RAD51-associated protein 1 (RAD51AP1) plays an integral role in homologous recombination by activating RAD51 recombinase. Homologous recombination is essential for preserving genome integrity and RAD51AP1 is critical for D-loop formation, a key step in homologous recombination. Although RAD51AP1 is involved in maintaining genomic stability, recent studies have shown that RAD51AP1 expression is significantly upregulated in human cancers. However, the functional role of RAD51AP1 in tumor growth and the underlying molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been fully understood. Here, we use Rad51ap1-knockout mice in genetically engineered mouse models of breast cancer to unravel the role of RAD51AP1 in tumor growth and metastasis. RAD51AP1 gene transcript was increased in both luminal estrogen receptor–positive breast cancer and basal triple-negative breast cancer, which is associated with poor prognosis. Conversely, knockdown of RAD51AP1 (RADP51AP1 KD) in breast cancer cell lines reduced tumor growth. Rad51ap1-deficient mice were protected from oncogene-driven spontaneous mouse mammary tumor growth and associated lung metastasis. In vivo, limiting dilution studies provided evidence that Rad51ap1 plays a critical role in breast cancer stem cell (BCSC) self-renewal. RAD51AP1 KD improved chemotherapy and radiotherapy response by inhibiting BCSC self-renewal and associated pluripotency. Overall, our study provides genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis by increasing BCSC self-renewal and may serve as a novel target for chemotherapy- and radiotherapy-resistant breast cancer.

Significance:

This study provides in vivo evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating breast cancer stem cell self-renewal.

Genome stability is dependent on the accurate repair of DNA double-strand breaks (DSB). DSBs can arise either from exogenous sources like DNA-damaging agents and ionizing radiation, or endogenous sources like reactive oxygen species, which are generated either during cellular metabolism or DNA replication (1). One major mechanism by which DSBs are repaired is homologous recombination, which is essential for the preservation of genome integrity and maintenance of accurate genome duplication (2). Homologous recombination utilizes the sister chromatid as a template and is considered a comparatively faithful DNA repair pathway. Defects in homologous recombination lead to destabilization of the genome and represent a progenitor of the tumor phenotype in mammals. In eukaryotic cells, homologous recombination reaction is mediated by RAD51 or DMC1 recombinases that are orthologous to bacterial RecA (3, 4). In response to DNA damage, RAD51 and other DNA repair proteins form nuclear foci, which are thought to represent the cellular structures responsible for DNA repair (5). Upon DNA damage induction by ionizing radiation or by treatment with interstrand DNA cross-linking agents, an increase in the number of RAD51 foci is observed (5). This provides evidence that RAD51 plays a critical role in the regulation of genomic integrity.

In recent years, a variety of recombinase partner proteins that enhance homologous recombination reaction by activating RAD51 or DMC1 have been identified (6). RAD51-associated protein 1 (RAD51AP1) is one of such protein that has been shown to participate in the homologous recombination pathway (7, 8). Interaction of RAD51AP1, either with RAD51 or DMC1 recombinases, greatly enhances their recombinase activity and stimulates the D-loop formation (9), a key step for homologous recombination-mediated DNA repair in mitotic and meiotic cells. D-loop formation plays a critical role in mitochondrial DNA (mtDNA) function (10). In the past few years, somatic mtDNA mutations have been identified in several types of human cancers (11–13).

RAD51AP1 knockdown (KD) in human cells leads to increased levels of genomic instability and to decreased levels of homology-directed DNA repair (7, 8). Human cells with silenced RAD51AP1 are viable, but are sensitized to the cytotoxic effects of interstrand cross-linking agents and ionizing radiation. However, human cells with depleted RAD51AP1 are able to form RAD51-associated DNA damage foci with normal kinetics (7, 8), suggesting that RAD51AP1 is not essential for RAD51-associated foci formation. RAD51AP1 is also involved in growth-promoting signaling (14, 15), in which overexpression of RAD51AP1 has been observed in cholangiocarcinoma (16), hepatocellular carcinomas (17), and acute myeloid leukemia (18). However, the functional role of RAD51AP1 in tumor growth and the molecular mechanism(s) by which RAD51AP1 regulates tumorigenesis have not been clarified. In this study, we provide direct evidence that although RAD51AP1 plays an important role in maintaining genomic integrity in normal cells, the elevated levels of RAD51AP1 may be critical for early steps of neoplasia, where increased cell proliferation and replication stresses occur at higher than normal levels. Using Rad51ap1 knockout mice, we provide genetic and biochemical evidences that RAD51AP1 is critical for tumor growth and metastasis using genetically engineered mouse (GEM) models of breast cancer. We also provide evidence that the high expression of RAD51AP1 in human cancers is correlated with poor prognosis and, by increasing breast cancer stem cell (BCSC) self-renewal, is associated with chemotherapy and radiotherapy resistance.

Institutional compliance

The animal experiments reported in this study were approved by the Augusta University (Augusta, GA; AU) Institutional Animal Care Use Committee (IACUC) and Biosafety Committees. Similarly, human breast cancer tissue and the surrounding normal tissues were obtained from the AU tumor bank as per the approval of the institutional review board and human assurance committee.

Cell lines

The primary human normal mammary epithelial cell line, HMEC, was obtained from the Lonza. The human nontransformed normal mammary epithelial cell lines, MCF10A and MCF12A, were obtained from the ATCC in June 2010. HBL100 was kindly provided by Dr. Saraswati Sukumar (Johns Hopkins University, Baltimore, MD) in April 2003. Estrogen receptor–positive breast cancer (ER+BC) cell lines (MCF7, T47D, ZR75.1, BT474, BT483, MDA-MB-361, and MDA-MB-415), estrogen receptor–negative breast cancer, triple-negative breast cancer (TNBC) cell lines (MDA-MB-231, MDA-MB-468, and HCC1937), mouse nontransformed mammary epithelial cell line (HC11), and mouse mammary tumor cell line (4T1) were obtained from ATCC between September 2008 and July 2011. The human breast cancer cell line, CAL51, was obtained from the DSMZ (Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures) in November 2011. The mouse mammary tumor cell line, AT-3, was kindly provided by Dr. Estaban Celis (Georgia Cancer Center at the Augusta University, Augusta, GA) in April 2014. AT-3 cell line was derived from autologous mammary tumor cells originating in a MTAG (MMTV-PyMT/B6) transgenic mouse (19). Mammary tumors arising in MTAG mice closely mimic changes observed in human breast carcinomas over the course of the disease progression (20). AT-3 cell line is very useful to generate syngeneic transplant in C57BL/6 mouse and to study tumor physiology that is representative of typical disease states in humans. Cell lines from ATCC and DSMZ were thoroughly tested and authenticated using morphology-, karyotyping-, and PCR-based approaches to confirm the identity of the cell lines. The HMEC and MCF10A cells were grown in MEGM complete medium and MCF12A cells were grown in DMEM/F12 medium with 5% horse serum, 1% penicillin/streptomycin, 20 ng/mL human EGF, 100 ng/mL cholera toxin, 0.01 mg/mL bovine insulin, and 500 ng/mL hydrocortisone. HBL100 cells were grown in McCoy 5A with 10% FBS. MCF7, BT20, HC11, and AT-3 cells were grown in DMEM with 10% FBS. T47D, ZR75.1, BT474, BT485, HCC1937, and 4T1 cells were grown in RPMI1640 medium with 10% FBS. MDA-MB-231, -361, and MDA-MB-468 cells were grown in Leibovitz L-15 medium with 10% FBS. MDA-MB-415 cells were grown in Leibovitz L-15 medium with 15% FBS, 1% penicillin/streptomycin, and 0.01 mg/mL insulin. CAL51 cells were grown in DMEM supplemented with 20% FBS, and 1% penicillin/streptomycin. All these cell lines were routinely tested for Mycoplasma contamination using the Universal Mycoplasma Detection Kit obtained from ATCC, and the last Mycoplasma test was performed in October 2019. Mycoplasma-free cell lines were used in all of our experiments.

Animals

C57BL/6 (stock # 000664), MMTV-PyMT-Tg (stock #002374), MMTV-Neu-Tg (stock # 002376), C3(1)-SV40-Tg (stock #013591), and Rad51ap1 knockout [B6N(Cg)-Rad51ap1tm1.1(KOMP)Vlcg/J, stock # 025176] mice were obtained from The Jackson Laboratory. All these mice were bred and maintained at AU Animal Facility in accordance with the guidelines of the IACUC. All euthanasia protocols were performed in accordance with the regulations and guidelines presented by IACUC and Laboratory Animal Service of AU.

Mammary gland single-cell preparation

Mammary gland single-cell preparation was carried out as described previously (15, 21). Briefly, the thoracic and inguinal mammary glands were dissected from mice. The tissues were digested for 6–8 hours at 37°C in DMEM/F12 medium supplemented with 10% FBS and 1% penicillin/streptomycin and 750 U/mL Collagenase and 250 U/mL hyaluronidase. After this step, the organoids were collected by centrifugation and then subjected to ACK treatment for red blood cell lysis followed by trypsin (0.5%) and dispase (5 mg/mL) treatment separately. All reagents were obtained from Stemcell Technology unless stated otherwise.

Analysis of mammary stem/progenitor and BCSCs

For isolation of mammary stem/progenitor cells (MaSC) and BCSCs, we used CD24lo and CD49fhi as markers for normal MaSC, and CD44/CD24 double-positive for human BCSCs and CD49f/CD24 double-positive for mouse BCSCs, as described in our previous articles (15, 21) and studies from other laboratories (22, 23). We used the following antibodies for this study: CD49f and CD24 (Stemcell Technology and eBioscience), CD45, CD31, CD49f, and CD44 (BD Biosciences), and Ter119 and CD61 (eBioscience). Blocking was done for 10 minutes with rat serum. Cells were stained for 30 minutes on ice and washed with staining media. Finally, cells were resuspended in staining media containing 7-aminoactinomycin D (1 μg/mL) or 4′-6-diamidino-2-phenylindole (DAPI, 1 μg/mL) to stain dead cells. Cells were sorted through Mo flow cell sorter and flow cytometry data were analyzed using LSR II and FlowJo as described before in our articles (15, 21).

Immunoblot analysis

For immunoblot analysis, cell lysates were prepared from cell lines and mammary tissues as described previously (24, 25). Protein samples were fractionated on SDS-PAGE gels and transferred to Protran Nitrocellulose Membrane (Whatman GmbH). Membranes were blocked with 5% nonfat dry milk and exposed to primary antibody at 4°C overnight followed by treatment with appropriate secondary antibody, conjugated to horseradish peroxidase at room temperature for 1 hour, and developed by enhanced chemiluminescence SuperSignal western system and the signals captured on an X-ray film.

Methods for: (i) generation of RAD51AP1shRNA-expressing stable cell lines; (ii) clonogenic assays; (iii) generation of paclitaxel-resistant cell lines; (iv) generation of Rad51ap1−/−-MMTV-PyMT-Tg, Rad51ap1−/−-MMTV-Neu-Tg, and Rad51ap1−/−-C3(1)-SV40-Tg mice; (v) in vivo limiting dilution assay; (vi) mouse genotyping; (vii) mouse xenograft; (viii) mouse syngeneic transplants; (ix) generation of mammospheres and tumorospheres; (x) RNA isolation and PCR analysis; and (xi) reverse transcriptase PCR are provided in the Supplementary Materials and Methods.

Statistical analysis

Statistical analysis was done using one-way ANOVA followed by Bonferroni multiple comparison test and also using Student t test with two-tail distribution. The software used was GraphPad Prism, version 8.0. A value of P < 0.05 was considered statistically significant. Kaplan–Meier analyses (http://kmplot.com/analysis/) were used to assay group differences in tumor-free survival. The Cancer Genome Atlas (TCGA) database, UCGC genome, and DAVID (http://david.abcc.ncifcrf.gov) were also used to analyze RAD51AP1 expression. GraphPad and Excel programs were used to draw figures.

RAD51AP1 expression is upregulated in human breast cancer and is associated with a poor prognosis

Oncomine database analysis (TCGA and GOBO) provides evidence that RAD51AP1 (human) gene expression is increased in human breast cancer (15). To validate this observation, we analyzed RAD51AP1 gene expression in human primary breast tumor tissues, which are ER+BC and TNBC, with respective controls. Irrespective of hormone receptor status, RAD51AP1 expression is significantly elevated in human breast cancer tissues when compared with normal tissues (Fig. 1A). Quantitative real-time PCR (qPCR) analysis provided evidence that RAD51AP1 expression was significantly higher in human basal TNBC when compared with luminal ER+BC (Fig. 1B). This observation was further confirmed by the TCGA database analysis (Fig. 1C). We then analyzed the functional correlation between RAD51AP1 expression and the overall survival of patients with breast cancer using Kaplan–Meier plotter analysis. We found that high RAD51AP1 expression was negatively associated with relapse-free survival (RFS) in both luminal ER+BC and basal TNBC (Fig. 1D), distant metastasis-free survival, and overall survival (Supplementary Fig. S1A and S1B). We also analyzed RAD51AP1 expression in human normal immortalized mammary epithelial cell lines, luminal ER+BC, and basal TNBC cell lines and recapitulated the similar findings, which were seen in human breast tumor tissues (Fig. 1E and F; Supplementary Fig. S1C).

Figure 1.

RAD51AP1 expression in normal and breast tumor tissues and cell lines. A,RAD51AP1 gene transcript levels were analyzed by semiquantitative RT-PCR in human breast tumor tissues (T), both luminal ER+BC (n = 5) and basal TNBC (n = 6), and respective normal (N) tissues. B, Real-time RT-PCR (qPCR) analysis showing the relative mRNA expression of RAD51AP1 in human normal and breast tumor tissues. C, TCGA database analysis confirms increased RAD51AP1 expression in human luminal ER+BC (n = 358) and basal TNBC (n = 98) when compared with normal (n = 114). D, Kaplan–Meier plots of RFS of patients with breast cancer in whole datasets for luminal ER+BC and basal TNBC stratified by RAD51AP1 expression. The P value was calculated using log-rank test. E and F, Semiquantitative and qPCR analyses show increased RAD51AP1 gene transcript levels in human luminal ER+BC (n = 7) and basal TNBC (n = 4) cell lines when compared with nontransformed normal mammary epithelial cell line (n = 4).

Figure 1.

RAD51AP1 expression in normal and breast tumor tissues and cell lines. A,RAD51AP1 gene transcript levels were analyzed by semiquantitative RT-PCR in human breast tumor tissues (T), both luminal ER+BC (n = 5) and basal TNBC (n = 6), and respective normal (N) tissues. B, Real-time RT-PCR (qPCR) analysis showing the relative mRNA expression of RAD51AP1 in human normal and breast tumor tissues. C, TCGA database analysis confirms increased RAD51AP1 expression in human luminal ER+BC (n = 358) and basal TNBC (n = 98) when compared with normal (n = 114). D, Kaplan–Meier plots of RFS of patients with breast cancer in whole datasets for luminal ER+BC and basal TNBC stratified by RAD51AP1 expression. The P value was calculated using log-rank test. E and F, Semiquantitative and qPCR analyses show increased RAD51AP1 gene transcript levels in human luminal ER+BC (n = 7) and basal TNBC (n = 4) cell lines when compared with nontransformed normal mammary epithelial cell line (n = 4).

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RAD51AP1 KD in human breast cancer cells reduces tumor growth

As shown in Fig. 1, RAD51AP1 expression is amplified in human breast tumor tissues and cell lines. We then asked whether RAD51AP1 KD in these cell lines would reduce tumor growth. RAD51AP1 KD in CAL51, a TNBC cell line, was confirmed by expression analyses (Fig. 2A; Supplementary Fig. S2A). RAD51AP1 KD significantly reduced cell growth, which was measured by colony formation assays, as described previously (Fig. 2B; refs. 25, 26). This observation was recapitulated in a mouse xenograft assay, as describe previously (24, 27). RAD51AP1 KD significantly reduced tumor size and mass (Fig. 2C–E). IHC analysis revealed that RAD51AP1 KD reduced tumor growth by inhibiting tumor cell proliferation assessed by Ki67 staining (Fig. 2F; Supplementary Fig. S2B). These observations were also recapitulated in two more human breast cancer cell lines ZR75.1 (a human luminal ER+BC cell line) and MB231 (a human basal TNBC cell line; Supplementary Fig. S2C–S2J). Altogether, these results provide evidence that (i) RAD51AP1 expression is significantly elevated in human breast cancer tissues and cell lines; (ii) this increased RAD51AP1 expression is associated with reduced overall survival; and (iii) RAD51AP1 ablation leads to reduced tumor growth by inhibiting cell proliferation.

Figure 2.

RAD51AP1 KD in human breast cancer cells reduces tumor growth. A,RAD51AP1 KD in CAL51 cell line was confirmed by semiquantitative RT-PCR and Western blot analyses. B, Representative images of colony formation assay of CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#1 and #2) stable cell lines show that RAD51AP1 KD significantly reduces colony formation. The resulting colonies were stained with Giemsa dye, following which the bound Giemsa dye was dissolved and quantified by spectrophotometer analysis. Data represent mean ± SD from three independent experiments. C, Tumor growth measurement in mouse xenograft derived from CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#1 and #2) shows that RAD51AP1 KD significantly reduces tumor growth. (n = 10 mice in each group). D, Representative images of mammary tumor tissues derived from CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) mouse xenograft (n = 10 mice in each group). E, Tumor weight measurement shows that RAD51AP1 KD significantly reduces tumor weight (grams; n = 10 mice in each group). F, Hematoxylin and eosin (H&E)- and Ki67-stained images of tumor tissues derived from CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) show a significantly reduced tumor burden in RAD51AP1-KD cell lines by inhibition of tumor cell proliferation. Images were taken at 40×; scale bar, 100 μm (n = 10 mice in each group).

Figure 2.

RAD51AP1 KD in human breast cancer cells reduces tumor growth. A,RAD51AP1 KD in CAL51 cell line was confirmed by semiquantitative RT-PCR and Western blot analyses. B, Representative images of colony formation assay of CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#1 and #2) stable cell lines show that RAD51AP1 KD significantly reduces colony formation. The resulting colonies were stained with Giemsa dye, following which the bound Giemsa dye was dissolved and quantified by spectrophotometer analysis. Data represent mean ± SD from three independent experiments. C, Tumor growth measurement in mouse xenograft derived from CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#1 and #2) shows that RAD51AP1 KD significantly reduces tumor growth. (n = 10 mice in each group). D, Representative images of mammary tumor tissues derived from CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) mouse xenograft (n = 10 mice in each group). E, Tumor weight measurement shows that RAD51AP1 KD significantly reduces tumor weight (grams; n = 10 mice in each group). F, Hematoxylin and eosin (H&E)- and Ki67-stained images of tumor tissues derived from CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) show a significantly reduced tumor burden in RAD51AP1-KD cell lines by inhibition of tumor cell proliferation. Images were taken at 40×; scale bar, 100 μm (n = 10 mice in each group).

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Rad51ap1 KD reduced tumor growth in syngeneic mouse models

To confirm the prosurvival potential of RAD51AP1, we used mouse models of breast cancer. First, we analyzed Rad51ap1 (mouse) expression in normal immortalized mouse mammary epithelial cell line (HC11) and the mouse mammary tumor cell lines (AT-3 and 4T1). Rad51ap1 expression was significantly elevated in mouse mammary tumor cell lines when compared with a normal immortalized cell line (Supplementary Fig. S3A and S3B). To determine the role of Rad51ap1 in mouse mammary tumor growth, we knocked down Rad51ap1 in the AT-3 cell line and confirmed it by expression analyses (Fig. 3A; Supplementary Fig. S3C). Rad51ap1 ablation led to reduced cell growth in vitro (Fig. 3B and C). This observation was recapitulated in an in vivo syngeneic mouse model. We subcutaneously implanted pLKO.1- and Rad51ap1shRNA-expressing AT-3 cells into the mammary fat pad of C57BL/6 mice and allowed them to grow for 7 weeks. We measured tumor size and weight. Rad51ap1 KD significantly reduced tumor size and weight (Fig. 3DF). Histologic sections provided evidence that Rad51ap1 KD significantly reduced tumor growth by inhibiting tumor cell proliferation (Fig. 3G; Supplementary Fig. S3D).

Figure 3.

Rad51ap1 KD reduced tumor growth in syngeneic mouse model. A,Rad51ap1 KD in AT-3 cell line was confirmed by Western blot analysis (n = 3 replicates). B and C, Representative images of colony formation assay of AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#3 and #4) stable cell lines show that Rad51ap1 KD significantly reduces colony formation. The resulting colonies were stained with Giemsa dye and bound Giemsa dye was dissolved and quantified by spectrophotometer analysis. Data represents mean ± SD from three independent experiments. D, Tumor growth measurement in mouse syngeneic transplant from AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#4) shows that Rad51ap1 KD significantly reduces tumor growth (n = 10 mice in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Representative images of mammary tumors derived from AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#4) mouse syngeneic transplant (n = 10 mice in each group). F, Tumor weight measurement shows that Rad51ap1 KD significantly reduces tumor weight (grams; n = 10 mice in each group). G, Representative images of hematoxylin and eosin (H&E) and Ki67 staining of pLKO.1 and Rad51ap1shRNA (#4) paraffin-embedded tumor sections show that Rad51ap1 KD significantly reduced tumor burden by inhibiting cell proliferation. Images were taken at 60×; scale bar, 100 μm.

Figure 3.

Rad51ap1 KD reduced tumor growth in syngeneic mouse model. A,Rad51ap1 KD in AT-3 cell line was confirmed by Western blot analysis (n = 3 replicates). B and C, Representative images of colony formation assay of AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#3 and #4) stable cell lines show that Rad51ap1 KD significantly reduces colony formation. The resulting colonies were stained with Giemsa dye and bound Giemsa dye was dissolved and quantified by spectrophotometer analysis. Data represents mean ± SD from three independent experiments. D, Tumor growth measurement in mouse syngeneic transplant from AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#4) shows that Rad51ap1 KD significantly reduces tumor growth (n = 10 mice in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. E, Representative images of mammary tumors derived from AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#4) mouse syngeneic transplant (n = 10 mice in each group). F, Tumor weight measurement shows that Rad51ap1 KD significantly reduces tumor weight (grams; n = 10 mice in each group). G, Representative images of hematoxylin and eosin (H&E) and Ki67 staining of pLKO.1 and Rad51ap1shRNA (#4) paraffin-embedded tumor sections show that Rad51ap1 KD significantly reduced tumor burden by inhibiting cell proliferation. Images were taken at 60×; scale bar, 100 μm.

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Rad51ap1 knockout mice confer resistance to tumor growth

The biological role of RAD51AP1 in tumor growth and progression was further analyzed in wild-type (Rad51ap1+/+) and Rad51ap1 knockout (Rad51ap1−/−) mice. Rad51ap1 knockout was confirmed by genotyping and expression analysis (Fig. 4A; Supplementary Fig. S4A and S4B). Rad51ap1−/− mice are normal, viable with no overt phenotype. To test the role of Rad51ap1 in tumor growth and progression, we repeated the syngeneic transplant of AT-3 cells, which has functional Rad51ap1, into the mammary fat pad of Rad51ap1+/+ and Rad51ap1−/− mice and monitored tumor growth for 7 weeks. Although AT-3 tumor cells were able to elicit rapid tumorigenesis in Rad51ap1+/+ mice, the tumor growth was significantly compromised in Rad51ap1−/− mice (Fig. 4B). The reduced tumor growth was also evidenced in tumor size and weight (Fig. 4C and D). Histologic examination of tumor tissues revealed that Rad51ap1 deletion reduced tumor growth by inhibiting tumor cell proliferation (Fig. 4E and F). Together, these data provide strong evidence that Rad51ap1 plays a critical role in the tumor microenvironment and that Rad51ap1 deficiency produces a microenvironment, which is not conducive for tumor cells to grow.

Figure 4.

Rad51ap1 knockout mice confer resistance to tumor growth. A, Representative Western blot image of RAD51AP1 provides evidence that Rad51ap1 was knocked out in mammary glands derived from wild-type (Rad51ap1+/+) and Rad51ap1 knockout (Rad51ap1−/−) mice. n = 4 in each genotype. B, Tumor growth measurement in mouse syngeneic transplant derived from Rad51ap1+/+ and Rad51ap1−/− mice shows that Rad51ap1 deficiency significantly reduced tumor growth (n = 10 mice in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. C and D, Representative images of mammary tumor tissues (C) and tumor weight (grams; D) driven by syngeneic transplant of AT-3 cell lines in Rad51ap1+/+ and Rad51ap1−/− mice provide evidence that Rad51ap1 deficiency significantly reduces tumor growth (n = 10 mice in each genotype). E, Representative images of hematoxylin and eosin (H&E) and Ki67 staining in tumor tissues of Rad51ap1+/+ and Rad51ap1−/− mice provide evidence that Rad51ap1 deletion significantly reduced tumor growth by inhibiting tumor cell proliferation. Images are taken at 60×; scale bar, 100 μm. F, Total number of Ki67-positive cells' analysis provide evidence that Ki67-positive cells were reduced in Rad51ap1−/− mice when compared with Rad51ap1+/+ mice.

Figure 4.

Rad51ap1 knockout mice confer resistance to tumor growth. A, Representative Western blot image of RAD51AP1 provides evidence that Rad51ap1 was knocked out in mammary glands derived from wild-type (Rad51ap1+/+) and Rad51ap1 knockout (Rad51ap1−/−) mice. n = 4 in each genotype. B, Tumor growth measurement in mouse syngeneic transplant derived from Rad51ap1+/+ and Rad51ap1−/− mice shows that Rad51ap1 deficiency significantly reduced tumor growth (n = 10 mice in each group). *, P < 0.05; **, P < 0.01; ***, P < 0.001. C and D, Representative images of mammary tumor tissues (C) and tumor weight (grams; D) driven by syngeneic transplant of AT-3 cell lines in Rad51ap1+/+ and Rad51ap1−/− mice provide evidence that Rad51ap1 deficiency significantly reduces tumor growth (n = 10 mice in each genotype). E, Representative images of hematoxylin and eosin (H&E) and Ki67 staining in tumor tissues of Rad51ap1+/+ and Rad51ap1−/− mice provide evidence that Rad51ap1 deletion significantly reduced tumor growth by inhibiting tumor cell proliferation. Images are taken at 60×; scale bar, 100 μm. F, Total number of Ki67-positive cells' analysis provide evidence that Ki67-positive cells were reduced in Rad51ap1−/− mice when compared with Rad51ap1+/+ mice.

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Rad51ap1 deficiency impedes tumor growth in spontaneous mammary tumorigenesis

On the basis of the relative expression of RAD51P1 in normal and tumor tissues and cell lines, RAD51AP1 overexpression has been reported in human cancers (15–18). Conversely, a recent study has reported the reduced RAD51AP1 expression (due to the CpG methylation of RAD51AP1 promoter) in prostate cancer cells (28). However, there is no direct evidence to show whether RAD51AP1 functions either as a tumor suppressor or a promoter of tumorigenesis. Hence, to test the relative contribution of RAD51AP1 in tumorigenesis, first, we analyzed the cell type–specific expression of Rad51ap1 in the mammary epithelium. As shown in Supplementary Fig. S5A, single-cell suspensions derived from the normal control mammary glands showed three distinct populations namely basal myoepithelial stem cells (LinCD49fhiCD24lo), luminal progenitor (LinCD49floCD24hi), and stromal (LinCD49fCD24) cells. Using FACS sorter, we isolated these three different cell types separately and analyzed Rad51ap1 gene expression. We found a relatively higher expression of Rad51ap1 in basal myoepithelial stem cells compared with the luminal progenitor and stromal cells (Supplementary Fig. S5A–S5C). Analysis of Rad51ap1 expression in normal and tumor tissues obtained from three different spontaneous mouse mammary tumor models (MMTV-PyMT-Tg, MMTV-Neu-Tg, and C3(1)-SV40-Tg) revealed that Rad51ap1 expression was significantly elevated in tumor tissues compared with normal (Fig. 5A and B). To determine the functional role of Rad51ap1 in mammary tumorigenesis, we crossed Rad51ap1−/− mice with MMTV-PyMT-Tg (PyMT-Tg) and MMTV-Neu-Tg (Neu-Tg), both of which mimic human luminal progenitor (29, 30), and C3(1)-SV40-Tg (C3(1)-Tg) mice, which mimics human basal TNBC (31). Age-matched littermates of Rad51ap1+/+ and Rad51ap1−/− in PyMT-Tg, Neu-Tg, and C3(1)-Tg background (n = 25 in each), respectively, were allowed to form spontaneous tumors and monitored closely for signs of tumor development. Percentage of animals in which tumors developed and time of tumor appearance in each genotype were monitored. Time until mouse sacrifice either due to the tumor burden or disease-free survival was also noted and used to calculate percent survival. After euthanizing the mice, total number of primary tumors formed in each genotype and lung metastatic nodules were also recorded. We found that there was no significant difference in the percent of tumor incidence between Rad51ap1+/+-PyMT-Tg and Rad51ap1−/−-PyMT-Tg mice as well as Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice (Fig. 5C and E). Irrespective of Rad51ap1 genotypes, almost 100% of Rad51ap1-PyMT-Tg and Rad51ap1-C3(1)-Tg mice developed tumors. Although no significant difference was observed in tumor incidence between wild-type and Rad51ap1 knockout mice, the tumor latency was significantly longer with reduced number of lung nodules, which led to increased overall survival of Rad51ap1-deficient mice when compared with the wild-type littermates (Fig. 5C and E). However, significant differences were observed in the percent of tumor incidence, time of tumor formation, number of lung nodules, and overall survival between Rad51ap1+/+-Neu-Tg and Rad51ap1−/−-Neu-Tg mice (Fig. 5D). Altogether, these results provide strong and direct evidence that Rad51ap1 plays a critical role in mammary tumor growth and progression, and that Rad51ap1 deficiency significantly abrogated tumor formation with extended overall survival.

Figure 5.

Rad51ap1 deficiency impedes tumor growth in spontaneous mammary tumorigenesis. Representative image of semiquantitative RT-PCR (A) and qPCR analyses (B) reveal high Rad51ap1 expression in tumor tissues derived from PyMT-Tg, Neu-Tg, and C3(1)-Tg mice. n = 2 mice in each. C and E, Although Rad51ap1 deletion does not affect the overall tumor incidence in PyMT-Tg and C3(1)-Tg mice, it significantly delayed the time of tumor onset and reduced the number of lung nodules, resulting in increased overall survival. n = 24 mice in each genotype. D,Rad51ap1 deficiency significantly reduced tumor incidence, time of tumor onset, and number of lung nodules in Neu-Tg mice, with increased overall survival (n = 24 mice in each genotype).

Figure 5.

Rad51ap1 deficiency impedes tumor growth in spontaneous mammary tumorigenesis. Representative image of semiquantitative RT-PCR (A) and qPCR analyses (B) reveal high Rad51ap1 expression in tumor tissues derived from PyMT-Tg, Neu-Tg, and C3(1)-Tg mice. n = 2 mice in each. C and E, Although Rad51ap1 deletion does not affect the overall tumor incidence in PyMT-Tg and C3(1)-Tg mice, it significantly delayed the time of tumor onset and reduced the number of lung nodules, resulting in increased overall survival. n = 24 mice in each genotype. D,Rad51ap1 deficiency significantly reduced tumor incidence, time of tumor onset, and number of lung nodules in Neu-Tg mice, with increased overall survival (n = 24 mice in each genotype).

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RAD51AP1 deficiency reduces cancer stem cell self-renewal signaling

As shown in Supplementary Figs. S1–S5, RAD51AP1 expression is amplified in human and mouse mammary tumors, and RAD51AP1 deficiency has a significant impact on tumor growth and metastasis retardation. Therefore, we attempted to elucidate the molecular mechanism(s) by which RAD51AP1 deficiency reduces tumor growth. We analyzed genes that are involved in epithelial cell differentiation, BCSC formation, pluripotency, and epithelial-to-mesenchymal transition (EMT)/metastasis using vector control (pLKO.1)- and RAD51AP1shRNA-transfected CAL51 cell line. RAD51AP1 KD significantly increased the expression of epithelial differentiation marker CDH1 (E-cadherin) with a significant reduction in BCSC markers (CD44 and CD49f) and pluripotent stem cell markers (NANOG and KLF4; Fig. 6A). These results were recapitulated in tumor tissues derived from the AT-3-pLKO.1 and AT-3-Rad51ap1shRNA as well as Rad51ap1−/−-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice (Fig. 6B and C). These findings suggest that RAD51AP1 plays a critical role in BCSC self-renewal signaling.

Figure 6.

RAD51AP1 KD reduces CSC self-renewal signaling. A, Heatmap generated from the qPCR dataset for epithelial differentiation, BCSC marker, pluripotency, and EMT/metastasis of CAL51-pLKO.1 and CAL51-RAD51AP1(#2) cells (n = 3 independent clones with triplicates = 9 dataset in each). B, Relative expression of BCSC self-renewal genes in AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#3) by qPCR analysis (n = 3 independent clones with triplicates = 9 dataset in each). C, Relative expression of BCSC self-renewal genes in Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg by qPCR analysis (n = 3 mice with triplicates = 9 dataset in each). D and E, Representative FACS dot plots and quantification of BCSC (CD49f+CD24+) in CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) cells. F and G, Representative FACS dot plots and quantification of BCSC in Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice. H and I, Representative images of tumorospheres generated form Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice and quantification of primary, secondary, and tertiary tumorospheres number (n = 3–5 mice in each). *, P < 0.01; ***, P < 0.0001.

Figure 6.

RAD51AP1 KD reduces CSC self-renewal signaling. A, Heatmap generated from the qPCR dataset for epithelial differentiation, BCSC marker, pluripotency, and EMT/metastasis of CAL51-pLKO.1 and CAL51-RAD51AP1(#2) cells (n = 3 independent clones with triplicates = 9 dataset in each). B, Relative expression of BCSC self-renewal genes in AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (#3) by qPCR analysis (n = 3 independent clones with triplicates = 9 dataset in each). C, Relative expression of BCSC self-renewal genes in Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg by qPCR analysis (n = 3 mice with triplicates = 9 dataset in each). D and E, Representative FACS dot plots and quantification of BCSC (CD49f+CD24+) in CAL51-pLKO.1 and CAL51-RAD51AP1shRNA (#2) cells. F and G, Representative FACS dot plots and quantification of BCSC in Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice. H and I, Representative images of tumorospheres generated form Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice and quantification of primary, secondary, and tertiary tumorospheres number (n = 3–5 mice in each). *, P < 0.01; ***, P < 0.0001.

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These observations are supported by the previous studies, in which RAD51AP1 expression is positively associated with stem cell self-renewal (15) and stem cells are highly reliant on DNA repair mechanisms to maintain the pluripotency (32). Therefore, to provide more direct evidence for the role of RAD51AP1 in BCSC self-renewal, we analyzed BCSC populations in CAL51-pLKO.1- and CAL51-RAD51AP1shRNA–expressing cells. As shown in Supplementary Fig. S5A, the normal mammary gland shows three distinct populations namely, luminal (LinCD49floCD24hi), basal (LinCD49fhiCD24lo), and stromal (LinCD49fCD24). However, we have recently shown that tumor tissues derived from the MMTV-Neu-Tg (15, 21) and MMTV-Hras-Tg (24) mice showed only two distinct populations: tumorigenic (LinCD49f+CD24+) and nontumorigenic (LinCD49fCD24). As described previously by Al-Hajj and colleagues (22), we used CD44 and CD24 as cell surface markers for human and CD49f and CD24 for mouse (15, 21). Analysis of CAL51-pLKO.1 showed only two distinct populations [CD44hi/(+) CD24hi/(+)] and [CD44lo/(-) CD24lo/(-); Fig. 6D]. Thus, we used CD44 and CD24 double-positive cells as human BCSCs and CD49f and CD24 double-positive cells as mouse BCSCs, as described previously (15, 21). We found that RAD51AP1 KD significantly reduced the BCSC pool (CD44+CD24+) when compared with control vector–transfected cells (Fig. 6D and E). We sorted the BCSCs (CD44+CD24+) by FACS and generated tumorospheres using stem cell enrichment medium, as described previously (15, 21). RAD51AP1 KD significantly reduced the size and number of tumorospheres (primary, secondary, and tertiary) when compared with pLKO.1-expressing CAL51 cells (Supplementary Fig. S6A and S6B). These observations were recapitulated in tumor tissues derived from the syngeneic transplant of AT-3-pLKO.1 and AT-3-Rad51ap1shRNA (Supplementary Fig. S6C–S6F). We also analyzed the BCSC populations in tumor tissues obtained from Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice. We found that Rad51ap1 deficiency not only reduced the BCSC pool, but also reprogramed BCSCs (Fig. 6F and G) and reduced the size and number of tumorospheres (primary, secondary, and tertiary; Fig. 6H and I). To test the role of Rad51ap1 in BCSC self-renewal further, we performed the limiting dilution of FACS sorted CD49f+CD24+ double-positive (BCSCs) and CD49fCD24 double-negative (nonstem) cells from the tumor tissues derived from Rad51ap1+/+-MMTV-Neu-Tg and Rad51ap1−/−-MMTV-Neu-Tg mice. First, we analyzed Rad51ap1 expression in BCSCs and nonstem cells and found significantly increased Rad51ap1 expression (>28-fold) in BCSCs than the nonstem cells (Supplementary Fig. S6G). Then, we injected BCSCs and nonstem cells (from 1 × 101 to 1 × 106 cells) in the mammary fat pad of NOD/SCID mice. Rad51ap1 deficiency significantly reduced the tumor forming potential of CD49f+CD24+ double-positive cells, while CD49fCD24 double-negative cells were unable to form tumors even in 1 × 106 cells (Supplementary Fig. S6H). These results demonstrate the importance of RAD51AP1 in stem cell self-renewal and gene expression.

RAD51AP1 deficiency potentiates chemotherapy and radiotherapy response by inhibiting stem cell self-renewal signaling

As shown in Fig. 6, RAD51AP1 plays a critical role in BCSC self-renewal and RAD51AP1 deficiency not only reduced the BCSC pool but also reprogramed BCSCs (three distinct population as seen in normal control mammary gland). Because cancer stem cells (CSC) have been implicated in conventional chemotherapy and radiotherapy resistance (33), we explored the clinical utility of RAD51AP1 KD in inducing sensitivity to chemo- and radiotherapy. CSCs are a small population of cells (1%–2%) with unique characteristics such as self-renewal, high proliferation rate, and the ability to generate heterogenic lineages of cancer cells (22, 23). Therefore, there are various types of CSCs within a tumor, with different mutational profiles and in different stages of cell cycling. Typically, within a tumor, there is one dominant population driving tumor progression, but there are always other minor populations waiting for an opportunity to expand. This opportunity often comes when the dominant CSC population is destroyed during chemotherapy or ionizing radiation. The smaller CSC population, which was either in a quiescent state during treatment, or has acquired resistance to therapeutics, can emerge and promote new tumor growth. The recurring tumor is often more aggressive and resistant to previous therapeutics, thereby increasing the risk for metastasis and requiring a new treatment strategy to be devised. As shown in Fig. 6, RAD51AP1 appears to play an important role in CSC maintenance and self-renewal and, thus, we asked whether RAD51AP1 deficiency will potentiate chemotherapy (paclitaxel) and radiotherapy (ionizing radiation) response in cancer cells. RAD51AP1 KD (RAD51AP1shRNA) in human breast cancer cell line (CAL51) and mouse mammary tumor cell line (AT-3) significantly potentiated both paclitaxel- and ionizing radiation–induced apoptosis when compared with vector-transfected cells (Fig. 7A and B). To provide more concrete evidence for the role of RAD51AP1 in drug resistance, we isolated mammary epithelial cells from the tumor tissues obtained from Rad51ap1+/+-C3(1)-Tg and Rad51ap1−/−-C3(1)-Tg mice and then treated with paclitaxel and ionizing radiation. We found that Rad51ap1 deficiency potentiates paclitaxel and ionizing radiation response by increasing apoptotic cell death (Fig. 7C). These data demonstrate that RAD51AP1 deficiency indeed leads to increased cellular sensitivity to chemotherapy and radiotherapy. We then asked whether paclitaxel resistance is associated with increased RAD51AP1 expression and whether RAD51AP1 KD would resensitize the CAL51 cells to chemotherapy response. Thus, we generated paclitaxel-resistant (PTX-R) CAL51 cell line by increasing paclitaxel concentration (10–250 nmol/L) as described previously (34). We analyzed RAD51AP1 expression in paclitaxel-sensitive (PTX-S) and PTX-R cell lines and found that RAD51AP1 expression was significantly increased in PTX-R cells compared with PTX-S cells (Fig. 7D). Then, we took the CAL51-PTX-R cell line and infected with pLKO.1 and RAD51AP1shRNA. RAD51AP1 KD was confirmed by qPCR analysis (Fig. 7E). We found that RAD51AP1 depletion resensitizes CAL51-PTX-R cells for paclitaxel as evidenced by increased apoptotic cell death (Fig. 7F). We also found that RAD51AP1 deficiency is associated with a reduced BCSC pool (Fig. 7G and H). We next analyzed the expression of stem cell self-renewal genes like NANOG, KLF4, SOX2, and OCT4 and found that RAD51AP1 KD in PTX-R cells significantly reduced NANOG and KLF4 expression (Fig. 7I). These data provide direct evidence that RAD51AP1 plays a critical role in resistance to chemotherapy and radiotherapy, in that RAD51AP1 deficiency not only potentiates chemotherapy response, but also resensitizes the resistant cancer cells to chemotherapy. Overall, our results provide evidence that RAD51AP1 plays a critical role in breast cancer growth and metastasis by regulating BCSC self-renewal, and is involved in tumor growth and metastasis, drug resistance, and reduced overall survival. Thus, RAD51AP1 is an attractive target for the prevention and treatment of breast cancer.

Figure 7.

RAD51AP1 KD potentiates chemotherapy and radiotherapy response by inhibiting stem cell self-renewal signaling. A, Percent apoptosis (Annexin V+/PI) was calculated in CAL51-pLKO.1 and CAL51-RAD51AP1shRNA transfected cells with paclitaxel (PTX; 10 nmol/L) and ionizing radiation (IR; 10 Gy), respectively. B and C, Similarly, percent apoptosis was calculated in AT-3-pLKO.1 and AT-3-RAD51AP1shRNA transfected cells, as well as cells isolated from the mammary tumor tissues obtained from Rad51ap1+/+-C3(1)-Tg and Rad51ap1+/+-C3(1)-Tg mice with paclitaxel (10 nmol/L) and ionizing radiation (10 Gy), respectively. Values are expressed as mean ± SD of three independent experiments or three mice in each with triplicated values = 9 datasets. D,RAD51AP1 mRNA and RAD51AP1 protein expression in PTX-S and PTX-R CAL51 cell lines as measured by qPCR and Western blotting, respectively. n = 4 independent clones. E, Relative RAD51AP1 transcript expression in CAL51-PTX-R cells transfected with pLKO.1 and RAD51AP1shRNA. F, Percent apoptosis was calculated in PTX-R-pLKO.1 and PTX-R-RAD51AP1shRNA transfected cells treated with and without paclitaxel (10 nmol/L for 48 hours). G and H, Representative FACS contour plots and quantification of BCSC (CD49f+CD24+) in CAL51-PTX-R-pLKO.1 and CAL51-PTX-R-RAD51AP1shRNA cells. I, Relative expression of stem cell self-renewal genes in CAL51-PTX-R-pLKO.1 and CAL51-PTX-R-RAD51AP1shRNA cells.

Figure 7.

RAD51AP1 KD potentiates chemotherapy and radiotherapy response by inhibiting stem cell self-renewal signaling. A, Percent apoptosis (Annexin V+/PI) was calculated in CAL51-pLKO.1 and CAL51-RAD51AP1shRNA transfected cells with paclitaxel (PTX; 10 nmol/L) and ionizing radiation (IR; 10 Gy), respectively. B and C, Similarly, percent apoptosis was calculated in AT-3-pLKO.1 and AT-3-RAD51AP1shRNA transfected cells, as well as cells isolated from the mammary tumor tissues obtained from Rad51ap1+/+-C3(1)-Tg and Rad51ap1+/+-C3(1)-Tg mice with paclitaxel (10 nmol/L) and ionizing radiation (10 Gy), respectively. Values are expressed as mean ± SD of three independent experiments or three mice in each with triplicated values = 9 datasets. D,RAD51AP1 mRNA and RAD51AP1 protein expression in PTX-S and PTX-R CAL51 cell lines as measured by qPCR and Western blotting, respectively. n = 4 independent clones. E, Relative RAD51AP1 transcript expression in CAL51-PTX-R cells transfected with pLKO.1 and RAD51AP1shRNA. F, Percent apoptosis was calculated in PTX-R-pLKO.1 and PTX-R-RAD51AP1shRNA transfected cells treated with and without paclitaxel (10 nmol/L for 48 hours). G and H, Representative FACS contour plots and quantification of BCSC (CD49f+CD24+) in CAL51-PTX-R-pLKO.1 and CAL51-PTX-R-RAD51AP1shRNA cells. I, Relative expression of stem cell self-renewal genes in CAL51-PTX-R-pLKO.1 and CAL51-PTX-R-RAD51AP1shRNA cells.

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In this study, we provide direct evidence that RAD51AP1 plays a critical role in tumor growth and chemotherapy resistance by regulating CSC self-renewal and Rad51ap1 deficiency abrogates tumor growth and resensitizes cancer cells for chemotherapy by inhibiting CSC self-renewal signaling. Although RAD51AP1 plays a central role in homologous recombination and genome integrity, it also plays an important role in growth by promoting signaling in the early stages of tumor growth where replication stress occurs at higher than normal levels. Previous studies have shown increased expression of RAD51AP1 in human tumor tissues (15–18), which can shift the balance from a precancerous milieu to cancerous one. There was, however, no direct evidence that RAD51AP1 is directly involved in tumor growth and CSC self-renewal. Our investigation, for the first time, uncovered direct evidence showing that RAD51AP1 expression is increased in different breast cancer subtypes and is inversely associated with overall survival. Furthermore, Rad51ap1 deficiency abrogated tumor growth and metastasis in GEM models of breast cancer. However, Rad51ap1 depletion did not affect the normal epithelial homeostasis in the mammary gland, suggesting that Rad51ap1 is dispensable for normal growth. Therefore, functional inactivation of RAD51AP1 would have far-reaching implications in cancer prevention and treatment without affecting normal cells. Given the overexpression of RAD51AP1 in patients with breast cancer, especially in TNBC, RAD51AP1 could be explored as a potential biomarker for this breast cancer subtype.

Furthermore, we found a significant discrepancy between results obtained from the RAD51AP1 KD in human breast cancer cell line (CAL51)-driven mouse xenograft, mouse mammary tumor cell line (AT-3)-driven syngeneic transplant, and Rad51ap1 knockout–driven spontaneous mammary tumor models. RAD51AP1 KD was associated with a significant reduction in tumor growth (tumor size and volume) and cell proliferation (Ki67 staining), while Rad51ap1 knockout had significant impact on inhibiting tumor metastasis rather than reducing primary tumor formation. This discrepancy was mainly due to the less metastatic potential of CAL51 and AT-3 cell lines, which very occasionally metastasize to the lung. Thus, we could not see the metastatic phenotype and were unable to study the role of RAD51AP1 in tumor metastasis in these mouse models. However, GEM models', especially PyMT-Tg and C3(1)-Tg, tumors develop very aggressively within a very short period of time (2–5 months) and multiple signaling pathway(s) are involved in tumor development in these GEM models. Although there was no significant difference in the overall tumor incidence between wild-type and Rad51ap1-deficient mice, the time of tumor formation, number of tumors developed, and percent of lung metastasis were significantly reduced with increased overall survival in Rad51ap1 knockout mice when compared with the wild-type mice (Fig. 5C and E). However, we could see a clear decrease in the percent of tumor incidence, tumor growth, time of tumor formation, percent of lung metastasis, and overall survival between Rad51ap1-replete (+/+) and knockout (−/−) mice in Neu-Tg model (Fig. 5D). Therefore, RAD51AP1 plays significant role in both breast tumor growth and metastasis, but slightly differs depending on the animal model used to study.

Our study provides mechanistic evidence that RAD51AP1 deficiency–associated reduction in tumor growth and metastasis can be attributed to targeting signaling pathways that are involved in BCSCs self-renewal and maintenance. Although RAD51AP1 plays a critical role in homologous recombination and genomic stability, our study, for the first time, provides evidence that RAD51AP1 also plays a critical role in BCSC self-renewal by targeting genes that are involved in BCSC self-renewal and pluripotency. We have shown that RAD51AP1 expression is significantly increased in BCSCs than non-BCSCs (Supplementary Fig. S5). We have also shown that RAD51AP1 KD in human breast cancer cell lines and Rad51ap1 deletion in GEM models shows significantly reduced BCSC self-renewal, as evidenced by the reduction in tumorospheres size and number (primary, secondary, and tertiary) and reduction in the expression of self-renewal and pluripotent stem cell markers, which are the commonly and routinely used functional assays for the BCSC self-renewal (Fig. 6). In our previous study, we isolated BCSCs (CD49f and CD24 double-positive) and nonstem cells (CD49f and CD24 double-negative) and analyzed tumor-forming potential of these cells by serial dilution, and found that only CD49f and CD24 double-positive cells were able to form tumors, but not CD49f and CD24 double-negative cells (15, 21). However, in this study, we are unable to provide direct evidence whether RAD51AP1 deficiency–associated tumor growth retardation is associated only with BCSCs and not with nonstem cell population. A definitive answer to this question would require in vivo serial dilution–based tumor initiation experiments using BCSCs isolated from wild-type and Rad51ap1-knockout mice, which were beyond the scope of this article. Furthermore, a recent study has shown that RAD51AP1 plays a critical role in lengthening of telomeres (35). Although human somatic cells lack telomerase activity, somatic stem and progenitor cells express detectable levels of telomerase, which contributes to the prolonged proliferative capacity of these cells. Thus, it is possible that the observed phenotypic changes in Rad51ap1-deficient mice might also be associated with other signaling pathways, such as regulating telomere lengthening.

CSCs are believed to initiate and propagate tumorigenesis and are responsible for chemotherapy and radiotherapy resistance, metastasis, and tumor recurrence (35–38). With chemotherapeutic drugs targeting only the bulk of the cancer cells, but not CSCs, the survival of CSCs reinitiates tumor formation in about 20%–45% of patients within years or decades after treatment. This is a key challenge for clinicians who are trying to treat patients and palliate the cancer to extend overall patient survival. Targeting CSCs is an attractive and potential strategy for cancer prevention and treatment. CSCs retain higher levels of DNA damage repair than normal healthy cells. Because of their high frequency of replication, understanding the mechanisms that promote successful repair and clearance of DNA strand breaks will allow us to determine the best way to undermine the system to either make cells more sensitive to conventional therapies or force them into apoptosis on their own. In this study, we have demonstrated the importance of RAD51AP1 in stem cell maintenance and also provided sufficient evidence showing that RAD51AP1 deficiency has a profound impact on stem cell self-renewal by inhibiting genes involved in pluripotency (NANOG, KLF4, OCT4, and SOX2). Among these, NANOG, a transcription factor, serves as the master regulator of stem cell self-renewal (39). Similarly, OCT4, another transcription factor, has been shown to play a similar role in lung CSC (40). OCT4 KD decreased lung CSC self-renewal and increased chemosensitivity in lung cancer model (41). Thus, reduced expression of NANOG and OCT4 in RAD51AP1-KD cells and Rad51ap1-deficient mice could provide possible explanations for the reduced self-renewal, tumor growth, and metastasis in Rad51ap1-deficient mouse models of breast cancer. RAD51AP1 should be seen as a novel target for cancer treatment that is applicable to a wide variety of cancer subtypes.

One of the important and significant findings of this study is that Rad51ap1 deficiency significantly reduced tumor metastasis in GEM models of breast cancer (Fig. 5). Tumor metastasis is associated with profound changes in the cell adhesion, polarity, and migratory properties of tumor cell and is collectively known as an EMT. Loss of functional E-cadherin (CHD1) is an essential event for EMT and is considered as a hallmark of the process (42). Interestingly, RAD51AP1 KD in human breast cancer cell lines and Rad51ap1 deficiency in GEM models of breast cancer significantly rescued the CHD1 expression. Furthermore, the EMT markers SNAI1 (Snail) and SNAI2 (Slug) have been associated with repression of E-cadherin and also activation of local and distant metastasis, tumor recurrence, and poor prognosis (43, 44). The reduced expression of SNAI1 and SNAI2 observed in RAD51AP1-KD breast cancer cells and Rad51ap1-deficient mice provided further evidence for the reduced tumor metastasis observed in Rad51ap−/−-PyMT-Tg, Rad51ap−/−-Neu-Tg, and Rad51ap−/−-C3(1)-Tg mice (Fig. 5). In addition, the reduced expression of vimentin (VIM), which is selectively expressed in aggressive breast cancer cells (45), in RAD51AP1-KD cells provided further evidence for the reduced metastasis observed in Rad51ap1-deficient cells. However, the molecular mechanism(s) by which RAD51AP1 regulates the EMT and associated tumor metastasis is not known, and a separate study is warranted to provide more mechanistic evidence to explore the role of RAD51AP1 in the regulation of EMT and associated tumor metastasis.

Finally, our study clearly demonstrates that RAD51AP1 plays an important role in chemotherapy and radiotherapy resistance, and that functional inactivation of this gene can resensitize tumor cells to chemotherapy and radiotherapy response. Many cancer drugs produce DNA lesions at replication fork, and cancer cells utilize RAD51AP1 and other homologous recombination proteins to repair DSBs to maintain the cellular integrity. It has been shown that a cell's ability to repair DSBs significantly affects the outcome of cancer treatment; cells deficient in homologous recombination and DNA damage repair process are hypersensitive to drug-induced cell death, resulting in a better chemotherapeutic response and outcome. Although the role of RAD51AP1 in the repair of various types of DNA damages has not been fully deciphered yet, we know for sure that its function is critical for homologous recombination-mediated repair of DNA strand breaks. We, therefore, believe that RAD51AP1 function imparts sensitivity to most, if not all, chemotherapeutic drugs that induce DNA strand breaks. Therefore, the increased sensitivity of chemotherapy and radiotherapy response in RAD51AP1-KD cells could be associated to compromise in DNA damage repair function. This effect is not unique for human breast cancer cells and we have observed a similar effect with 5-flurouracil–resistant human colon cancer cells too. The connection between DNA repair and CSCs in chemotherapy and radiotherapy resistance was first established in high-grade primary brain tumors. The CD133+ glioblastoma stem cells activate ATM and CHK1 more promptly than the CD133 cells. This molecular response enabled CD133+ cells to survive during ionizing radiation, while CD133 population underwent cell death (46). Interestingly, subsequent studies have shown that there are significant increases in the expression of DNA repair genes in pancreatic, prostate, colon, and lung CSCs when exposed to standard chemotherapeutic agents and radiotherapy (47–49). Similarly, enhanced DNA repair ability has been described in BCSCs (48). BCSCs exhibit higher level of DNA repair genes (BRCA1, RAD51, ATM, ATR, and CHK1) when compared with differentiated tumor cells (50). Along with the DNA repair potential of RAD51AP1, it also regulates CSC self-renewal. This is, in turn, involved in chemotherapy and radiotherapy response. Altogether, our study provides direct evidence that RAD51AP1 could become a novel target for cancer prevention and treatment and overcoming drug resistance.

B.L. Lokeshwar reports grants from U.S. Veterans Administration (grant no. 5I01BX003862-02) during the conduct of the study. No potential conflicts of interest were disclosed by the other authors.

A.E. Bridges: Investigation, methodology, writing-original draft. S. Ramachandran: Formal analysis, investigation, methodology. R. Pathania: Investigation. U. Parwal: Investigation, visualization. A. Lester: Investigation, visualization. P. Rajpurohit: Investigation. D.S. Morera: Investigation, visualization. N. Patel: Investigation, writing-review and editing. N. Singh: Investigation, writing-review and editing. H. Korkaya: Writing-review and editing. S. Manicassamy: Investigation, writing-review and editing. P.D. Prasad: Investigation, visualization, writing-review and editing. V.B. Lokeshwar: Writing-review and editing. B.L. Lokeshwar: Writing-review and editing. V. Ganapathy: Writing-review and editing. M. Thangaraju: Conceptualization, resources, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing-original draft, project administration, writing-review and editing.

We thank Penny P. Roon, Donna Kumiski, and Dr. Brendan Marshall in the Electron Microscopy and Histology core facility, Augusta University Cancer Center, for their help in histologic analysis. This work was supported by Augusta University Intramural PSRP Pilot Award (IGPP00004), Start-up, and Bridge funding (to M. Thangaraju).

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.

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