Blagden, SP

Buluwela, L

Chettle, J

Fischer, R

Kessler, B

Maghami, SG

Mura, M

Stavraka, C

Thezenas, M-L

Cancer Research

Spiral - Imperial College Digital Repository

Protein	interactions	involving	LARP1	in	chemotherapy	resistant	ovarian	cancer	cells	1*	Chara	Stavraka,	1*	Manuela	Mura,	2	Roman	Fischer,	2	Marie-Laetitia	Thezenas,	1	Sadaf-Ghaem	Maghami,	3James	Chettle,	1	Laki	Buluwela,		2	Benedikt	Kessler,3	Sarah	P.	Blagden	1	Department	of	Surgery	and	Cancer,	Imperial	College	London,	UK	2	Target	Discovery	Institute,	NDMRB,	University	of	Oxford,	UK	3	Department	of	Oncology,	University	of	Oxford,	UK	Ovarian	 cancer	 (OC)	 is	 the	 most	 lethal	 gynecological	 malignancy	 accounting	 for	 152,000	 deaths	annually	 worldwide.	 Due	 to	 the	 lack	 of	 a	 validated	 diagnostic	 tool,	 patients	 usually	 present	 with	disseminated	 disease	 and	 the	 majority	 will	 develop	 resistance	 to	 platinum	 chemotherapy.	Resensitizng	 OC	 to	 cisplatin-based	 chemotherapy	 is	 an	 unmet	 clinical	 need.	 	 The	 RNA-binding	protein	 LARP1	 is	highly	expressed	 in	OC	and	 is	 a	post-transcriptional	 regulator	of	 cell	 survival	 and	tumorigenesis.	 LARP1	 depletion	 using	 RNA	 interference	 (RNAi)	 restores	 platinum	 sensitivity	 in	cisplatin-resistant	 OC	 cell	 lines	 and	 shows	 a	 synergistic	 anti-tumour	 effect	 with	 cisplatin.	 	 We	explored	the	effect	of	 this	resensitization	on	the	efficiency	of	protein	translation	using	the	Surface	Sensing	 of	 Translation	 (SUnSET)	 method.	 Whilst	 cisplatin-induced	 genotoxic	 stress	 attenuated	 de	novo	 protein	 synthesis	 in	 platinum	 sensitive	 (OVCAR3)	 cells,	 this	 was	 not	 observed	 in	 platinum-resistant	 OVCAR8	 cells	 where	 residual	 protein	 synthesis	 was	 maintained.	 However	 this	 residual	translation	 was	 completely	 abolished	 after	 RNAi	 to	 LARP1	 suggesting	 that,	 in	 the	 OVCAR8	 cells,	LARP1	maintains	protein	synthesis	during	genotoxic	stress.	Although	LARP1	is	known	to	bind	Poly	A	binding	protein	 (PABP)	we	questioned	whether	other	 interacting	partners	might	also	 regulate	 this	response.	 Using	 immunoprecipitation	 followed	 by	 ultra-high	 performance	 liquid	 chromatography	tandem	mass	spectrometry	(UHPLC-MS/MS)	in	OVCAR3	and	OVCAR8	cells	before	and	after	cisplatin	treatment,	we	 identified	 PABPC1,	 PABPC4	 and	 YB-1	 as	 strong	 interactors	with	 LARP1.	 By	western	blotting,	 Duolink	 Proximity	 Ligation	 Assay	 and	 Immunofluorescence	 we	 then	 confirmed	 these	candidates	as	being	complexed	with	LARP1.		We	are	currently	elucidating	the	functional	role	of	these	interactions	 on	 the	 LARP1-mediated	 genotoxic	 stress	 response	with	 the	 overall	 aim	 of	 identifying	therapeutic	strategies	to	overcome	platinum-resistance	in	ovarian	cancer.	

Protein interactions involving LARP1 in chemotherapy resistant ovarian cancer cells

Protein interactions involving LARP1 in chemotherapy resistant ovarian cancer cells

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