Cancer-associated mesothelial cells promote ovarian cancer chemoresistance through paracrine osteopontin signaling

Ovarian cancer is the leading cause of gynecological malignancy-related deaths, due to its widespread intraperitoneal metastases and acquired chemoresistance. Mesothelial cells are an important cellular component of the ovarian cancer microenvironment that promote metastasis. However, their role in chemoresistance is unclear. Here, we investigated whether cancer-associated mesothelial cells promote ovarian cancer chemoresistance and stemness in vitro and in vivo. We found that osteopontin is a key secreted factor that drives mesothelial-mediated ovarian cancer chemoresistance and stemness. Osteopontin is a secreted glycoprotein that is clinically associated with poor prognosis and chemoresistance in ovarian cancer. Mechanistically, ovarian cancer cells induced osteopontin expression and secretion by mesothelial cells through TGF-β signaling. Osteopontin facilitated ovarian cancer cell chemoresistance via the activation of the CD44 receptor, PI3K/AKT signaling, and ABC drug efflux transporter activity. Importantly, therapeutic inhibition of osteopontin markedly improved the efficacy of cisplatin in both human and mouse ovarian tumor xenografts. Collectively, our results highlight mesothelial cells as a key driver of ovarian cancer chemoresistance and suggest that therapeutic targeting of osteopontin may be an effective strategy for enhancing platinum sensitivity in ovarian cancer

Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice

Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer

ATM kinase activity restricts DNA-PK localization and activation at SV40 Tag foci.

<p>(A, B) Micrographs of chromatin-bound NHEJ factors at 48 hpi from SV40-infected BSC40 cells treated with DMSO, ATRi, or ATMi from 40–48 hpi. Scale bars represent 10 µm. Fluorescence signals along the line in the merged imaged are graphed in the right panel. (C–E) DNA-PK_cs pS2056 (C), DNA-PK_cs (D), or KU80 (E) fluorescence intensities at a minimum of 70 (C) or 100 (D, E) SV40 DNA replication centers from images described in (A) and (B). The average and median are shown with dashed and solid lines, respectively. The boxes encompass the 25th–75th quartiles of intensities. Minimum and maximum intensities are portrayed by the whiskers.</p

ATM or ATR inhibition increases broken replication forks and linear viral DNA replication products.

<p>(A) Diagram of 2D gel electrophoresis of undigested circular dsDNA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004536#ppat.1004536-Lucas1" target="_blank">[86]</a>. (B, C, D, E) Southern blots of the first dimension of a neutral 1D gel (top panel) or 2D gel (middle panel) from SV40-infected BSC40 cells exposed to DMSO (B), ATRi (C), ATMi (D), or ATRi and ATMi (E) during the last 28 h of a 48 h SV40 infection. Arrow on 1D gel points to the location of the ∼20 kb replication product. Bottom panel: Enlargement of the picture within the boxed area in middle panel. Exposure of the bottom panel was increased to enhance visualization of θ and σ replication intermediates shown in (A). Arrow in lower panels points to location of the σ arc.</p

ATR and DNA-PK_cs prevent viral genome concatemer formation when ATM is inhibited.

<p>(A) Southern blot of DNA extracted from SV40-infected BSC40 cells treated with combinations of DMSO, ATRi, ATMi, and/or Nu7026 during the final 28 h of a 48 h infection. The middle panel shows a longer exposure of a portion of the Southern blot pictured in the top panel. To emphasize SV40 replication intermediates and aberrant products, equal amounts of total SV40 DNA were loaded into each lane. (B) Quantification of monomer accumulation when PIKK(s) are inhibited from Southern blots as shown in (A). (C) Graph of total viral or SV40 monomer DNA signals normalized to SV40 DNA replicated in the presence of DMSO from Southern blots as shown in (A). (D) Graph of aberrant structure(s) accumulated as a result of single or multiple PIKK inhibition from Southern blots as shown in (A). In (B–D), bars for DMSO, ATRi, and ATMi show the average of 6 to 7 independent experiments. In the same panels, the bar for Nu-7026 shows the average of 4 independent experiments; whereas bars for combinations of inhibitors (ATMi/ATRi, Nu7026/ATRi, and ATMi/Nu7026) show the average of 3 independent experiments.</p

Model of the role of DNA double strand break repair during SV40 DNA replication.

<p>(A) Tag translocates toward a nick in the DNA (orange dashed arrow), unwinding the helix. (B) Helicase activity moves past the nick and generates a one-sided DSB that recruits MRN and KU to the DSB termini. Wild-type conditions (blue arrow): (C) MRN binding recruits and activates ATM at the DSB. ATM phosphorylates CtIP to create a stable interaction with NBS1 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004536#ppat.1004536-Wang1" target="_blank">[59]</a> at the DSB. ATM-phosphorylated MRN-CtIP catalyzes the initial 5′ to 3′ end resection of the DSB resulting in loss of KU. Further phosphorylation of BLM, DNA2, and EXO1 by ATM facilitates mobilization of each protein to broken DNA and more robust end resection leading to the loss of KU at viral DNA. (D) The single-stranded DNA generated by end resection is bound by RPA. Replacement of RPA on resected 3′ tail with RAD51 facilitates HDR, and the DSB is repaired to yield two intact unit length viral genomes at the end of the replication cycle (E). ATM inhibited conditions (red arrow): (F) MRN binding recruits ATM to DSB termini. However, failure of ATM to phosphorylate and create binding sites for CtIP, BLM/DNA2, or EXO1 causes stable KU binding to blunt or short single-stranded DNA at the DSB termini. Bound KU recruits DNA-PK_cs to the DSB. DNA-PK_cs kinase activity is activated to commence NHEJ. The DSB is not repaired efficiently by NHEJ resulting in rolling circle replication and concatemer formation (G).</p

Localization of 5′ to 3′ DNA end resection proteins is promoted by ATM kinase.

<p>(A, C) Representative images at 48 hpi of ATRi-treated, ATMi-treated, or un-inhibited SV40-infected BSC40 cells probed for chromatin-bound Nbs1 and Tag (A) or CtIP, BLM, and Tag (C). DMSO, ATRi, or ATMi was present in the media from 40–48 hpi. Scale bars represent 10 µm. In the right panel, the fluorescence signals along the line in the merged image are graphed. (B, D, E) Nbs1 (B), CtIP (D), or BLM (E) fluorescence intensity signals at greater than or equal to 100 SV40 DNA replication centers from micrographs described in (A) and (C), respectively. The average and median are denoted by the dashed and solid lines, respectively. The 25th–75th quartiles of intensities are enclosed within the boxes. Minimum and maximum intensities are indicated by the whiskers.</p

ATM or ATR inhibition does not affect incorporation of nucleotides into SV40 DNA.

<p>(A) Southern blot of DNA extracted from SV40-infected BSC40 cells treated with DMSO, ATRi, or ATMi from 40 to 48 hpi and labeled with 20 µM EdU for 30 minutes prior to DNA extraction. Left Panel: phosphorimager scan of Southern blot probed for mitochondrial or SV40 DNA. Right Panel: same Southern blot as in left panel but probed for EdU with an anti-BrdU antibody. (B, C) Western blots of cells lysates from SV40-infected BSC40 cells treated with DMSO, ATRi, or ATMi during the final 8 h of a 48 h SV40 infection. (D) Immunofluorescence microscopy of the indicated factors from SV40- or mock-infected BSC40 cells treated with DMSO, ATRi, or ATMi during the final 8 h of a 48 h SV40 infection. Prior to fixation at 48 hpi, cells were pulsed with 20 µM EdU for 5 minutes and non-chromatin bound proteins were extracted from cells. The fluorescence intensity along the line shown in the merged image is graphed in the right panel. Scale bars represent 10 µm. (E, F) Fluorescence intensity signals of Rad51 (E) or EdU (F) at a minimum of 100 SV40 DNA replication centers from micrographs as in (D). Dashed and solid lines within boxes represent the average and median, respectively. Boxes contain the 25th–75th quartiles of intensities, whereas whiskers show minimum and maximum intensities.</p

SV40 DNA replication centers colocalize with HDR proteins.

<p>(A–C) Images of chromatin-bound Tag and the indicated HDR factors from SV40-infected BSC40 (A, C) or U2OS (B) cells at 48 hours post infection. Vectors for expression of HA-topoisomerase IIIα or Flag-RMI2 were transfected 24 h prior to infection. In (C), 20 µM EdU was present in the media of SV40-infected BSC40 cells during the final 5 minutes prior to fixation. Scale bar in all images represents 10 µm.</p

Abdominal FLASH irradiation reduces radiation-induced gastrointestinal toxicity for the treatment of ovarian cancer in mice.

Radiation therapy is the most effective cytotoxic therapy for localized tumors. However, normal tissue toxicity limits the radiation dose and the curative potential of radiation therapy when treating larger target volumes. In particular, the highly radiosensitive intestine limits the use of radiation for patients with intra-abdominal tumors. In metastatic ovarian cancer, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of abdominal metastases. However, TAI fell out of favor due to high toxicity of the intestine. Here we utilized an innovative preclinical irradiation platform to compare the safety and efficacy of TAI ultra-high dose rate FLASH irradiation to conventional dose rate (CONV) irradiation in mice. We demonstrate that single high dose TAI-FLASH produced less mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and spared cell death in crypt base columnar cells compared to TAI-CONV irradiation. Importantly, TAI-FLASH and TAI-CONV irradiation had similar efficacy in reducing tumor burden while improving intestinal function in a preclinical model of ovarian cancer metastasis. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of abdominal radiotherapy, with potential application to metastatic ovarian cancer