Targeting proliferating cell nuclear antigen (PCNA) for cancer therapy.

Proliferating cell nuclear antigen (PCNA) is an essential scaffold protein in many cellular processes. It is best known for its role as a DNA sliding clamp and processivity factor during DNA replication, which has been extensively reviewed by others. However, the importance of PCNA extends beyond its DNA-associated functions in DNA replication, chromatin remodelling, DNA repair and DNA damage tolerance (DDT), as new non-canonical roles of PCNA in the cytosol have recently been identified. These include roles in the regulation of immune evasion, apoptosis, metabolism, and cellular signalling. The diverse roles of PCNA are largely mediated by its myriad protein interactions, and its centrality to cellular processes makes PCNA a valid therapeutic anticancer target. PCNA is expressed in all cells and plays an essential role in normal cellular homeostasis; therefore, the main challenge in targeting PCNA is to selectively kill cancer cells while avoiding unacceptable toxicity to healthy cells. This chapter focuses on the stress-related roles of PCNA, and how targeting these PCNA roles can be exploited in cancer therapy.

The bacterial DNA sliding clamp, ß-clamp: structure, interactions, dynamics and drug discovery.

DNA replication is a tightly coordinated event carried out by a multiprotein replication complex. An essential factor in the bacterial replication complex is the ring-shaped DNA sliding clamp, ß-clamp, ensuring processive DNA replication and DNA repair through tethering of polymerases and DNA repair proteins to DNA. ß -clamp is a hub protein with multiple interaction partners all binding through a conserved clamp binding sequence motif. Due to its central role as a DNA scaffold protein, ß-clamp is an interesting target for antimicrobial drugs, yet little effort has been put into understanding the functional interactions of ß-clamp. In this review, we scrutinize the ß-clamp structure and dynamics, examine how its interactions with a plethora of binding partners are regulated through short linear binding motifs and discuss how contexts play into selection. We describe the dynamic process of clamp loading onto DNA and cover the recent advances in drug development targeting ß-clamp. Despite decades of research in ß-clamps and recent landmark structural insight, much remains undisclosed fostering an increased focus on this very central protein.

PCNA regulates primary metabolism by scaffolding metabolic enzymes.

The essential roles of proliferating cell nuclear antigen (PCNA) as a scaffold protein in DNA replication and repair are well established, while its cytosolic roles are less explored. Two metabolic enzymes, alpha-enolase (ENO1) and 6-phosphogluconate dehydrogenase (6PGD), both contain PCNA interacting motifs. Mutation of the PCNA interacting motif APIM in ENO1 (F423A) impaired its binding to PCNA and resulted in reduced cellular levels of ENO1 protein, reduced growth rate, reduced glucose consumption, and reduced activation of AKT. Metabolome and signalome analysis reveal large consequences of impairing the direct interaction between PCNA and ENO1. Metabolites above ENO1 in glycolysis accumulated while lower glycolytic and TCA cycle metabolite pools decreased in the APIM-mutated cells; however, their overall energetic status were similar to parental cells. Treating haematological cancer cells or activated primary monocytes with a PCNA targeting peptide drug containing APIM (ATX-101) also lead to a metabolic shift characterized by reduced glycolytic rate. In addition, we show that ATX-101 treatments reduced the ENO1 - PCNA interaction, the ENO1, GAPDH and 6PGD protein levels, as well as the 6PGD activity. Here we report for the first time that PCNA acts as a scaffold for metabolic enzymes, and thereby act as a direct regulator of primary metabolism.

Targeting the non-canonical roles of PCNA modifies and increases the response to targeted anti-cancer therapy.

Receptor tyrosine kinases (RTKs), such as HER2 and/or EGFR are important therapeutic targets in multiple cancer cells. Low and/or short response to targeted therapies are often due to activation of compensatory signaling pathways, and therefore a combination of kinase inhibitors with other anti-cancer therapies have been proposed as promising strategies. PCNA is recently shown to have non-canonical cytosolic roles, and targeting PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM is shown to mediate changes in central signaling pathways such as PI3K/Akt and MAPK, acting downstream of multiple RTKs. In this study, we show how targeting PCNA increased the anti-cancer activity of EGFR/HER2/VEGFR inhibition in vitro as well as in vivo. The combination treatment resulted in reduced tumor load and increased the survival compared to either single agent treatments. The combination treatment affected multiple cellular signaling responses not seen by EGFR/HER2/VEGFR inhibition alone, and changes were seen in pathways determining protein degradation, ER-stress, apoptosis and autophagy. Our results suggest that targeting the non-canonical roles of PCNA in cellular signaling have the potential to improve targeted therapies.

Anti-Cancer Potential of Homemade Fresh Garlic Extract Is Related to Increased Endoplasmic Reticulum Stress.

The use of garlic and garlic-based extracts has been linked to decreased incidence of cancer in epidemiological studies. Here we examine the molecular and cellular activities of a simple homemade ethanol-based garlic extract (GE). We show that GE inhibits growth of several different cancer cells in vitro, as well as cancer growth in vivo in a syngeneic orthotopic breast cancer model. Multiple myeloma cells were found to be especially sensitive to GE. The GE was fractionated using solid-phase extractions, and we identified allicin in one GE fraction; however, growth inhibitory activities were found in several additional fractions. These activities were lost during freeze or vacuum drying, suggesting that the main anti-cancer compounds in GE are volatile. The anti-cancer activity was stable for more than six months in −20 °C. We found that GE enhanced the activities of chemotherapeutics, as well as MAPK and PI3K inhibitors. Furthermore, GE affected hundreds of proteins involved in cellular signalling, including changes in vital cell signalling cascades regulating proliferation, apoptosis, and the cellular redox balance. Our data indicate that the reduced proliferation of the cancer cells treated by GE is at least partly mediated by increased endoplasmic reticulum (ER) stress.

APIM-peptide targeting PCNA improves the efficacy of docetaxel treatment in the TRAMP mouse model of prostate cancer.

Docetaxel is the chemotherapeutic choice for metastatic hormone-refractory prostate cancer, however, it only marginally improves the survival rate. The purpose of the present study was to examine if a peptide targeting the cellular scaffold protein PCNA could improve docetaxel's efficacy. We found that docetaxel given in combination with a cell penetrating peptide containing the AlkB homolog 2 PCNA interacting motif (APIM-peptide), reduced the prostate volume and limited prostate cancer regrowth in vivo in the immunocompetent transgenic adenocarcinoma model of prostate cancer (TRAMP). In accordance with this, we found that the APIM-peptide enhanced the efficacy of docetaxel in vitro. Gene expression analysis on prostate cancer cell lines indicated that the combination of docetaxel and APIM-peptide alters expression of genes involved in cellular signaling, apoptosis, and prostate cancer development. These changes were not detected in single agent treated cells. Our results suggest that targeting PCNA and thereby affecting multiple cellular pathways simultaneously has the potential to improve docetaxel therapy of advanced prostate cancer.