Development of drug resistance is a major factor limiting the continued success of cancer chemotherapy. To overcome drug resistance, understanding the underlying mechanism(s) is essential. We found that HOXC10 is overexpressed in primary carcinomas of the breast, and even more significantly in distant metastasis arising after failed chemotherapy. High HOXC10 expression correlates with shorter recurrence-free and overall survival in patients with estrogen receptor–negative breast cancer undergoing chemotherapy. We found that HOXC10 promotes survival in cells treated with doxorubicin, paclitaxel, or carboplatin by suppressing apoptosis and upregulating NF-κB. Overexpressed HOXC10 increases S-phase–specific DNA damage repair by homologous recombination (HR) and checkpoint recovery in cells at three important phases. For double-strand break repair, HOXC10 recruits HR proteins at sites of DNA damage. It enhances resection and lastly, it resolves stalled replication forks, leading to initiation of DNA replication following DNA damage. We show that HOXC10 facilitates, but is not directly involved in DNA damage repair mediated by HR. HOXC10 achieves integration of these functions by binding to, and activating cyclin-dependent kinase, CDK7, which regulates transcription by phosphorylating the carboxy-terminal domain of RNA polymerase II. Consistent with these findings, inhibitors of CDK7 reverse HOXC10-mediated drug resistance in cultured cells. Blocking HOXC10 function, therefore, presents a promising new strategy to overcome chemotherapy resistance in breast cancer. Cancer Res; 76(15); 4443–56. ©2016 AACR.

Many chemotherapeutic agents induce DNA damage, triggering cells to activate their DNA damage repair machinery. Nucleotide excision repair (NER) is a major DNA repair pathway that mediates repair of DNA adducts that cause inter- or intrastrand cross links (ICL) and the formation of pyrimidine dimers. Alkylating- and platinum-based agents often induce bulky DNA damage that is repaired via NER.

Large scale interaction screens as well as reciprocal affinity purifications have been instrumental in identifying several HOX proteins that interact with proteins critical for DNA damage repair processes (reviewed in ref. 1). HOXB7 associates with Ku70, Ku80, and DNA-Pkcs (2), and with PARP1 (3), whose polyADP-ribosylation activity enhances the kinase activity of DNA-PK at the initiation of non-homologous end joining (NHEJ). Recent evidence supports HOX protein participation in the cell 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 HOXC10 expression was implicated in the development of resistance to estrogen response modulators in estrogen receptor (ER)–positive breast cancer (8). At the present time, the function of HOXC10 in breast cancer remains poorly understood.

Here, we report that HOXC10 expression is frequently higher in ER-negative breast carcinomas that are also chemotherapy resistant. We determined that HOXC10 contributes to chemoresistance through suppression of apoptosis and enhanced DNA damage repair, mediated through direct interaction with, and activation of CDK7. We also show that CDK7 inhibition can reverse chemotherapy resistance.

Cell lines, constructs, and reagents

All cell lines were purchased from the ATCC and passages used were within 6 months of purchase. Stable MCF10A-Ras cells were established by transfection of LXSN-K-Ras vector into MCF10A cells; MCF7 wt and drug-resistant sublines were from Dr. A.M. Parissenti, Laurentian University, Sudbury, ON, Canada (9). DR-95 is a human fibroblast cell line stably expressing a pDR-GFP plasmid containing a mutated GFP gene with an 18 bp I-SceI endonuclease cleavage site and in-frame termination codon (10). Retrovirus and lentivirus were produced in HEK 293T cells. Human HOXC10 cDNA (Thermo Scientific) was cloned into the EcoRI and ClaI sites of pLPCX for retroviral production for creating HOXC10-expressing cell lines. Mutated HOXC10 constructs were generated with full-length myc-tagged HOXC10 cloned in pCDNA3.1-Neo at EcoRI and XbaI sites. The following reagents were used: doxorubicin (Sigma), paclitaxel, and gemcitabine (Tocris Bioscience) and carboplatin, BS-181 and SNS-032 (gifted by Selleckchem; ref. 11), TRC lentiviral Human HOXC10 shRNA (set of 4; Thermo Scientific); FlexiTube siRNA for HOXC10 (SI04296621), E2F1 (SI00300083), or CDK7 (SI02664795) (Qiagen). All other reagents were from Sigma.

Human tissue samples

Fresh frozen primary human tissues were used following approval of the Johns Hopkins Institutional Review Board.

Survival analysis in patients with breast cancer

Kaplan–Meier analyses were performed using 4,117 breast cancer patients. http://kmplot.com/analysis (12).

Real-time quantitative PCR analysis

qRT-PCR was conducted using the MaximaSYBR Green/ROX Master Mix (Fermentas) per manufacturer protocol. The ΔΔCt method was used, with GAPDH expression for normalization (13).

Soft agar colony formation assay and Matrigel invasion assay

Six-well plates containing a 0.6% agar layer were overlaid with 3 × 103 cells i n 0.3% agar layer. Colonies were counted after 7 days. Cell invasion assay was performed using The BD BioCoat Matrigel Invasion Chamber assay system (13).

Tumor xenograft studies

Approved by Johns Hopkins Institutional Animal Care and Use Committee (IACUC), BALB/c nu/nu athymic mice (Sprague–Dawley–Harlan) received subcutaneous injections of 3 million cells/100 μL PBS/Matrigel (1:1), and were treated with doxorubicin (4 mg/kg BW/i.v./weekly) or paclitaxel (10 mg/kg BW/i.p./weekly).

Growth assay

Cells were grown in 12-well plates (2,000 cells/well), fixed with formalin, and stained with crystal violet. To quantitate growth, the dye was solubilized by 10% acetic acid, and absorbance was measured at 560 nmol/L.

Flow cytometry analysis

Cells at 70% to 80% confluence or after double thymidine synchronization were collected, permeabilized, pelleted, and resuspended in an isotonic-buffered propidium iodide (PI) staining solution containing RNase A (0.1 mg/mL) and PI (20 μg/mL). Samples were run on the BD FACSCalibur system (Becton Dickinson), and data analyzed using WinMDI 2.9 software.

Luciferase assay

A total of 1 × 105 cells were seeded in 12-well cell culture plates, and transfected with a total of 1.6 μg of plasmids, including reporter, expression, and pCMV-β-galactosidase plasmids using Lipofectamine 2000 (14). NF-κB-luc (Igκ2-IFN-luc wt or mut) was a kind gift from Dr. Joel L. Pomerantz, Johns Hopkins University School of Medicine, Baltimore, MD (15). The TOPFlash plasmid (16) was purchased from Addgene.

Clonogenic cell survival assay

Exponentially growing cells were exposed to drugs or ultraviolet light (9). Twenty-four hours later, cells were reseeded (1–2 × 103/well) in triplicate in 6-well plates. Viable colonies were fixed, stained with crystal violet, and counted 1 to 2 weeks later.

MTT assay

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 MTT assay was performed (17). Values are expressed as percent survival of the vehicle-treated control.

Caspase-3/7 activity

Caspase-3 and -7 activities were measured with the Caspase- Glo Assay Kit (Promega) according to the manufacturer's instructions. Values were expressed as the percentage of activity of the vehicle-treated control.

Western blot analysis

Cells were lysed with RIPA buffer and processed as described previously (17). The following antibodies were used: anti-CDK7 and anti-XPD (Santa Cruz Biotechnology) and BCL-xL and BIRC3 (Abcam). All other antibodies were from Cell Signaling Technology.

Host-cell reactivation assay

The assay was performed (18) with some modifications. pGL3-basic (Promega) was exposed to UV light [Stratalinker UV-Crosslinker 1800 (Stratagene)] or treated with 100 to 1,000 nmol/L cisplatin. To measure HR-mediated repair, 1 μg of vector digested with HindIII was transfected into cells with pCMV-β-galactosidase. Luciferase and β-galactosidase activities were measured after 48 hours.

Comet assay

The alkaline Comet assay to measure DNA strand breaks in single cells was performed according to the manufacturer's protocol (CometAssay Kit; Trevigen). Comet tail moments of ≥100 cells were measured and quantified using the CometScore software.

Chromosomal aberration analysis at metaphase

Chromosomal aberration analysis was performed as described previously (18, 19). Exponentially growing cells were gamma irradiated (3 G) and analyzed for metaphase aberrations after 12 hours (20). Cisplatin-induced chromosome aberrations were analyzed as described previously (1,9).

Detection of γ-H2AX Foci

Cells growing in chamber slides (Nunc Lab-Tek II) were treated with Dox (200 nmol/L) or gemcitabine (50 nmol/L) for 24 hours. After fixation and permeabilization, cells were probed with an antibody against phosphorylated H2AX-Ser139 (Upstate Biotechnology); H2AX foci were visualized using a Zeiss Axio Scope fluorescent microscope and scored with the ImageJ software (v1.47, NIH, Bethesda, MD). One-hundred cells or more were evaluated.

I-Sce1 assay for homologous recombination repair activity

DR-95 cells were transfected with pI-Sce1, pEGFP, or pCMV (21, 22), harvested after 72 hours, and %GFP-expressing cells was measured by flow cytometry. Frequency of recombination events = mean %GFP-positive cells transfected with pI-Sce1/Mean %GFP-positive cells transfected with pEGFP.

Recruitment of HR protein at double-strand break sites

DR95 cells were electroporated with I-pCBASce. ChIP was done using antibodies to Rad51 (Abcam); BRCA1 (Cell Signaling Technology); KU80 (Cell Signaling Technology; ref. 20). Quantification of ChIP DNA was carried out by real-time PCR in triplicate using the LightCycler Fast Start DNA Master SYBR Green I (Roche Applied Sciences).

DNA fiber assay

DNA fiber spreads were prepared (19) with minor modifications. Cells in exponential phase were labeled for sites of ongoing replication with 5-iododeoxyuridine (IdUrd; 50 μmol/L) followed by exposure to hydroxyurea (4 mmol/L), washed, and labeled with 5-chlorodeoxyuridine (CldUrd; 50 μmol/L). Fibers were analyzed using ImageJ software.

Coimmunoprecipitation to detect HOXC10/CDK7

One milligram of protein lysate was subjected to immunoprecipitation overnight at 4°C with CDK7 antibody (C-19; Santa Cruz Biotechnology) or normal rabbit IgG control (sc-2027; Santa Cruz Biotechnology); immunoblotting was performed using: Myc-Tag (9B11; Cell Signaling Technology), RNA Polymerase II (CTD4H8; Millipore), and XPD [sc-101174, Santa Cruz Biotechnology; ref. 14).

CDK7 kinase activity

Three hundred micrograms of protein extract from drug-treated cells was used for immunoprecipitation with CDK7 antibody. The immunoprecipitate was resuspended in 40 μL of kinase buffer (50 mmol/L HEPES, pH 7.5, 10 mmol/L MgCl2, 250 μmol/L EGTA, 10 mmol/L β- glycerophosphate, 1 mmol/L DTT), 10 μL of 50 nmol/L ATP, and 20 ng GST-CDT peptide (P4016, Proteinone) as substrate, incubated for 1 hour at 30°C, added to an equal volume of kinase-GLO reagent (Promega), and incubated for 15 minutes at room temperature. Control reactions lacked the CTD peptide substrate. Luminescence was recorded and expressed as relative RLU to the untreated control cells.

Statistical analysis

Results were expressed as mean ± SEM of at least three independent experiments. Paired Student t test or ANOVA tests were performed for data analysis. All statistical analyses were performed using GraphPad Prism version 4.03 (GraphPad Software, Inc.). In all figures, *, P < 0.05; **, P < 0.01; and ***, P < 0.001.

Details of constructs, materials, and experimental procedures are provided in Supplementary Materials and Methods online.

HOXC10 overexpression is linked to chemotherapy resistance

By HOX tiling mRNA expression array, compared with normal breast, HOXC10 was among the top five overexpressed genes in breast cancer (23), and higher than all 39 HOX genes in distant metastasis (Fig. 1A). In Oncomine (24), HOXC10 was among the top 1% transcripts in breast compared with other carcinomas (Fig. 1B), and in TCGA (https://tcga-data.nci.nih.gov/), was equally prevalent in all breast carcinomas (Fig. 1C). qRT-PCR analysis of our primary tissue panel validated this finding; median HOXC10 expression was 10-fold higher in primary invasive carcinomas and 30- fold higher in distant metastatic tissues (Fig. 1D). No correlation of HOXC10 expression with relapse-free survival (RFS) or overall survival (OS) was observed in breast cancer patients in the GEO (http://www.ncbi.nlm.nih.gov/geo) and the METABRIC cohorts (Fig. 1E, a and F, a; ref. 25). In chemotherapy treated patients, high HOXC10 expression correlated with short RFS (Fig. 1E, b and Supplementary Fig. S1A and S1B) and short OS (Fig. 1F, b and Supplementary Fig. S1C), and even more consistently in the subset of ER-negative patients treated with chemotherapy (Fig. 1E, c and F, c). Cox multivariate regression analysis, taking all clinical parameters into account, revealed a highly significant (P = 0.00013) inverse correlation between HOXC10 expression and RFS.

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 = 3.18 e−40. C, invasive ductal and lobular breast carcinoma. D, qRT-PCR of HOXC10 expression in normal breast organoids (n = 12), invasive ductal carcinoma (n = 31), and distant metastasis (n = 49). Kaplan-M plots of correlation of HOXC10 expression with RFS (E) and OS (F) in all patients with breast cancer (n = 4117; a), chemotherapy-treated (b), chemotherapy-treated ER-negative (c) breast cancer.

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 = 3.18 e−40. C, invasive ductal and lobular breast carcinoma. D, qRT-PCR of HOXC10 expression in normal breast organoids (n = 12), invasive ductal carcinoma (n = 31), and distant metastasis (n = 49). Kaplan-M plots of correlation of HOXC10 expression with RFS (E) and OS (F) in all patients with breast cancer (n = 4117; a), chemotherapy-treated (b), chemotherapy-treated ER-negative (c) breast cancer.

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HOXC10 is induced during development of chemotherapy resistance

As a first step to investigating the contribution of HOXC10 to drug resistance, we analyzed HOXC10 expression in chemotherapy-resistant MCF7-sublines (9) that displayed resistance (>250-fold) to epirubicin, paclitaxel and docetaxel. HOXC10 was expressed at 2- to 8-fold higher levels in MCF-7- R sublines (Fig. 2A) and in newly developed MDA-MB-231-R sublines that emerged (>30 days) following exposure to drugs (Supplementary Fig. S2A). Depleting HOXC10 levels in MCF-7-Tax-R subline restored its response to paclitaxel (Fig. 2B). Further studies were carried out in eight breast cancer cell lines with varying levels of HOXC10 mRNA (Supplementary Fig. S2B). On exposure of cells in culture to drugs, induction of HOXC10 expression was rapid, starting at day 1 in MDA-MB-231 (Fig. 2C), SUM159 (Fig. 2D), and SUM149 (Supplementary Fig. S2C). Basal levels of HOXC10 expression determined response to Dox. Mouse xenografts of low HOXC10-expressing MDA-MB-231 (Fig. 2E) responded to Dox significantly better than high HOXC10-expressing SUM159 (Fig. 2F) or HCC1954 (Supplementary Fig. S2D). By qRT-PCR, MDA-MB-231 (Fig. 2G) and SUM149 (Supplementary Fig. S2E) tumors that resumed growth during treatment showed significantly higher HOXC10 expression compared with SUM159 (Fig. 2H) and HCC1954 (Supplementary Fig. S2F) supporting the argument that upregulated HOXC10 expression correlated with resistance to chemotherapy.

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 μmol/L). Growth in vivo of xenografts of MDA-MB-231 (n = 12; E) and SUM159 (n = 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.

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 μmol/L). Growth in vivo of xenografts of MDA-MB-231 (n = 12; E) and SUM159 (n = 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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HOXC10 overexpression decreases susceptibility to chemotherapy

To characterize the effects of HOXC10 overexpression, we derived stable clones of MCF10A-Ras [stably expressing K-ras (Gly12-Val)] and MDA-MB-231–overexpressing myc-tagged HOXC10, and SUM159 and SUM149 cells depleted of HOXC10 by 50% to 70% (immunoblot in Fig. 3A) using specific shRNAs. Properties of anchorage-independent growth (Supplementary Fig. S3A and S3B), colony formation (Supplementary Fig. S3C), invasion through Matrigel (Supplementary Fig. S3D), proliferation (Supplementary Fig. SE), quantified in (Supplementary Fig. S3F), and tumor growth in mice (Fig. 3B and Supplementary Fig. S3G) were all significantly decreased by depleting HOXC10 and enhanced by overexpressing HOXC10 in tumor cells. By colony survival assays, Dox, gemcitabine, carboplatin, and Tax were more cytotoxic in SUM159-shC10 (Fig. 3C and Supplementary Fig. S3H), MCF7-shC10 and SUM149-shC10 (Supplementary Fig. S3H) than in MCF10A-Ras-C10 cells (Supplementary Fig. S3I). This body of data, supporting an increased oncogenicity and aggressiveness of HOXC10-expressing breast cancer cells, was further corroborated by MTT assays in SUM159-shC10 (Supplementary Fig. S3J) and in MCF10A-Ras-10 cells (Supplementary Fig. S3K).

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 μmol/L), gemcitabine (1 μmol/L), taxol (0.5 μmol/L), docetaxel (0.5 μmol/L), or carboplatin (100 μmol/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 = 6) and MCF10A-Ras-C10 (n = 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.

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 μmol/L), gemcitabine (1 μmol/L), taxol (0.5 μmol/L), docetaxel (0.5 μmol/L), or carboplatin (100 μmol/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 = 6) and MCF10A-Ras-C10 (n = 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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The activation of caspase-3/7 is a reliable marker of cells undergoing apoptosis (26). Susceptibility to the drugs was reflected by an increase in caspase 3/7 activity in SUM159-shC10 cells (Fig. 3D) and a decrease in MCF10A-Ras-C10 cells (Fig. 3E). Apoptotic cells were identified on the basis of DNA content frequency histograms as cells with fractional "sub-G1" DNA content (27). In response to Dox, there was a decrease in sub-G1 population in SUM159shC10 cells (Fig. 3F) and an increase in MCF10A-Ras-C10 cells (Fig. 3G). Also, several pro- and antiapoptotic mRNAs were modulated in SUM159-shC10 and MCF10A-Ras (Supplementary Fig. S3L and S3M), and further intensified by drug treatment (Fig. 3H and Supplementary Fig. S3N). Western blot analysis of SUM159-shC10 cells showed that antiapoptotic genes, hsp27, Birc 3, PI3K, and BCL2 levels were decreased, whereas proapoptotic genes, p21, and BID levels were increased by depletion of HOXC10 (Fig. 3I). Dox-treated MCF10A-Ras-vec xenografts regressed by week 4, whereas MCF10A-Ras-C10 tumors grew after week 2 (Fig. 3J). Consistent with this observation, MCF10A-Ras-C10 tumors showed high mRNA expression of many antiapoptotic genes (Fig. 3K).

Given that the antiapoptotic genes examined are known direct targets of NF-κB (28), we measured NF-κB activity using the NF-κB–responsive reporter Igκ2-IFN-LUC, with and without Dox treatment. The basal activity of NF-κB was lower in SUM159-shC10 (Supplementary Fig. S3O) and high in MCF10A-Ras-C10 (Supplementary Fig. S3P) compared with their respective controls, and further enhanced by Dox treatment. These data are consistent with findings that the basal activity of NF-κB is much higher, and maintained as such, in Dox-resistant MCF7 cells (29). Thus, HOXC10 upregulates NF-κB to achieve both activation of antiapoptotic pathways, and increases cell survival following stress.

HOXC10 enhances DNA damage repair and checkpoint recovery

Several homeodomain-related proteins have been functionally related to DNA damage repair (1, 2, 30, 31). Overexpression of HOXA5 in S. cerevisiae upregulates components of the mismatch repair (MMR) system, important for the detection and repair of DNA damage (32). Hence, we investigated whether HOXC10 participates directly in DNA repair and if so, the type of damage and cell-cycle stage of repair.

A host–cell reactivation assay was used to assess cellular ability to repair exogenously damaged DNA. A pGL3 (luciferase-reporter) plasmid was exposed to UV or cisplatin, or digested with Hind III (to generate double-strand breaks, DSB) followed by transfection into SUM159-shC10 cells or MCF-10A-Ras-C10 cells and their respective controls. SUM159-shC10 cells displayed a significantly decreased ability to repair DNA damage (Fig. 4A), whereas the reverse was shown for UV-induced DNA damage in MCF-10A-Ras-C10 cells (Fig. 4B). Clonogenic cell survival assays in both MCF10A-Ras-C10 and SUM159-shC10 cells exposed to UV (Fig. 4C, quantified in S4A) supported these findings. However, repair of DNA DSBs caused by Hind III digestion (Fig. 4A) remained unchanged in SUM159-shC10 cells, suggesting no involvement of the NHEJ pathway. Furthermore, ionizing radiation (IR) induced G1-chromosome damage repair was not affected in SUM159-shC10 cells (Supplementary Fig. S4B), wherein DSB repair by NHEJ is predominant. On the other hand, SUM159-shC10 cells had a higher frequency of S-phase–specific IR-induced chromosomal aberrations (Supplementary Fig. S4C), suggesting that HOXC10 expression enhances repair, possibly by the homologous recombination (HR) pathway. MCF10A-Ras-C10 cells treated with cisplatin showed significantly reduced number of cells with chromosomal aberrations at metaphase (Supplementary Fig. S4D) compared with the vector control cells. Collectively, the data strongly support the hypothesis that HOXC10 is an important participant in DNA DSB repair by HR.

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 γ-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 (+) 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, χ2 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.

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 γ-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 (+) 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, χ2 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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We had noted that HOXC10 expression had a dramatic effect on survival following exposure to UV light or the platinum drugs (Fig. 4A–C and Supplementary S4A). DNA damage induced by such agents is repaired by nucleotide excision repair (NER) and HR. Therefore, we measured the activity of the NER pathway by the alkaline comet assay (Fig. 4D), a single-cell gel electrophoresis method in which the intensity of the comet tail of the migrating cells relative to the head reflects the number of DNA breaks (33). Cells depleted of HOXC10 had significantly more residual UV-, Dox- and gemcitabine-induced DNA damage 24 hours after treatment (Fig. 4E). Furthermore, time-course analysis after UV treatment showed that DNA damage in SUM159-scr cells had returned to baseline levels in 24 hours, but was still 4-fold higher in SUM159-shC10 cells (Fig. 4E), revealing inefficient DNA repair in the absence of HOXC10. Improved response to DNA damage was evident in MCF10A-Ras-C10 cells compared with the vector-control cells (Fig. 4B, C, and F). The reduced survival of SUM159-shC10 cells in colony formation assays (Fig. 4C) also correlated with higher levels of residual DSBs.

DNA damage that results in DSBs is always followed by the phosphorylation of the histone, H2AX (34). Newly formed phosphorylated protein γ-H2AX is the initial step of damage detection to recruit repair proteins (34). We quantified the number of residual γ-H2AX foci 24 hours after treatment of SUM159-shC10 cells with Dox or gemcitabine. On average, 20% to 30% more γ-H2AX foci accumulated in SUM159-shC10 cells after drug treatment (Fig. 4G) with almost 50% to 60% more cells displaying ≥100 foci/nucleus (Supplementary Fig. S4E). A time-course analysis of γ-H2AX protein after UV or Dox treatment showed no differences in the initial induction of γ-H2AX (10 minutes to 8 hours; Supplementary Fig. S4F and S4G). Residual γ-H2AX measured 24 hours after treatment, and not the initial kinetics of γ-H2AX formation, is a better predictor of cell viability (35, 36). These results indicated that HOXC10 might be more involved in DNA damage processing rather than in damage sensing. No major difference was observed in the phosphorylation status of known DNA damage sensors of the ATR/ATM pathways after Dox or gemictabine treatment of SUM159-shC10 (Supplementary Fig. S4H), thus confirming that HOXC10 is not involved in DNA damage sensing.

These findings were further substantiated by the HR-DSB repair assay of lesions induced by I-Sce1 endonuclease performed in DR-95 cells engineered to stably express a pDR-GFP plasmid containing a mutated GFP gene with an 18 bp I-Sce1 endonuclease cleavage site and an in-frame termination codon (10, 20). Efficient repair of the Sce-1 cleavage site restores expression of GFP. DR-95 cells, with and without HOXC10 silencing (using siRNA) were transfected with the I-Sce1 expression plasmid. DR95-siHOXC10 showed reduced repair of I-Sce-1–induced lesions. Consequently, restoration of GFP expression occurred at a significantly lower efficiency than in control cells (Fig. 4H). Reduction of GFP expression was comparable with cells with reduced expression of BRCA-1, a known DNA repair protein, used as a positive control (Fig. 4H). Thus, repair of DSB by HR is impaired in cells lacking HOXC10 expression.

To investigate a role of HOXC10 in the recruitment of HR repair proteins at the site of a single DNA DSB, we performed chromatin immunoprecipitation (ChIP) in DR-95 cells (10, 20). We compared the levels of BRCA1, RAD51, KU80, and HOXC10 at different distances from an I-Sce1–induced DSB site using ChIP analysis with specific primers (10) in cells with and without expression of HOXC10. Depletion of HOXC10 using siRNAs did not decrease KU80 levels in close proximity to the DSB, whereas levels of RAD51 and BRCA1 at the break site were significantly reduced (Fig. 4I). This finding in HOXC10-depleted cells strongly supported the role of HOXC10 in facilitating the recruitment of proteins involved in HR. However, HOXC10 itself was undetectable at the I-Sce1 cleavage site, ruling out a direct role for HOXC10 in DSB repair.

We therefore considered the possibility that HOXC10 has a role in the initiation of new origins of DNA replication. Single-molecule examination of replication dynamics by DNA fiber analysis (19) after hydroxyurea (HU) treatment in SUM159-shC10 and MCF10A-Ras-C10 cells showed that HOXC10 is required for the initiation of new origins, but not for the accumulation of stalled forks (Fig. 4J and K).

Collectively, the data suggest that HOXC10, as a transcriptional factor, affects the chromatin status and sets the stage for HR by, (i) decreasing S-phase chromosome aberrations, (ii) enhancing resection as shown by γ-H2AX foci, (iii) increasing HR based on l-Scel endonuclease–induced DSB repair, and (iv) resolving stalled replication forks as shown by DNA fiber analysis, possibly leading to initiation of DNA replication following DNA damage.

HOXC10 binds to CDK7 in vivo after chemotherapy treatment

We had thus far observed that HOXC10 enhances cell survival by affecting apoptosis, NF-kB activity, and damage repair, mainly by the HR pathways. One key protein that links all three pathways is cyclin-dependent kinase 7 (CDK7). In metazoans, CDK7 has essential roles in transcription as a component of the general transcription factor, TFIIH. CDK7 is an effector kinase, which also phosphorylates RNA Pol II and other proteins during transcription after the pausing of the TFIIH complex following DNA damage (37). Highly relevant to this study, HOXC10 was found to be the tightest binding protein linking the CAK (Cdk-activating kinase, CDK7) complex to TFIIH in a yeast 4-hybrid system (38). We therefore tested for an association of HOXC10 with CDK7 by coimmunoprecipitation. A weak interaction between the two proteins increased significantly upon treatment with Dox and gemcitabine (Fig. 5A). This interaction was reduced significantly upon addition of a pharmacologic inhibitor of CDK7 activity, SNS-032 (Fig. 5A), or by expressing the dominant negative, kinase-dead mutant CDK7 (D155A) in the cells (Fig. 5B). We concluded that CDK7 and HOXC10 formed a complex in breast cancer cells, which became more abundant upon exposure to drugs.

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 μmol/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.

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 μmol/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.

Close modal

Different sets of proteins are phosphorylated by CDK7 depending on its function in cell division (i.e., CDK1, 2, 4, 6) or in transcription as a complex with TFIIH (37). We therefore investigated whether binding of HOXC10 to CDK7 altered its substrate preference. Also, elongating RNA Pol II can be arrested by endogenous and exogenous DNA lesions such as UV-induced pyrimidine dimers, adducts induced by anticancer drugs, and DSBs. These transcription-blocking lesions located on the transcribed strand are primarily repaired by transcription-coupled repair, the NER pathway (39). XPD helicase is a key member of the human TFIIH complex that anchors CAK kinase (cyclin H, MAT1, and CDK7) to TFIIH and unwinds DNA for transcription and for repair of duplex warping damage by nucleotide excision repair (NER), thereby maintaining genomic integrity (40). We therefore examined whether HOXC10 affects the composition of the TFIIH–XPD–CAK complex through its protein/protein interaction with CDK7. The binding of XPD to CDK7 was reduced in SUM159-shC10 cells (Fig. 5C) and was increased in MCF10A-Ras- C10 cells, compared with their respective control cells (Fig. 5D). This finding suggested that HOXC10 may have a role in anchoring CAK to XPD, thus maintaining the integrity of the holoenzyme TFIIH during response to DNA damage. This finding is also consistent with reports that in repair-deficient cells, the association of CAK kinase, but not of XPD, to damaged DNA was reduced (41).

Next, we investigated whether the association of CDK7 with the RNA Pol II is modulated by HOXC10. We found that the association between CDK7 and RNA Pol II in drug-treated cells was significantly stronger in the presence of HOXC10 in MCF10A-Ras-C10 (Fig. 5E) and weaker in the absence of HOXC10 in SUM150-shC10 (Fig. 5F). Furthermore, the kinase activity of CDK7 on the C-terminal domain (CTD) of RNA Pol II was significantly reduced in SUM159-shC10 cells after DNA damage (Fig. 5G). MCL1 is an antiapoptotic protein that is rapidly depleted upon inhibition of RNA Pol II, permitting measures of its mRNA to serve as a surrogate for CDK7 activity (42). The lack of CDK7 activity is likely to impede recovery of the transcriptional machinery, thereby promoting cell death after DNA damage as was reflected by MCL1 expression (Fig. 5H). Conversely, the enhanced interaction between Pol II and CDK7 in MCF10A-Ras-C10 cells (Fig. 5E) promotes cell survival following DNA damage (Fig. 5I). Consistent with this data, newly emerging drug-resistant MCF7 sublines in cell culture showed increases in both MCL1 and HOXC10 mRNA expression (Supplementary Fig. S5).

Inhibiting CDK7 reverses HOXC10-dependent chemoresistance

Inhibiting CDK7 is of special interest in cancer therapeutics as it affects multiple signaling pathways (43). Two known selective CDK7 inhibitors are BS-181 (44) and SNS-032 (11); the latter is in phase I studies (http://clinicaltrials.gov/). Combining SNS-032 or BS-181 at minimally cytotoxic doses (∼20%) with chemotherapy significantly improved the response of both MCF10-Kras-C10 cells (Fig. 6A and B) and SUM159-shC10 cells (Supplementary Fig. S6A and S6B). In taxol and epirubicin-resistant MCF7 sublines, Western blot analysis showed that CDK7 (pThr170) and CTD (pSer5) were activated (Fig. 6C) with no change in cell-cycle kinases, CDK1 and CDK2. qRT-PCR analysis showed an increase in both HOXC10 and MCL1 mRNAs (Fig. 6D). Treatment with taxol combined with BS-181 restored drug susceptibility of Tax-R-MCF-7 cells, as seen in colony formation assays (Fig. 6E), and caused cell kill in parental MCF-7 cells as shown by MTT assays (Fig. 6F). Similarly, BS-181 restored sensitivity of two MDA-MB-231-Tax-R sublines to taxol (Supplementary Fig. S6C and S6D), an effect not achieved using inhibitors to CDK1/2/9, AZD5438 (45) and CDK4/6, PD0332991 (Supplementary Fig. S6E and S6F; ref. 46). These data suggest a specific role for CDK7 in resistance to chemotherapeutic drugs.

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) or BS-181 (5 μmol/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 μmol/L) for 7 days. F, MTT assay of MCF7-Tax-R treated with taxol (0–250 nmol/L) ± BS-181 (10 μmol/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.

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) or BS-181 (5 μmol/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 μmol/L) for 7 days. F, MTT assay of MCF7-Tax-R treated with taxol (0–250 nmol/L) ± BS-181 (10 μmol/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.

Close modal

In summary, our data support a model of HOXC10 action wherein it is intimately involved in multiple steps in the process of HR-mediated DNA repair following chemotherapy exposure of breast cancer cells and in the development of drug resistance in the long term. HOXC10 is an integral component of the CAK complex, which allows restart of transcription following DNA damage (Fig. 7).

Figure 7.

A model for HOXC10 action in breast cancer cells. Upon exposure to chemotherapy or ionizing radiation (IR), DNA damage mediator genes such as γH2AX/MDC1/53BP1/BRCA1 are activated, triggering DNA DSB repair. HOXC10 is upregulated following chemotherapy or ionizing radiation in ER-negative breast cancer. HOXC10, as part of the CAK complex, participates in the late stages of DNA repair that involves restart of transcription for recovery and acts as an inhibitor of apoptosis.

Figure 7.

A model for HOXC10 action in breast cancer cells. Upon exposure to chemotherapy or ionizing radiation (IR), DNA damage mediator genes such as γH2AX/MDC1/53BP1/BRCA1 are activated, triggering DNA DSB repair. HOXC10 is upregulated following chemotherapy or ionizing radiation in ER-negative breast cancer. HOXC10, as part of the CAK complex, participates in the late stages of DNA repair that involves restart of transcription for recovery and acts as an inhibitor of apoptosis.

Close modal

Chemotherapy failure in breast cancer claims at least 80,000 lives each year worldwide. Here, we present evidence that HOXC10 overexpression is common and functionally important for onset of resistance to chemotherapy in ER-negative breast cancer and is also prognostic of poor outcome primarily in patients treated with chemotherapy.

Upregulation of HOXC10 is likely to be an important cancer adaptation mechanism. Ectopic expression of HOXC10 leads to decreased drug susceptibility, while decreasing its levels enhances the cytotoxic effects of chemotherapy in both in vitro and in vivo models of breast cancer. Mechanistically, HOXC10 is involved in activating different survival pathways, including driving checkpoint recovery after DNA damage, a key pathway that allows cancer cells to overcome damage response arrest (Fig. 4 and Supplementary Fig. S4; ref. 47). HOXC10 does not affect the sensing of DDR but is required at later stages of the DNA damage repair by NER and HR (Fig. 4 and Supplementary Fig. S4). Cells expressing high levels of HOXC10 repaired DNA damage more efficiently and resumed transcription and growth; whereas their low-HOXC10–expressing counterparts eventually committed to apoptosis. Part of this response, we propose, may be attributed to the observed association of HOXC10 with the CAK complex.

The CAK complex along with TFIIH can participate in diverse functions, including transcription, DNA repair (NER), and cell-cycle regulation. We confirmed 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 complexes, enhancing the recovery process after DNA damage. Indeed, treatment of drug-resistant MCF-7 cells with CDK7 inhibitors restored their susceptibility to chemotherapy (Fig. 6). The importance of current findings to breast cancer therapy stems from the recent attention given to CDK inhibition, including CDK7, in clinical trials either as single agents or in combination with chemo- or targeted therapies to overcome resistance (43, 48).

The second survival pathway that is activated by HOXC10 is shifting the balance between growth and apoptosis to allow continuous proliferation and survival under adverse conditions. Our data showed that HOXC10 facilitates the transition from G1 to S phase and progression through the S-phase during the cell cycle (Fig. 3). As a consequence, cells with high HOXC10 levels have a growth advantage, especially under nonideal conditions, and restart their replication to overcome stressful conditions. At the same time, the increase in NF-κβ activity and the consequent increase in the levels of many antiapoptotic proteins likely decrease cell sensitivity to many stressors.

Because of HOXC10 involvement in survival and proliferation of cancer cells despite exposure to chemotherapy, HOXC10 is an attractive target to reverse chemotherapeutic resistance in breast 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.

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/advisory board member for AVON Foundation. No potential conflicts of interest were disclosed by the other authors.

Conception and design: H. Sadik, R. Kumar, S. Park, N. Shah, H.Y. Chang, T.K. Pandita, S. Sukumar

Development of methodology: H. Sadik, R. Kumar, M. Hedayati, S. Park, T.G. Munoz, N. Shah, R.K. Pandita, J.C. Chang, H.Y. Chang, T.K. Pandita, S. Sukumar

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): H. Sadik, P. Korangath, N.K. Nguyen, M. Hedayati, W.W. Teo, N. Shah, J.C. Chang, T. DeWeese, H.Y. Chang, S. Sukumar

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): H. Sadik, B. Gyorffy, R. Kumar, M. Hedayati, H. Panday, T.G. Munoz, N. Shah, J.C. Chang, S. Sukumar

Writing, review, and/or revision of the manuscript: H. Sadik, B. Gyorffy, M. Hedayati, N. Shah, T.K. Pandita, S. Sukumar

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): H. Panday, O. Menyhart, R.K. Pandita, T.K. Pandita, S. Sukumar

Study supervision: T.K. Pandita, S. Sukumar

We thank Dr. Joel L. Pomerantz for providing us their responsive reporter plasmids NF-κB-luc, Dr. Amadeo M Parissenti for the drug-resistant MCF7 cell lines, and Dr. Alan Rein for reviewing the article.

This work was supported by funding from the DOD-BCRP BC093970 (H. Sadik), NCI-P50-CA88843 and P30-CA006973 (S. Sukumar) and NIH 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 accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1.
Rezsohazy
R
. 
Non-transcriptional interactions of Hox proteins: inventory, facts, and future directions
.
Dev Dyn
2014
;
243
:
117
31
.
2.
Rubin
E
,
Wu
X
,
Zhu
T
,
Cheung
JC
,
Chen
H
,
Lorincz
A
, et al
A role for the HOXB7 homeodomain protein in DNA repair
.
Cancer Res
2007
;
67
:
1527
35
.
3.
Wu
X
,
Ellmann
S
,
Rubin
E
,
Gil
M
,
Jin
K
,
Han
L
, et al
ADP ribosylation by PARP-1 suppresses HOXB7 transcriptional activity
.
PLoS ONE
2012
;
7
:
e40644
.
4.
Miotto
B
,
Graba
Y
. 
Control of DNA replication: a new facet of Hox proteins?
Bioessays
2010
;
32
:
800
7
.
5.
Marchetti
L
,
Comelli
L
,
D'Innocenzo
B
,
Puzzi
L
,
Luin
S
,
Arosio
D
, et al
Homeotic proteins participate in the function of human-DNA replication origins
.
Nucleic Acids Res
2010
;
38
:
8105
19
.
6.
de Stanchina
E
,
Gabellini
D
,
Norio
P
,
Giacca
M
,
Peverali
FA
,
Riva
S
, et al
Selection of homeotic proteins for binding to a human DNA replication origin
.
J Mol Biol
2000
;
299
:
667
80
.
7.
Christen
B
,
Beck
CW
,
Lombardo
A
,
Slack
JM
. 
Regeneration-specific expression pattern of three posterior Hox genes
.
Dev Dyn
2003
;
226
:
349
55
.
8.
Pathiraja
TN
,
Nayak
SR
,
Xi
Y
,
Jiang
S
,
Garee
JP
,
Edwards
DP
, et al
Epigenetic reprogramming of HOXC10 in endocrine-resistant breast cancer
.
Sci Transl Med
2014
;
6
:
229ra41
.
9.
Hembruff
SL
,
Laberge
ML
,
Villeneuve
DJ
,
Guo
B
,
Veitch
Z
,
Cecchetto
M
, et al
Role of drug transporters and drug accumulation in the temporal acquisition of drug resistance
.
BMC Cancer
2008
;
8
:
318
.
10.
Rodrigue
A
,
Lafrance
M
,
Gauthier
MC
,
McDonald
D
,
Hendzel
M
,
West
SC
, et al
Interplay between human DNA repair proteins at a unique double-strand break in vivo
.
EMBO J
2006
;
25
:
222
31
.
11.
Chen
R
,
Wierda
WG
,
Chubb
S
,
Hawtin
RE
,
Fox
JA
,
Keating
MJ
, et al
Mechanism of action of SNS-032, a novel cyclin-dependent kinase inhibitor, in chronic lymphocytic leukemia
.
Blood
2009
;
113
:
4637
45
.
12.
Gyorffy
B
,
Lanczky
A
,
Eklund
AC
,
Denkert
C
,
Budczies
J
,
Li
Q
, et al
An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients
.
Breast Cancer Res Treat
2010
;
123
:
725
31
.
13.
Lo
PK
,
Lee
JS
,
Liang
X
,
Han
L
,
Mori
T
,
Fackler
MJ
, et al
Epigenetic inactivation of the potential tumor suppressor gene FOXF1 in breast cancer
.
Cancer Res
2010
;
70
:
6047
58
.
14.
Shah
N
,
Jin
K
,
Cruz
LA
,
Park
S
,
Sadik
H
,
Cho
S
, et al
HOXB13 mediates tamoxifen resistance and invasiveness in human breast cancer by suppressing ERalpha and inducing IL-6 expression
.
Cancer Res
2013
;
73
:
5449
58
.
15.
Pomerantz
JL
,
Denny
EM
,
Baltimore
D
. 
CARD11 mediates factor-specific activation of NF-kappaB by the T-cell receptor complex
.
EMBO J
2002
;
21
:
5184
94
.
16.
Veeman
MT
,
Slusarski
DC
,
Kaykas
A
,
Louie
SH
,
Moon
RT
. 
Zebrafish prickle, a modulator of noncanonical Wnt/Fz signaling, regulates gastrulation movements
.
Curr Biol
2003
;
13
:
680
5
.
17.
Alanee
S
,
Shah
S
,
Vijai
J
,
Schrader
K
,
Hamilton
R
,
Rau-Murthy
R
, et al
Prevalence of HOXB13 mutation in a population of Ashkenazi Jewish men treated for prostate cancer
.
Fam Cancer
2013
;
12
:
597
600
.
18.
Pandita
RK
,
Sharma
GG
,
Laszlo
A
,
Hopkins
KM
,
Davey
S
,
Chakhparonian
M
, et al
Mammalian Rad9 plays a role in telomere stability, S- and G2-phase-specific cell survival, and homologous recombinational repair
.
Mol Cell Biol
2006
;
26
:
1850
64
.
19.
Singh
M
,
Hunt
CR
,
Pandita
RK
,
Kumar
R
,
Yang
CR
,
Horikoshi
N
, et al
Lamin A/C depletion enhances DNA damage-induced stalled replication fork arrest
.
Mol Cell Biol
2013
;
33
:
1210
22
.
20.
Gupta
A
,
Hunt
CR
,
Hegde
ML
,
Chakraborty
S
,
Udayakumar
D
,
Horikoshi
N
, et al
MOF phosphorylation by ATM regulates 53BP1-mediated double-strand break repair pathway choice
.
Cell Rep
2014
;
8
:
177
89
.
21.
Dungey
FA
,
Caldecott
KW
,
Chalmers
AJ
. 
Enhanced radiosensitization of human glioma cells by combining inhibition of poly(ADP-ribose) polymerase with inhibition of heat shock protein 90
.
Mol Cancer Ther
2009
;
8
:
2243
54
.
22.
Pierce
AJ
,
Johnson
RD
,
Thompson
LH
,
Jasin
M
. 
XRCC3 promotes homology-directed repair of DNA damage in mammalian cells
.
Genes Dev
1999
;
13
:
2633
8
.
23.
Gupta
RA
,
Shah
N
,
Wang
KC
,
Kim
J
,
Horlings
HM
,
Wong
DJ
, et al
Long non-coding RNA HOTAIR reprograms chromatin state to promote cancer metastasis
.
Nature
2010
;
464
:
1071
6
.
24.
Rhodes
DR
,
Kalyana-Sundaram
S
,
Mahavisno
V
,
Varambally
R
,
Yu
J
,
Briggs
BB
, et al
Oncomine 3.0: genes, pathways, and networks in a collection of 18,000 cancer gene expression profiles
.
Neoplasia
2007
;
9
:
166
80
.
25.
Curtis
C
,
Shah
SP
,
Chin
SF
,
Turashvili
G
,
Rueda
OM
,
Dunning
MJ
, et al
The genomic and transcriptomic architecture of 2,000 breast tumours reveals novel subgroups
.
Nature
2012
;
486
:
346
52
.
26.
Degterev
A
,
Yuan
J
. 
Expansion and evolution of cell death programmes
.
Nat Rev Mol Cell Biol
2008
;
9
:
378
90
.
27.
Kajstura
M
,
Halicka
HD
,
Pryjma
J
,
Darzynkiewicz
Z
. 
Discontinuous fragmentation of nuclear DNA during apoptosis revealed by discrete "sub-G1" peaks on DNA content histograms
.
Cytometry A
2007
;
71
:
125
31
.
28.
Oeckinghaus
A
,
Ghosh
S
. 
The NF-kappaB family of transcription factors and its regulation
.
Cold Spring Harb Perspect Biol
2009
;
1
:
a000034
.
29.
Gangadharan
C
,
Thoh
M
,
Manna
SK
. 
Inhibition of constitutive activity of nuclear transcription factor kappaB sensitizes doxorubicin-resistant cells to apoptosis
.
J Cell Biochem
2009
;
107
:
203
13
.
30.
Ramdzan
ZM
,
Pal
R
,
Kaur
S
,
Leduy
L
,
Berube
G
,
Davoudi
S
, et al
The function of CUX1 in oxidative DNA damage repair is needed to prevent premature senescence of mouse embryo fibroblasts
.
Oncotarget
2015
;
6
:
3613
26
.
31.
Bowen
C
,
Gelmann
EP
. 
NKX3.1 activates cellular response to DNA damage
.
Cancer research
2010
;
70
:
3089
97
.
32.
Duriseti
S
,
Winnard
PT
 Jr
,
Mironchik
Y
,
Vesuna
F
,
Raman
A
,
Raman
V
. 
HOXA5 regulates hMLH1 expression in breast cancer cells
.
Neoplasia
2006
;
8
:
250
8
.
33.
Nandhakumar
S
,
Parasuraman
S
,
Shanmugam
MM
,
Rao
KR
,
Chand
P
,
Bhat
BV
. 
Evaluation of DNA damage using single-cell gel electrophoresis (Comet Assay)
.
J Pharmacol Pharmacother
2011
;
2
:
107
11
.
34.
Mah
LJ
,
El-Osta
A
,
Karagiannis
TC
. 
gammaH2AX: a sensitive molecular marker of DNA damage and repair
.
Leukemia
2010
;
24
:
679
86
.
35.
Olive
PL
,
Banath
JP
. 
Kinetics of H2AX phosphorylation after exposure to cisplatin
.
Cytometry B Clin Cytom
2009
;
76
:
79
90
.
36.
Banath
JP
,
Klokov
D
,
MacPhail
SH
,
Banuelos
CA
,
Olive
PL
. 
Residual gammaH2AX foci as an indication of lethal DNA lesions
.
BMC Cancer
2010
;
10
:
4
.
37.
Fisher
RP
. 
The CDK Network: linking cycles of cell division and gene expression
.
Genes Cancer
2012
;
3
:
731
8
.
38.
Sandrock
B
,
Egly
JM
. 
A yeast four-hybrid system identifies Cdk-activating kinase as a regulator of the XPD helicase, a subunit of transcription factor IIH
.
The J Biol Chem
2001
;
276
:
35328
33
.
39.
Tornaletti
S
. 
Transcription arrest at DNA damage sites
.
Mutat Res
2005
;
577
:
131
45
.
40.
Fuss
JO
,
Tainer
JA
. 
XPB and XPD helicases in TFIIH orchestrate DNA duplex opening and damage verification to coordinate repair with transcription and cell cycle via CAK kinase
.
DNA Repair
2011
;
10
:
697
713
.
41.
Arab
HH
,
Wani
G
,
Ray
A
,
Shah
ZI
,
Zhu
Q
,
Wani
AA
. 
Dissociation of CAK from core TFIIH reveals a functional link between XP-G/CS and the TFIIH disassembly state
.
PLoS ONE
2010
;
5
:
e11007
.
42.
Conroy
A
,
Stockett
DE
,
Walker
D
,
Arkin
MR
,
Hoch
U
,
Fox
JA
, et al
SNS-032 is a potent and selective CDK 2, 7 and 9 inhibitor that drives target modulation in patient samples
.
Cancer Chemother Pharmacol
2009
;
64
:
723
32
.
43.
Wesierska-Gadek
J
,
Kramer
MP
. 
The impact of multi-targeted cyclin-dependent kinase inhibition in breast cancer cells: clinical implications
.
Expert Opin Investig Drugs
2011
;
20
:
1611
28
.
44.
Ali
S
,
Heathcote
DA
,
Kroll
SH
,
Jogalekar
AS
,
Scheiper
B
,
Patel
H
, et al
The development of a selective cyclin-dependent kinase inhibitor that shows antitumor activity
.
Cancer Res
2009
;
69
:
6208
15
.
45.
Byth
KF
,
Thomas
A
,
Hughes
G
,
Forder
C
,
McGregor
A
,
Geh
C
, et al
AZD5438, a potent oral inhibitor of cyclin-dependent kinases 1, 2, and 9, leads to pharmacodynamic changes and potent antitumor effects in human tumor xenografts
.
Mol Cancer Ther
2009
;
8
:
1856
66
.
46.
Fry
DW
,
Harvey
PJ
,
Keller
PR
,
Elliott
WL
,
Meade
M
,
Trachet
E
, et al
Specific inhibition of cyclin-dependent kinase 4/6 by PD 0332991 and associated antitumor activity in human tumor xenografts
.
Mol Cancer Ther
2004
;
3
:
1427
38
.
47.
Peng
A
. 
Working hard for recovery: mitotic kinases in the DNA damage checkpoint
.
Cell Biosci
2013
;
3
:
20
.
48.
Gallorini
M
,
Cataldi
A
,
di Giacomo
V
. 
Cyclin-dependent kinase modulators and cancer therapy
.
BioDrugs
2012
;
26
:
377
91
.