"Two hits - one stone"; increased efficacy of cisplatin-based therapies by targeting PCNA's role in both DNA repair and cellular signaling.

Low response rate and rapid development of resistance against commonly used chemotherapeutic regimes demand new multi-targeting anti-cancer strategies. In this study, we target the stress-related roles of the scaffold protein PCNA with a cell-penetrating peptide containing the PCNA-interacting motif APIM. The APIM-peptide increased the efficacy of cisplatin-based therapies in a muscle-invasive bladder cancer (MIBC) solid tumor model in rat and in bladder cancer (BC) cell lines. By combining multiple omics-levels, from gene expression to proteome/kinome and metabolome, we revealed a unique downregulation of the EGFR/ERBB2 and PI3K/Akt/mTOR pathways in the APIM-peptide-cisplatin combination treated cells. Additionally, the combination treatment reduced the expression of anti-apoptotic proteins and proteins involved in development of resistance to cisplatin. Concurrently, we observed increased levels of DNA breaks in combination treated cells, suggesting that the APIM-peptide impaired PCNA - DNA repair protein interactions and reduced the efficacy of repair. This was also seen in cisplatin-resistant cells, which notably was re-sensitized to cisplatin by the APIM-peptide. Our data indicate that the increased efficacy of cisplatin treatment is mediated both via downregulation of known oncogenic signaling pathways and inhibition of DNA repair/translesion synthesis (TLS), thus the APIM-peptide hits both nuclear and cytosolic functions of PCNA. The novel multi-targeting strategy of the APIM-peptide could potentially improve the efficacy of chemotherapeutic regiments for treatment of MIBC, and likely other solid tumors.

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.

Increased Anticancer Efficacy of Intravesical Mitomycin C Therapy when Combined with a PCNA Targeting Peptide.

Non-muscle-invasive bladder cancers (NMIBCs) are tumors confined to the mucosa or the mucosa/submucosa. An important challenge in treatment of NMIBC is both high recurrence and high progression rates. Consequently, more efficacious intravesical treatment regimes are in demand. Inhibition of the cell's DNA repair systems is a new promising strategy to improve cancer therapy, and proliferating cell nuclear antigen (PCNA) is a new promising target. PCNA is an essential scaffold protein in multiple cellular processes including DNA replication and repair. More than 200 proteins, many involved in stress responses, interact with PCNA through the AlkB homologue 2 PCNA-interacting motif (APIM), including several proteins directly or indirectly involved in repair of DNA interstrand crosslinks (ICLs). In this study, we targeted PCNA with a novel peptide drug containing the APIM sequence, ATX-101, to inhibit repair of the DNA damage introduced by the chemotherapeutics. A bladder cancer cell panel and two different orthotopic models of bladder cancer in rats, the AY-27 implantation model and the dietary BBN induction model, were applied. ATX-101 increased the anticancer efficacy of the ICL-inducing drug mitomycin C (MMC), as well as bleomycin and gemcitabine in all bladder cancer cell lines tested. Furthermore, we found that ATX-101 given intravesically in combination with MMC penetrated the bladder wall and further reduced the tumor growth in both the slow growing endogenously induced and the rapidly growing transplanted tumors. These results suggest that ATX-101 has the potential to improve the efficacy of current MMC treatment in NMIBC.

Targeting proliferating cell nuclear antigen and its protein interactions induces apoptosis in multiple myeloma cells.

Multiple myeloma is a hematological cancer that is considered incurable despite advances in treatment strategy during the last decade. Therapies targeting single pathways are unlikely to succeed due to the heterogeneous nature of the malignancy. Proliferating cell nuclear antigen (PCNA) is a multifunctional protein essential for DNA replication and repair that is often overexpressed in cancer cells. Many proteins involved in the cellular stress response interact with PCNA through the five amino acid sequence AlkB homologue 2 PCNA-interacting motif (APIM). Thus inhibiting PCNA's protein interactions may be a good strategy to target multiple pathways simultaneously. We initially found that overexpression of peptides containing the APIM sequence increases the sensitivity of cancer cells to contemporary therapeutics. Here we have designed a cell-penetrating APIM-containing peptide, ATX-101, that targets PCNA and show that it has anti-myeloma activity. We found that ATX-101 induced apoptosis in multiple myeloma cell lines and primary cancer cells, while bone marrow stromal cells and primary healthy lymphocytes were much less sensitive. ATX-101-induced apoptosis was caspase-dependent and cell cycle phase-independent. ATX-101 also increased multiple myeloma cells' sensitivity against melphalan, a DNA damaging agent commonly used for treatment of multiple myeloma. In a xenograft mouse model, ATX-101 was well tolerated and increased the anti-tumor activity of melphalan. Therefore, targeting PCNA by ATX-101 may be a novel strategy in multiple myeloma treatment.

Mutation screening in a Norwegian cohort with pheochromocytoma.

Pheochromocytomas (PHEOs) are neuroendocrine tumours, originating from chromaffin cells in the adrenal medulla. They are either sporadic or hereditary. It is important to identify the hereditary cases, so that patients and relatives with germline mutations can be offered regular surveillance. The objective of this study was the detection of pathogenic germline mutations in a cohort of Norwegian PHEO patients. Blood samples and/or formalin-fixed, paraffin-embedded tissue specimens, were collected from 60 patients who were operated upon between 1986 and 2004 at two university hospitals in Norway. DNA mutation analyses were performed successfully in the 42 blood samples and in one of the paraffin-embedded tissue specimen in VHL, RET, SDHB, SDHC, SDHD and NF1. In all, 32 different DNA variants were observed, of which 8 were classified as pathogenic (19 %), or possibly pathogenic; three in NF1, two in RET and VHL and one in SDHB. Two variants were observed in one patient, one in SDHB and one in NF1. Three of these variants are, to the best of our knowledge, new ones; two in NF1 [c.950_51insGCTGA, (p.Glu318LeufsX59) and c.1588G > A, (p.Val530Ile)] and one in VHL (c.308C > T, p.Pro103Leu). In conclusion the overall incidence of germline mutations in genes associated with familial PHEO was found to be of the same order of magnitude in the present Norwegian series as in those from other countries. Two new NF1 variants and one new VHL gene variant were detected.