Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction

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Downregulation of class II phosphoinositide 3-kinase PI3K-C2β delays cell division and potentiates the effect of docetaxel on cancer cell growth

Abstract Background Alteration of signalling pathways regulating cell cycle progression is a common feature of cancer cells. Several drugs targeting distinct phases of the cell cycle have been developed but the inability of many of them to discriminate between normal and cancer cells has strongly limited their clinical potential because of their reduced efficacy at the concentrations used to limit adverse side effects. Mechanisms of resistance have also been described, further affecting their efficacy. Identification of novel targets that can potentiate the effect of these drugs or overcome drug resistance can provide a useful strategy to exploit the anti-cancer properties of these agents to their fullest. Methods The class II PI3K isoform PI3K-C2β was downregulated in prostate cancer PC3 cells and cervical cancer HeLa cells using selective siRNAs and the effect on cell growth was determined in the absence or presence of the microtubule-stabilizing agent/anti-cancer drug docetaxel. Mitosis progression was monitored by time-lapse microscopy. Clonogenic assays were performed to determine the ability of PC3 and HeLa cells to form colonies upon PI3K-C2β downregulation in the absence or presence of docetaxel. Cell multi-nucleation was assessed by immunofluorescence. Tumour growth in vivo was assessed using a xenograft model of PC3 cells upon PI3K-C2β downregulation and in combination with docetaxel. Results Downregulation of PI3K-C2β delays mitosis progression in PC3 and HeLa cells, resulting in reduced ability to form colonies in clonogenic assays in vitro. Compared to control cells, PC3 cells lacking PI3K-C2β form smaller and more compact colonies in vitro and they form tumours more slowly in vivo in the first weeks after cells implant. Stable and transient PI3K-C2β downregulation potentiates the effect of low concentrations of docetaxel on cancer cell growth. Combination of PI3K-C2β downregulation and docetaxel almost completely prevents colonies formation in clonogenic assays in vitro and strongly inhibits tumour growth in vivo. Conclusions These data reveal a novel role for the class II PI3K PI3K-C2β during mitosis progression. Furthermore, data indicate that blockade of PI3K-C2β might represent a novel strategy to potentiate the effect of docetaxel on cancer cell growth. </jats:sec

Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease. Author summary Identifying the genetic basis of rare disorders can provide insight into gene function, susceptibility to disease, guide the development of new therapeutics, improve opportunities for genetic counseling, and help clinicians evaluate and potentially treat complicated clinical presentations. However, it is estimated that the genetic basis of approximately one-half of all rare genetic disorders remains unknown. We describe one such rare disorder based on genetic and clinical evaluations of individuals from 3 unrelated consanguineous families with a similar constellation of features including short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations including stroke, among other findings. We discovered that these features were due to deficiency of the PIK3C2A enzyme. PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of the lipids phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2 that are essential for a variety of cellular processes including cilia formation and vesicle trafficking. This syndrome is the first monogenic disorder caused by mutations in a class II PI3K family member and thus sheds new light on their role in human development

Downregulation of class II phosphoinositide 3-kinase PI3K-C2β delays cell division and potentiates the effect of docetaxel on cancer cell growth.

BACKGROUND: Alteration of signalling pathways regulating cell cycle progression is a common feature of cancer cells. Several drugs targeting distinct phases of the cell cycle have been developed but the inability of many of them to discriminate between normal and cancer cells has strongly limited their clinical potential because of their reduced efficacy at the concentrations used to limit adverse side effects. Mechanisms of resistance have also been described, further affecting their efficacy. Identification of novel targets that can potentiate the effect of these drugs or overcome drug resistance can provide a useful strategy to exploit the anti-cancer properties of these agents to their fullest. METHODS: The class II PI3K isoform PI3K-C2β was downregulated in prostate cancer PC3 cells and cervical cancer HeLa cells using selective siRNAs and the effect on cell growth was determined in the absence or presence of the microtubule-stabilizing agent/anti-cancer drug docetaxel. Mitosis progression was monitored by time-lapse microscopy. Clonogenic assays were performed to determine the ability of PC3 and HeLa cells to form colonies upon PI3K-C2β downregulation in the absence or presence of docetaxel. Cell multi-nucleation was assessed by immunofluorescence. Tumour growth in vivo was assessed using a xenograft model of PC3 cells upon PI3K-C2β downregulation and in combination with docetaxel. RESULTS: Downregulation of PI3K-C2β delays mitosis progression in PC3 and HeLa cells, resulting in reduced ability to form colonies in clonogenic assays in vitro. Compared to control cells, PC3 cells lacking PI3K-C2β form smaller and more compact colonies in vitro and they form tumours more slowly in vivo in the first weeks after cells implant. Stable and transient PI3K-C2β downregulation potentiates the effect of low concentrations of docetaxel on cancer cell growth. Combination of PI3K-C2β downregulation and docetaxel almost completely prevents colonies formation in clonogenic assays in vitro and strongly inhibits tumour growth in vivo. CONCLUSIONS: These data reveal a novel role for the class II PI3K PI3K-C2β during mitosis progression. Furthermore, data indicate that blockade of PI3K-C2β might represent a novel strategy to potentiate the effect of docetaxel on cancer cell growth.This work was supported by Prostate Cancer UK through a Project grant (grant PG13–029 to TM and MF). OC was supported by Prostate Cancer UK (grant PG13–029). IM was supported by MRC a MRC Doctoral Training Award to Barts and The London School of Medicine and Dentistry. FG is supported by Fondazione Italiana Ricerca Cancro (FIRC 19421). EH acknowledges funding from Associazione Italiana Ricerca Cancro (AIRC 21875)

Phenotypic characteristics of <i>PIK3C2A</i> deficient patients.

Phenotypic characteristics of PIK3C2A deficient patients.</p

PIK3C2A deficiency causes delayed proliferation rates in patient-derived fibroblasts.

Proliferation curve of primary fibroblasts isolated from PIK3C2A WT (+/+), heterozygous (+/-), and homozygous (-/-) individuals. Values are reported as the mean ± SEM from 3 independent experiments. * indicates p < 0.05, *** indicates p < 0.0001.</p

Protein and mRNA levels of PIK3C2A in patient-derived cells.

PIK3C2A mRNA levels were detected by qRT-PCR in patient derived fibroblasts from (A) Family I, (B) Family II, and (C) Family III. The WT sample in Fig 2A is based on n = 1 individual, the WT sample in Fig 2B is based on n = 3 individuals, and the WT sample in Fig 2C is based on n = 1 individual, totaling n = 5 different WT samples. qRT-PCR data is represented as mean ± SEM (n = 3–4 technical replicates per sample). (D, E) Whole cell lysates from fibroblasts of healthy controls (WT), heterozygous parents, and affected individuals from (D) Family III and (E) Families I and II were analyzed by Western blotting for PIK3C2A and the loading controls Actin or GAPDH. Immunogen of anti-PIK3C2A antibodies (AB1-AB4) are detailed in S5 Table. (F) Densitometry of Western blot results was performed using ImageJ. Individual samples are shown with the data combined from the four different PIK3C2A antibodies used, with the exception of the WT samples which includes fibroblasts from two individuals. * indicates p < 0.05. ** indicates p < 0.01. *** indicates p < 0.0001.</p

Ciliary defects due to PIK3C2A deficiency in patient-derived fibroblasts.

(A) Cilia length and (B) cilia number were determined in primary fibroblasts from two affected individuals and three unrelated controls. Data is represented as mean ± SEM (n>300/sample). Raw data for cilia length is provided in S6 Table. (C) Immunofluorescence analysis of RAB11 localization at the base of the primary cilium. Results showed that RAB11 is significantly reduced at the base of the primary cilium in -/- cells compared with +/+ and or +/- cells. n = 3 independent experiments and 15 cells per genotype for each experiment. (D) Immunofluorescence analysis of IFT88 localization within the primary cilia. Results showed that IFT88 is significantly increased along the primary cilium in -/- cells compared with +/+ and or +/- cells, suggesting a defective trafficking of ciliary components. n = 3 independent experiments and 15 cells per genotype for each experiment. Quantification of IFT88 and RAB11 were normalized on whole cell fluorescence. *** indicates p < 0.0001.</p

PIK3C2B levels are increased by PIK3C2A deficiency.

PIK3C2B mRNA levels were detected by qRT-PCR in (A) Family I, (B) Family II, and (C) Family III. qRT-PCR data is represented as mean ± SEM (n = 3 technical replicates per sample). The WT sample in Fig 6A is based on n = 1 and the WT sample in Fig 6B is based on n = 3, totaling n = 4 different WT samples. (D) PIK3C2B protein levels were detected by Western blotting in Family I from 2 independent experiments. (E) PIK3C2A and PIK3C2B protein levels were analyzed in HeLa cells by Western blotting following doxycycline inducible shRNA mediated knockdown of PIK3C2A. Data shown is representative from 3 independent experiments. * indicates p < 0.05, ** indicates p < 0.01. *** indicates p < 0.0001.</p