Proteomic screening reveals the PARylation landscape of base excision repair proteins after Beta‐Lapachone treatment in colorectal cancer cells

Abstract

Colorectal cancer (CRC) is a leading cause of cancer death and the third most common type of cancer worldwide. In early cases, surgery is the mainstay of treatment, but often patients are primarily diagnosed in an advanced stage of disease and may have distant metastases. Most solid tumors overexpress NAD(P)H;Quinone Oxidoreductase 1 (NQO1), which catalyzes a futile redox cycle of b‐lapachone during exposure, resulting in the formation of high levels of reactive oxygen species (ROS). Futile cycle b‐lapachone recycling produces elevated superoxide levels, resulting in massive levels of hydrogen peroxide that lead to substantial DNA damage and endoplasmic reticulum Ca2+ release. DNA damage and Ca+2 entry into the nucleus leads to the hyperactivation of poly(ADP‐ribose) polymerase 1 (PARP1). A majority of parylation is noted when hyperactivated PARP1 self‐parylates, forming extensive Poly(ADP) ribosylated PAR‐PARP1 levels are noted within 5 mins of exposure to 4 μM β‐lap in A549 cells, as noted by Western blotting using an antibody specific for PARylated moieties. PAR‐PARP1 is a protein post‐translational modification catalyzed by a family of enzymes known as PARPs. DNA single strand breaks along with base‐damage and abasic sites together with nuclear Ca2+ levels lead to hyperactivation of PARP1, a DNA repair enzyme that utilizes NAD+ to generate PAR moieties. This is followed by dramatic NAD+/ATP losses and tumor cells die of a special programmed necrosis process, referred to as NAD+‐keresis.PAR‐PARP1 is rapidly degraded by poly(ADP‐ribose) glycohydrolase (PARG) a exo‐ and endoglycosidase. PARG‐knockdown HCT116 cells were used to gain insight into the function of PARG and whether PARG depletion would enhance b‐lapachone induced cell death by blocking PAR recycling. Proteomic screening after b‐lapachone treatment in colorectal cancer cell line revealed the PARylation landscape of base excision, mismatch and nucleotide excision repair proteins. Twenty (20) of the proteins were found to overlap with H2O2 treatments (200 μM or 2 mM), while one protein (i.e., SPSF) was unique to b‐lapachone treatment. b‐Lapachone treatment affects BER pathways and qualitative analysis of the human Asp‐ and Glu‐ADP‐ribosylated proteome after b‐lapachone treatment indicate major alterations in BER pathways. We confirmed that b‐lapachone mediated cytotoxicity was independent of PARG knockdown or TOPO I or II protein losses. In contrast, RFC1 and PCNA siRNA‐mediated protein knock downs significantly enhanced b‐lapachone lethality, whereas knockdown of Topo I or Topo IIalpha did not affect b‐lapachone lethality. Taken together, these results demonstrated that knockdown of RFC1 enhances lethality of b‐lapachone and could serve as an effective treatment strategy in CRC. We are currently delineating the mechanism of enhanced lethality of β‐lapachone by loss of RFC1.Support or Funding InformationThis work was funded by NIH/NCI 5 R01 CA102792‐16 to DAB.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.