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Loss of INPP4B causes a DNA repair defect through loss of BRCA1, ATM and ATR and can be targeted with PARP inhibitor treatment.

Ip LR, Poulogiannis G, Viciano FC, Sasaki J, Kofuji S, Spanswick VJ, Hochhauser D, Hartley JA, Sasaki T, Gewinner CA - Oncotarget (2015)

Bottom Line: The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma.INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models.Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, UCL Cancer Institute, University College London, London, UK.

ABSTRACT
Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.

No MeSH data available.


Related in: MedlinePlus

INPP4B loss in human ovarian cancer cells results in a DNA repair defectA. Stable Ovca429 knockdown cell pools were irradiated (30 Gy) and comet assays conducted. Tail moments were measured 0 min, 15 min, 30 min, 45 min, 60 min and 90 min post-treatment. Representative pictures are shown. B. Ovca429 knockdown cell pools were fixed 0min, 30 min, 60 min and 180 min after etoposide treatment and γH2AX foci quantified. Representative pictures are shown. C. Ovca429 knockdown cell pools were fixed 6 h, 12 h or 24 h after etoposide treatment and RAD51 foci quantified. INPP4B knockdown cell pools displayed higher percentage of nuclei containing > 15 foci per nucleus compared to the control cells. Representative pictures are shown.
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Figure 2: INPP4B loss in human ovarian cancer cells results in a DNA repair defectA. Stable Ovca429 knockdown cell pools were irradiated (30 Gy) and comet assays conducted. Tail moments were measured 0 min, 15 min, 30 min, 45 min, 60 min and 90 min post-treatment. Representative pictures are shown. B. Ovca429 knockdown cell pools were fixed 0min, 30 min, 60 min and 180 min after etoposide treatment and γH2AX foci quantified. Representative pictures are shown. C. Ovca429 knockdown cell pools were fixed 6 h, 12 h or 24 h after etoposide treatment and RAD51 foci quantified. INPP4B knockdown cell pools displayed higher percentage of nuclei containing > 15 foci per nucleus compared to the control cells. Representative pictures are shown.

Mentions: Based on our findings that loss of INPP4B resembles features of BRCA1-negative tumors, we hypothesized that INPP4B deficiency may lead to defects in DNA repair. Stable knockdown cell pools of the human ovarian cancer cell lines Ovca429 and Ovca433 expressing shRNA hairpins directed against Renilla luciferase, INPP4B, PTEN and BRCA1 were generated. Cell pools over clones were chosen to best reflect heterogeneity of protein loss in the tumor environment. On average, a 50% knockdown efficiency for INPP4B was achieved (Supplemental Figure 1B). To investigate DNA repair efficiency single cell gel electrophoresis assays (Comet assays) under alkaline conditions were conducted (Figure 2A). Ovca429 knockdown cell pools were lysed 0, 15, 30, 45, 60 and 90 min after x-ray irradiation and the intensity and length of comet tails relative to head (tail moment) were analyzed after cell electrophoresis. Ovca429 shRNA-INPP4B cell pools revealed significantly increased tail moment at early time points of DNA repair compared to Renilla luciferase knockdown controls pointing to decreased DNA repair. At later time points (t = 45 min and t = 60 min) INPP4B knockdown cell pools continued to display a higher level of residual strand breaks. In contrast, we observed no abberation in olive tail moment in Ovca429 shRNA-PTEN expressing cell pools, contrary to the DNA repair deficiency associated with PTEN loss reported elsewhere (Supplemental Figure 4A) [20, 21].


Loss of INPP4B causes a DNA repair defect through loss of BRCA1, ATM and ATR and can be targeted with PARP inhibitor treatment.

Ip LR, Poulogiannis G, Viciano FC, Sasaki J, Kofuji S, Spanswick VJ, Hochhauser D, Hartley JA, Sasaki T, Gewinner CA - Oncotarget (2015)

INPP4B loss in human ovarian cancer cells results in a DNA repair defectA. Stable Ovca429 knockdown cell pools were irradiated (30 Gy) and comet assays conducted. Tail moments were measured 0 min, 15 min, 30 min, 45 min, 60 min and 90 min post-treatment. Representative pictures are shown. B. Ovca429 knockdown cell pools were fixed 0min, 30 min, 60 min and 180 min after etoposide treatment and γH2AX foci quantified. Representative pictures are shown. C. Ovca429 knockdown cell pools were fixed 6 h, 12 h or 24 h after etoposide treatment and RAD51 foci quantified. INPP4B knockdown cell pools displayed higher percentage of nuclei containing > 15 foci per nucleus compared to the control cells. Representative pictures are shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496374&req=5

Figure 2: INPP4B loss in human ovarian cancer cells results in a DNA repair defectA. Stable Ovca429 knockdown cell pools were irradiated (30 Gy) and comet assays conducted. Tail moments were measured 0 min, 15 min, 30 min, 45 min, 60 min and 90 min post-treatment. Representative pictures are shown. B. Ovca429 knockdown cell pools were fixed 0min, 30 min, 60 min and 180 min after etoposide treatment and γH2AX foci quantified. Representative pictures are shown. C. Ovca429 knockdown cell pools were fixed 6 h, 12 h or 24 h after etoposide treatment and RAD51 foci quantified. INPP4B knockdown cell pools displayed higher percentage of nuclei containing > 15 foci per nucleus compared to the control cells. Representative pictures are shown.
Mentions: Based on our findings that loss of INPP4B resembles features of BRCA1-negative tumors, we hypothesized that INPP4B deficiency may lead to defects in DNA repair. Stable knockdown cell pools of the human ovarian cancer cell lines Ovca429 and Ovca433 expressing shRNA hairpins directed against Renilla luciferase, INPP4B, PTEN and BRCA1 were generated. Cell pools over clones were chosen to best reflect heterogeneity of protein loss in the tumor environment. On average, a 50% knockdown efficiency for INPP4B was achieved (Supplemental Figure 1B). To investigate DNA repair efficiency single cell gel electrophoresis assays (Comet assays) under alkaline conditions were conducted (Figure 2A). Ovca429 knockdown cell pools were lysed 0, 15, 30, 45, 60 and 90 min after x-ray irradiation and the intensity and length of comet tails relative to head (tail moment) were analyzed after cell electrophoresis. Ovca429 shRNA-INPP4B cell pools revealed significantly increased tail moment at early time points of DNA repair compared to Renilla luciferase knockdown controls pointing to decreased DNA repair. At later time points (t = 45 min and t = 60 min) INPP4B knockdown cell pools continued to display a higher level of residual strand breaks. In contrast, we observed no abberation in olive tail moment in Ovca429 shRNA-PTEN expressing cell pools, contrary to the DNA repair deficiency associated with PTEN loss reported elsewhere (Supplemental Figure 4A) [20, 21].

Bottom Line: The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma.INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models.Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Biology, UCL Cancer Institute, University College London, London, UK.

ABSTRACT
Treatment options for ovarian cancer patients remain limited and overall survival is less than 50% despite recent clinical advances. The lipid phosphatase inositol polyphosphate 4-phosphatase type II (INPP4B) has been described as a tumor suppressor in the PI3K/Akt pathway with loss of expression found most pronounced in breast, ovarian cancer and melanoma. Using microarray technology we identified a DNA repair defect in INPP4B-deficient cells, which we further characterized by comet assays and quantification of γH2AX, RAD51 and 53BP1 foci formation. INPP4B loss resulted in significantly increased sensitivity towards PARP inhibition, comparable to loss of BRCA1 in two- and three-dimensional in vitro models, as well as in in vivo xenograft models. Mechanistically, we discovered that INPP4B forms a protein complex with the key players of DNA repair, ATR and BRCA1, in GST pulldown and 293T overexpression assays, and INPP4B loss affects BRCA1, ATM and ATR protein stability resulting in the observed DNA repair defect. Given that INPP4B loss has been found in 40% of ovarian cancer patients, this study provides the rationale for establishing INPP4B as a biomarker of PARP inhibitor response, and consequently offers novel therapeutic options for a significant subset of patients. Loss of the tumor suppressor inositol polyphosphate 4-phosphatase type II (INPP4B) results in a DNA repair defect due to concomitant loss of BRCA1, ATR and ATM and can be therapeutically targeted with PARP inhibitors.

No MeSH data available.


Related in: MedlinePlus