The Cancer/Testes (CT) Antigen HORMAD1 promotes Homologous Recombinational DNA Repair and Radioresistance in Lung adenocarcinoma cells

The Cancer/Testes (CT) Antigen HORMAD1 is germ cell-restricted and plays developmental roles in generation and processing of meiotic DNA Double Strand Breaks (DSB). Many tumors aberrantly overexpress HORMAD1 yet the potential impact of this CT antigen on cancer biology is unclear. We tested a potential role of HORMAD1 in genome maintenance in lung adenocarcinoma cells. We show that HORMAD1 re-distributes to nuclear foci and co-localizes with the DSB marker γH2AX in response to ionizing radiation (IR) and chemotherapeutic agents. The HORMA domain and C-term disordered oligomerization motif are necessary for localization of HORMAD1 to IR-induced foci (IRIF). HORMAD1-depleted cells are sensitive to IR and camptothecin. In reporter assays, Homologous Recombination (HR)-mediated repair of targeted ISce1-induced DSBs is attenuated in HORMAD1-depleted cells. In Non-Homologous End Joining (NHEJ) reporter assays, HORMAD1-depletion does not affect repair of ISce1-induced DSB. Early DSB signaling events (including ATM phosphorylation and formation of γH2AX, 53BP1 and NBS1 foci) are intact in HORMAD1-depleted cells. However, generation of RPA-ssDNA foci and redistribution of RAD51 to DSB are compromised in HORMAD1-depleted cells, suggesting that HORMAD1 promotes DSB resection. HORMAD1-mediated HR is a neomorphic activity that is independent of its meiotic partners (including HORMAD2 and CCDC36. Bioinformatic analysis of TCGA data show that similar to known HR pathway genes HORMAD1 is overexpressed in lung adenocarcinomas. Overexpression of HR genes is associated with specific mutational profiles (including copy number variation). Taken together, we identify HORMAD1-dependent DSB repair as a new mechanism of radioresistance and a probable determinant of mutability in lung adenocarcinoma.


Supplementary Figures
Supplementary Figure S1 (accompanies Fig. 1) Defining the effect of DNA damage on subcellular distribution of HORMAD1.
(a) Imaging analysis to empirically measure degree of co-localization between HORMAD1 and known DNA damage markers. The fluorescence intensities of HORMAD1 and H2AX signals were plotted using the Carl Zeiss ZEN software. The Pearson's correlation coefficient (PCC) for HORMAD1 localization in relation to H2AX was calculated using IMARIS microscopy image analysis software. A PCC value of 1 represents perfect co-localization and a PCC value of -1 represents complete mutual exclusivity. Therefore, the PCC values indicate co-localization of H2AX with HORMAD1 but not with the other CTAs tested. The histogram shows the mean PCC values obtained for CTA/H2AX colocalization obtained in three independent analyses and the error bars represent the range.
(b) Effect of ATMi on co-localization between HORMAD1 and 53BP1. HA-HORMAD1 was expressed in H1299 cells using a recombinant adenovirus. 24 h post-infection, some cultures were treated with 10 M KU55933 for 1 h. Control and KU55933-treated cells were conditionally irradiated (10 Gy) and 1 h later the subcellular distribution of HA-HORMAD1 in relation to 53BP1 was analyzed by confocal microscopy and PCCs were determined using IMARIS microscopy image analysis software. The PCC values indicate that the co-localization of HORMAD1 and 53BP1 (PCC=0.67) is impaired following ATM inhibition (PCC=0.14).
(c) HORMAD1 redistributes to IRIF in cancer cell lines. HA-HORMAD1 was expressed in replicate cultures of A549 lung adenocarcinoma and U2OS osteosarcoma cells using a recombinant adenovirus. 24 h post-infection, some cultures were irradiated (10 Gy) and 1 h later the subcellular distribution of HA-HORMAD1 in relation to H2AX was analyzed by confocal microscopy.  Fig. 3. (b) Multiple HORMAD1 siRNAs attenuate HR activity in lung adenocarcinoma cells. Replicate plates of H1299 cells harboring the stably-integrated DR-GFP reporter construct were transfected with the indicated siRNAs (against HORMAD1, BRCA1, or non-targeting siRNA). 24 h later the siRNA-treated cells were transfected with an ISceI expression plasmid (to induce DSB in the DR-GFP locus) or with an empty control vector. After 24 h cells were trypsinized and GFP-expressing populations (resulting from HR-mediated reconstitution of a silent GFP allele) were enumerated by flow cytometry. Error bars indicate the standard error of the mean from three independent experiments. The immunoblot shows relative of HORMAD1 expression in replicate plates of siRNA-transfected cells.

Supplementary
(c) Replicate plates of A549 cells harboring stably-integrated DR-GFP were transfected with the indicated siRNAs and tested for repair of ISceI-induced breaks as described for (A). Error bars indicate the standard error of the mean from three independent experiments. Figure S4 (to accompany Fig. 4) HORMAD1 facilitates HR but not impact NHEJ (a) HORMAD1 sustains chromatin-bound CtIP in cancer cell lines. Lung adenocarcinoma (H1299, A549, H358) and breast cancer (MDA-MB436) cell lines were transfected with siHORMAD1 or nontargeting control RNA (siCon). 48 h post-transfection cells were treated with IR (10 Gy) to induce DSB. Chromatin-bound proteins were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies.

Supplementary
(b) HORMAD1 sustains RPA foci in A549 cells. A549 cells were transfected with siHORMAD1, siCtIP, or with non-targeting siCon RNA. 48 later cells were irradiated (10 Gy) and 2 h later nuclei were fixed and analyzed for RPA foci using immunofluorescence confocal microscopy. The percentage of cells showing RPA IRIF are quantified.
(c) HORMAD1-depletion does not affect redistribution of NBS to IRIF. H1299 cells were transfected with siCon, siBRCA1 or siHORMAD1 RNAs. 48 h post-transfection cells were irradiated (10 Gy) and 1 h later nuclei were analyzed for H2AX and NBS1 foci using immunofluorescence confocal microscopy. 100 nuclei were analyzed for each experimental condition. The results of analyses are shown on the histogram and error bars indicate the standard deviation from three different experiments.