions induced by I-Sce1 endonuclease performed in DR- component of the general transcription factor, TFIIH. CDK7 95 cells engineered to stably express a pDR-GFP plasmid contain- is an effector kinase, which also phosphorylates RNA Pol II and ing a mutated GFP gene with an 18 bp I-Sce1 endonuclease other proteins during transcription after the pausing of the cleavage site and an in-frame termination codon (10, 20). Effi- TFIIH complex following DNA damage (37). Highly relevant cient repair of the Sce-1 cleavage site restores expression of GFP. to this study, HOXC10 was found to be the tightest binding DR-95 cells, with and without HOXC10 silencing (using siRNA) protein linking the CAK (Cdk-activating kinase, CDK7) com- were transfected with the I-Sce1 expression plasmid. DR95- plex to TFIIH in a yeast 4-hybrid system (38). We therefore siHOXC10 showed reduced repair of I-Sce-1–induced lesions. tested for an association of HOXC10 with CDK7 by coimmu- Consequently, restoration of GFP expression occurred at a signif- noprecipitation. A weak interaction between the two proteins icantly lower efficiency than in control cells (Fig. 4H). Reduction increased significantly upon treatment with Dox and gemci- of GFP expression was comparable with cells with reduced tabine (Fig. 5A). This interaction was reduced significantly expression of BRCA-1, a known DNA repair protein, used as a upon addition of a pharmacologic inhibitor of CDK7 activity, positive control (Fig. 4H). Thus, repair of DSB by HR is impaired SNS-032 (Fig. 5A), or by expressing the dominant negative, in cells lacking HOXC10 expression. kinase-dead mutant CDK7 (D155A) in the cells (Fig. 5B). We To investigate a role of HOXC10 in the recruitment of HR repair concluded that CDK7 and HOXC10 formed a complex in proteins at the site of a single DNA DSB, we performed chromatin breast cancer cells, which became more abundant upon expo- immunoprecipitation (ChIP) in DR-95 cells (10, 20). We com- sure to drugs. pared the levels of BRCA1, RAD51, KU80, and HOXC10 at Different sets of proteins are phosphorylated by CDK7 depend- different distances from an I-Sce1–induced DSB site using ChIP ing on its function in cell division (i.e., CDK1, 2, 4, 6) or in analysis with specific primers (10) in cells with and without transcription as a complex with TFIIH (37). We therefore inves- expression of HOXC10. Depletion of HOXC10 using siRNAs did tigated whether binding of HOXC10 to CDK7 altered its substrate not decrease KU80 levels in close proximity to the DSB, whereas preference. Also, elongating RNA Pol II can be arrested by endog- levels of RAD51 and BRCA1 at the break site were significantly enous and exogenous DNA lesions such as UV-induced pyrimi- reduced (Fig. 4I). This finding in HOXC10-depleted cells strongly dine dimers, adducts induced by anticancer drugs, and DSBs. supported the role of HOXC10 in facilitating the recruitment of These transcription-blocking lesions located on the transcribed proteins involved in HR. However, HOXC10 itself was undetect- strand are primarily repaired by transcription-coupled repair, the able at the I-Sce1 cleavage site, ruling out a direct role for HOXC10 NER pathway (39). XPD helicase is a key member of the human in DSB repair. TFIIH complex that anchors CAK kinase (cyclin H, MAT1, and

Figure 4. HOXC10 facilitates repair of DNA damage. A and B, host cell reactivation assay in SUM159-shC10 (A) and MCF10A-Ras-C10 (B) cells following UV and/or cisplatin exposure. C, colony survival assay of SUM159-shC10 and MCF10A-Ras-C10 cells, 7 days after UV exposure. D, alkaline comet assay measure of DSBs caused by UV or Dox in SUM159-shC10 cells. E and F, quantification of tail moments in SUM159 (E) and MCF10A-Ras (F) treated with Dox (200 nmol/L), gemcitabine (200 nmol/L), and UV. G, quantification of phosphorylated g-H2AX using fluorescent anti–phospho-H2AX Ser139 antibodies in SUM159-shC10 cells treated with Dox (200 nmol/L) or gemcitabine (50 nmol/L) for 24 hours. H, I-Sce1 assay for HR repair activity in DR95-siC10, 72 hours after transfection. HR frequencies are shown with (þ) or without () I-SceI induction. Positive control, BRCA-1 siRNA–transfected cells. I, ChIP analysis of recruitment of repair proteins to I-Sce I DSB site (, P < 0.05, c2 test). J, representative images of DNA fiber assay on SUM159-shC10 and MCF10-Ras-C10 at 21 hours. K, quantification of stalled forks (green) and new origins (red) in the DNA fiber assay showing correlation between HOXC10 expression and initiation of new origins of replication. Mean values SEM are plotted. , P < 0.05; , P < 0.01; , P < 0.001.

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A B

293T Cells 293T Cells

C D

F SUM159 E MCF10A-Ras 0 Dox Gemc V C10 V C10 V C10 RNA Pol II IP: Cdk7 Cdk7

RNA Pol II Input Cdk7

G H I 4.0 MCF10A-Ras SUM159 scr MCL1 MCL1 SUM159 v C10 shC10-1 1.6 * 3.2 2.0 scr ** shC10-2 * shC10 1.2 2.4 * * 1.5 0.8 1.6 * * 1.0 * ** 0.8 0.5 0.4 Relative CDK7 activity Relative Relative expression Relative expression

0.0 0.0 0.0 Control Dox Gemc 0DoxGemc 0DoxGemc

Figure 5. HOXC10 binds to CDK7 during DNA damage response. A, 293T cells were transfected with HOXC10 and treated with Dox (200 nmol/L), gemcitabine (Gemc; 200 nmol/L), taxol (50 nmol/L), or carboplatin (50 mmol/L) for 24 hours. Cell lysates were coimmunoprecipitated with CDK7 (D, doxorubicin; SNS-032, CDK7-inhibitor). B, coimmunoprecipitation of HOXC10 with CDK7 (wt) and its kinase mutant (D155A). C and D, coimmunoprecipitation of XPD by CDK7 in SUM159 cells (-scr and -shC10; C) and MCF10A-Ras (-vec and -C10; D) after treatment with Dox and gemcitabine for 24 hours. E and F, coimmunoprecipitation of RNA Pol II by CDK7 in SUM159 cells (-scr and -shC10; E) and MCF10A-Ras (-vec and -C10; F) after treatment with Dox and gemcitabine for 24 hours. G, kinase activity of CDK7 on recombinant GST-CTD substrate in SUM159-shC10 cells treated with Dox (100 nmol/L) or gemcitabine (100 nmol/L) for 24 hours. H and I, qRT-PCR analysis of MCL1 (as marker of CDK7 activity) in SUM159-shC10 (H) and MCF10A-Ras-C10 (I) cells treated with Dox (100 nmol/L) or gemcitabine (100 nmol/L) for 24 hours. Mean values SEM are plotted. , P < 0.05; , P < 0.01.

CDK7) to TFIIH and unwinds DNA for transcription and for repair fore examined whether HOXC10 affects the composition of the of duplex warping damage by nucleotide excision repair TFIIH–XPD–CAK complex through its protein/protein interaction (NER), thereby maintaining genomic integrity (40). We there- with CDK7. The binding of XPD to CDK7 was reduced in SUM159-

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A BCwtTax-R wt Epi-R MCF7 cells p-CTD (Ser5) MCF10A-Ras; SNS-032 MCF10A-Ras; BS-181 100 ** 100 ** t-RNA polll * 80 ns p-CDK7 (Thr 170) 75 * 60 ** * t-CDK7 ns 50 p-CDK2 (Thr160) 40 % Survival

% Survival 25 20 p-CDK1 (Thr161) t-CDK1 0 0 0 Dox 100 Gemc 10Carb 100 0Dox 25Gemc 100 β-Actin v v+SNS C10 C10+SNS v v+BS C10 C10+BS

D E F BS-181 Treatment-MCF7-Tax-R cells wt Tax-R 100 3.0 ** *** HOXC10 0 0 BS-181 * 2.5 MCL1 ** 2.0 ** 0

1.5 ** Tax-R-Veh Tax-R-BS, 10 μmol/L

1.0 % Survival MCF-7-Veh Tax

Relative expression 0.5 10 0.0 0 50 100 150 200 250 wt Tax-R Epi-R MCF-7 [Tax] nmol/L

Figure 6. Inhibiting CDK7 restores chemosensitivity to breast cancer cells. A and B, MTT assay of MCF10A-Ras-C10 cells treated with CDK7 inhibitors, SNS-032 (40 nmol/L; A)orBS-181(5mmol/L; B) in combination with Dox, gemcitabine (Gemc), and/or carboplatin for 48 hours. C, Western blot analysis of p-CDK7 and p-CTD, p-CDK1, p-CDK2 in parental MCF (WT) and drug-resistant sublines. D, qRT-PCR analysis of MCL1 and HOXC10 in MCF7 drug- resistant sublines, Tax-R and Epi-R. E, colony survival assay after treatment of MCF-7-Tax-R with taxol, 100 nmol/L BS-181 (20 mmol/L) for 7days.F, MTT assay of MCF7-Tax-R treated with taxol (0–250 nmol/L) BS-181 (10 mmol/L) for 48 hours. Parental MCF-7 cells served as a control for taxol susceptibility. Mean values SEM are plotted. , P < 0.05; , P < 0.01; , P < 0.001. shC10 cells (Fig. 5C) and was increased in MCF10A-Ras- C10 cells, Inhibiting CDK7 reverses HOXC10-dependent compared with their respective control cells (Fig. 5D). This chemoresistance finding suggested that HOXC10 may have a role in anchoring Inhibiting CDK7 is of special interest in cancer therapeutics as CAK to XPD, thus maintaining the integrity of the holoenzyme it affects multiple signaling pathways (43). Two known selective TFIIH during response to DNA damage. This finding is also CDK7 inhibitors are BS-181 (44) and SNS-032 (11); the latter is consistent with reports that in repair-deficient cells, the asso- in phase I studies (http://clinicaltrials.gov/). Combining SNS- ciation of CAK kinase, but notofXPD,todamagedDNAwas 032 or BS-181 at minimally cytotoxic doses (20%) with reduced (41). chemotherapy significantly improved the response of both Next, we investigated whether the association of CDK7 MCF10-Kras-C10 cells (Fig. 6A and B) and SUM159-shC10 with the RNA Pol II is modulated by HOXC10. We found that cells (Supplementary Fig. S6A and S6B). In taxol and epirubi- the association between CDK7 and RNA Pol II in drug-treated cin-resistant MCF7 sublines, Western blot analysis showed that cells was significantly stronger in the presence of HOXC10 in CDK7 (pThr170) and CTD (pSer5) were activated (Fig. 6C) with MCF10A-Ras-C10 (Fig. 5E) and weaker in the absence of no change in cell-cycle kinases, CDK1 and CDK2. qRT-PCR HOXC10 in SUM150-shC10 (Fig. 5F). Furthermore, the kinase analysis showed an increase in both HOXC10 and MCL1 activity of CDK7 on the C-terminal domain (CTD) of RNA Pol mRNAs (Fig. 6D). Treatment with taxol combined with BS- II was significantly reduced in SUM159-shC10 cells after DNA 181 restored drug susceptibility of Tax-R-MCF-7 cells, as seen in damage (Fig. 5G). MCL1 is an antiapoptotic protein that is colony formation assays (Fig. 6E), and caused cell kill in rapidly depleted upon inhibition of RNA Pol II, permitting parental MCF-7 cells as shown by MTT assays (Fig. 6F). Sim- measures of its mRNA to serve as a surrogate for CDK7 activity ilarly, BS-181 restored sensitivity of two MDA-MB-231-Tax-R (42). The lack of CDK7 activity is likely to impede recovery of sublines to taxol (Supplementary Fig. S6C and S6D), an effect the transcriptional machinery, thereby promoting cell death not achieved using inhibitors to CDK1/2/9, AZD5438 (45) and after DNA damage as was reflected by MCL1 expression (Fig. CDK4/6, PD0332991 (Supplementary Fig. S6E and S6F; ref. 46). 5H). Conversely, the enhanced interaction between Pol II and ThesedatasuggestaspecificroleforCDK7inresistanceto CDK7 in MCF10A-Ras-C10 cells (Fig. 5E) promotes cell sur- chemotherapeutic drugs. vival following DNA damage (Fig. 5I). Consistent with this In summary, our data support a model of HOXC10 action data, newly emerging drug-resistant MCF7 sublines in cell wherein it is intimately involved in multiple steps in the culture showed increases in both MCL1 and HOXC10 mRNA process of HR-mediated DNA repair following chemotherapy expression (Supplementary Fig. S5). exposure of breast cancer cells and in the development of drug

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Drug/IR-induced DNA damage in ER- (Fig. 4 and Supplementary Fig. S4; ref. 47). HOXC10 does not affect the sensing of DDR but is required at later stages of the negave breast cancer DNA damage repair by NER and HR (Fig. 4 and Supplementary Fig. S4). Cells expressing high levels of HOXC10 repaired DNA HOXC10 damage more efficiently and resumed transcription and growth; whereas their low-HOXC10–expressing counterparts eventually γH2AX/MDC1/53BP1/BRCA1 committed to apoptosis. Part of this response, we propose, may be attributed to the observed association of HOXC10 with the CAK complex. HOXC10 DNA DSB The CAK complex along with TFIIH can participate in diverse functions, including transcription, DNA repair (NER), fi RAD51 and cell-cycle regulation. We con rmed that HOXC10 binding to CDK7 enhances its kinase activity toward the CTD domain of RNAPII after DNA damage. We surmise that HOXC10 plays a role in bridging the gap between these 2 BRCA1-PALB2-BRCA2 complexes, enhancing the recovery process after DNA damage. Indeed, treatment of drug-resistant MCF-7 cells with HOXC10 CDK7 inhibitors restored their susceptibility to chemotherapy (Fig. 6). The importance of current findings to breast cancer HOXC10 therapy stems from the recent attention given to CDK inhi- HR Repair TFIIH bition, including CDK7, in clinical trials either as single Resoluon of agents or in combination with chemo- or targeted therapies XPD CDK7 to overcome resistance (43, 48). stalled replicaon The second survival pathway that is activated by HOXC10 is forks RNA Pol II shifting the balance between growth and apoptosis to allow continuous proliferation and survival under adverse conditions. Our data showed that HOXC10 facilitates the transition Complete repair from G1 to S phase and progression through the S-phase during Restart transcripon for recovery the cell cycle (Fig. 3). As a consequence, cells with high HOXC10 levels have a growth advantage, especially under Inhibit apoptosis nonideal conditions, and restart their replication to overcome stressfulconditions.Atthesametime,theincreaseinNF-kb Figure 7. activity and the consequent increase in the levels of many A model for HOXC10 action in breast cancer cells. Upon exposure to antiapoptotic proteins likely decrease cell sensitivity to many chemotherapy or ionizing radiation (IR), DNA damage mediator genes such as stressors. H2AX/MDC1/53BP1/BRCA1 are activated, triggering DNA DSB repair. g Because of HOXC10 involvement in survival and proliferation HOXC10 is upregulated following chemotherapy or ionizing radiation in ER-negative breast cancer. HOXC10, as part of the CAK complex, participates of cancer cells despite exposure to chemotherapy, HOXC10 is an in the late stages of DNA repair that involves restart of transcription for attractive target to reverse chemotherapeutic resistance in breast recovery and acts as an inhibitor of apoptosis. cancer. Future studies could focus on developing direct inhibitors of HOXC10 or indirect modulators of its function by targeting its downstream effectors, such as CDK7. resistance in the long term. HOXC10 is an integral component of the CAK complex, which allows restart of transcription Disclosure of Potential Conflicts of Interest following DNA damage (Fig. 7). H.Y. Chang has ownership interest (including patents) in Epinomics and is a consultant/advisory board member for RaNA Therapeutics. S. Sukumar reports receiving a commercial research grant from CEPHEID and is consultant/advi- Discussion sory board member for AVON Foundation. No potential conflicts of interest Chemotherapy failure in breast cancer claims at least 80,000 were disclosed by the other authors. lives each year worldwide. Here, we present evidence that HOXC10 overexpression is common and functionally important Authors' Contributions for onset of resistance to chemotherapy in ER-negative breast Conception and design: H. Sadik, R. Kumar, S. Park, N. Shah, H.Y. Chang, cancer and is also prognostic of poor outcome primarily in T.K. Pandita, S. Sukumar Development of methodology: H. Sadik, R. Kumar, M. Hedayati, S. Park, patients treated with chemotherapy. T.G. Munoz, N. Shah, R.K. Pandita, J.C. Chang, H.Y. Chang, T.K. Pandita, Upregulation of HOXC10 is likely to be an important cancer S. Sukumar adaptation mechanism. Ectopic expression of HOXC10 leads to Acquisition of data (provided animals, acquired and managed patients, decreased drug susceptibility, while decreasing its levels provided facilities, etc.): H. Sadik, P. Korangath, N.K. Nguyen, M. Hedayati, enhances the cytotoxic effects of chemotherapy in both in vitro W.W. Teo, N. Shah, J.C. Chang, T. DeWeese, H.Y. Chang, S. Sukumar and in vivo models of breast cancer. Mechanistically, HOXC10 is Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Sadik, B. Gyorffy, R. Kumar, M. Hedayati, involved in activating different survival pathways, including H. Panday, T.G. Munoz, N. Shah, J.C. Chang, S. Sukumar driving checkpoint recovery after DNA damage, a key pathway Writing, review, and/or revision of the manuscript: H. Sadik, B. Gyorffy, that allows cancer cells to overcome damage response arrest M. Hedayati, N. Shah, T.K. Pandita, S. Sukumar

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Administrative, technical, or material support (i.e., reporting or organizing Grant Support data, constructing databases): H. Panday, O. Menyhart, R.K. Pandita, This work was supported by funding from the DOD-BCRP BC093970 T.K. Pandita, S. Sukumar (H. Sadik), NCI-P50-CA88843 and P30-CA006973 (S. Sukumar) and NIH Study supervision: T.K. Pandita, S. Sukumar grants CA129537, CA154320, and GM109768 (T.K. Pandita). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in Acknowledgments accordance with 18 U.S.C. Section 1734 solely to indicate this fact. We thank Dr. Joel L. Pomerantz for providing us their responsive reporter plasmids NF-kB-luc, Dr. Amadeo M Parissenti for the drug-resistant MCF7 cell Received April 25, 2016; revised May 24, 2016; accepted May 25, 2016; lines, and Dr. Alan Rein for reviewing the article. published OnlineFirst June 14, 2016.

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OF14 Cancer Res; 76(15) August 1, 2016 Cancer Research

HOXC10 Expression Supports the Development of Chemotherapy Resistance by Fine Tuning DNA Repair in Breast Cancer Cells

Helen Sadik, Preethi Korangath, Nguyen K. Nguyen, et al.

Cancer Res Published OnlineFirst June 14, 2016.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-16-0774

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