Hitting the bull's eye: Novel directed cancer therapy through helicase‐targeted synthetic lethality

Designing strategies for anti‐cancer therapy have posed a significant challenge. One approach has been to inhibit specific DNA repair proteins and their respective pathways to enhance chemotherapy and radiation therapy used to treat cancer patients. Synthetic lethality represents an approach that exploits pre‐existing DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor‐associated repair deficiency. Since helicases play critical roles in the DNA damage response and DNA repair, particularly in actively dividing and replicating cells, it is proposed that the identification and characterization of synthetic lethal relationships of DNA helicases will be of value in developing improved anti‐cancer treatment strategies. In this review, we discuss this hypothesis and current evidence for synthetic lethal interactions of eukaryotic DNA helicases in model systems. J. Cell. Biochem. 106: 758–763, 2009. Published 2009 Wiley‐Liss, Inc.     

Cancer stem cells: A guide for skeptics

Cancer stem cells (CSC) were postulated to exist many years ago as cells within a tumor that regenerate the tumor following treatment. A stochastic clonal evolution model was used to explain observed tumor heterogeneity. Recently, xenotransplantation studies have demonstrated that prospectively identifiable subpopulations from human cancers can initiate tumors in immune deficient mice, and these results along with recent advances in stem cell biology have generated much excitement in the cancer field. The modern CSC theory posits a hierarchy of cells analogous to normal stem cell development. Some controversy remains, however, as to whether these tumor initiating cells truly represent CSC, and whether the modern CSC field can live up to the promise of providing improved cancer treatments based on a novel model of cancer biology. Recent data from CSC investigators are discussed critically. J. Cell. Biochem. 106: 745–749, 2009. © 2009 Wiley‐Liss, Inc.     

Intestinal epithelial cancer cell anoikis resistance: EGFR‐mediated sustained activation of Src overrides Fak‐dependent signaling to MEK/Erk and/or PI3‐K/Akt‐1

Herein, we investigated the survival roles of Fak, Src, MEK/Erk, and PI3‐K/Akt‐1 in intestinal epithelial cancer cells (HCT116, HT29, and T84), in comparison to undifferentiated and differentiated intestinal epithelial cells (IECs). We report that: (1) cancer cells display striking anoikis resistance, as opposed to undifferentiated/differentiated IECs; (2) under anoikis conditions and consequent Fak down‐activation, cancer cells nevertheless exhibit sustained Fak–Src interactions and Src/MEK/Erk activation, unlike undifferentiated/differentiated IECs; however, HCT116 and HT29 cells exhibit a PI3‐K/Akt‐1 down‐activation, as undifferentiated/differentiated IECs, whereas T84 cells do not; (3) cancer cells require MEK/Erk for survival, as differentiated (but not undifferentiated) IECs; however, T84 cells do not require Fak and HCT116 cells do not require PI3‐K/Akt‐1, in contrast to the other cells studied; (4) Src acts as a cornerstone in Fak‐mediated signaling to MEK/Erk and PI3‐K/Akt‐1 in T84 cells, as in undifferentiated IECs, whereas PI3‐K/Akt‐1 is Src‐independent in HCT116, HT29 cells, as in differentiated IECs; and (5) EGFR activity inhibition abrogates anoikis resistance in cancer cells through a loss of Fak–Src interactions and down‐activation of Src/MEK/Erk (T84, HCT116, HT29 cells) and PI3‐K/Akt‐1 (T84 cells). Hence, despite distinctions in signaling behavior not necessarily related to undifferentiated or differentiated IECs, intestinal epithelial cancer cells commonly display an EGFR‐mediated sustained activation of Src under anoikis conditions. Furthermore, such sustained Src activation confers anoikis resistance at least in part through a consequent sustenance of Fak–Src interactions and MEK/Erk activation, thus not only overriding Fak‐mediated signaling to MEK/Erk and/or PI3‐K/Akt‐1, but also the requirement of Fak and/or PI3‐K/Akt‐1 for survival. J. Cell. Biochem. 107: 639–654, 2009. © 2009 Wiley‐Liss, Inc.     

Osteoprotegerin production by breast cancer cells is suppressed by dexamethasone and confers resistance against TRAIL‐induced apoptosis

Osteoprotegerin (OPG) is a decoy receptor for receptor activator of NF‐κB ligand (RANKL) and TNF‐related apoptosis‐inducing ligand (TRAIL). While RANKL is essential for osteoclastogenesis and facilitates breast cancer migration into bone, TRAIL promotes breast cancer apoptosis. We analyzed the expression of OPG and TRAIL and its modulation in estrogen receptor‐positive MCF‐7 cells and receptor‐negative MDA‐MB‐231 cells. In both cells, OPG mRNA levels and protein secretion were dose‐ and time‐dependently enhanced by interleukin (IL)‐1β and suppressed by dexamethasone. In contrast to MCF‐7 cells, MDA‐MB‐231 abundantly expressed TRAIL mRNA, which was enhanced by IL‐1β and inhibited by dexamethasone. TRAIL activated pro‐apoptotic caspase‐3, ‐7, and poly‐ADP‐ribose polymerase and decreased cell numbers of MDA‐MB‐231, but had no effect on MCF‐7 cells. Gene silencing siRNA directed against OPG resulted in a 31% higher apoptotic rate compared to non‐target siRNA‐treated MDA‐MB‐231 cells. Furthermore, TRAIL induced significantly less apoptosis in cells cultured in conditioned media (containing OPG) compared to cells exposed to TRAIL in fresh medium lacking OPG (P < 0.01) and these protective effects were reversed by blocking OPG with its specific ligand RANKL (P < 0.05). The association between cancer cell survival and OPG production by MDA‐MB‐231 cells was further supported by the finding, that modulation of OPG secretion using IL‐1β or dexamethasone prior to TRAIL exposure resulted in decreased and increased rate of apoptosis, respectively (P < 0.05). Thus, OPG secretion by breast cancer cells is modulated by cytokines and dexamethasone, and may represent a critical resistance mechanism that protects against TRAIL‐induced apoptosis. J. Cell. Biochem. 108: 106–116, 2009. © 2009 Wiley‐Liss, Inc.     

9‐bromonoscapine‐induced mitotic arrest of cigarette smoke condensate‐transformed breast epithelial cells

In the present investigation, we determined the chemotherapeutic efficacy of 9‐bromonoscapine (Br‐Nos), a more potent noscapine analog, on MCF10A, spontaneously immortalized human normal breast epithelial cells and MCF10A‐CSC3, cigarette smoke condensate (CSC)‐transformed cells. The results from cytogenetic analysis showed that Br‐Nos induced polyploidy and telomeric association in MCF10A‐CSC3 cells, while MCF10A cells remained unaffected. Our immunofluorescence data further demonstrated that MCF10A‐CSC3 cells were susceptible to mitotic catastrophe on exposure to Br‐Nos and failed to recover after drug withdrawal. MCF10A‐CSC3 cells exhibited Br‐Nos‐induced aberrant multipolar spindle formation, which irreversibly impaired the alignment of replicated chromosome to the equatorial plane and finally culminated in cell death. Although MCF10A cells upon Br‐Nos treatment showed bipolar spindles with some uncongressed chromosomes, these cells recovered fairly well after drug withdrawal. Our flow‐cytometry analysis data reconfirmed that MCF10A‐CSC3 cells were more susceptible to cell death compared to MCF10A cells. Furthermore, our results suggest that decreased levels of cdc2/cyclin B1 and cdc2 kinase activity are responsible for Br‐Nos‐induced mitotic cell arrest leading to cell death in MCF10A‐CSC3 cells. This study thus explores the underlying mechanism of Br‐Nos‐induced mitotic catastrophe in CSC‐transformed MCF10A‐CSC3 cells and its potential usefulness as a chemotherapeutic agent for prevention of cigarette smoke‐induced breast cancer growth. J. Cell. Biochem. 106: 1146–1156, 2009. © 2009 Wiley‐Liss, Inc.     

Role of hypoxia in the hallmarks of human cancer

Hypoxia has been recognized as one of the fundamentally important features of solid tumors and plays a critical role in various cellular and physiologic events, including cell proliferation, survival, angiogenesis, immunosurveillance, metabolism, as well as tumor invasion and metastasis. These responses to hypoxia are at least partially orchestrated by activation of the hypoxia‐inducible factors (HIFs). HIF‐1 is a key regulator of the response of mammalian cells to oxygen deprivation and plays critical roles in the adaptation of tumor cells to a hypoxic microenvironment. Hypoxia and overexpression of HIF‐1 have been associated with radiation therapy and chemotherapy resistance, an increased risk of invasion and metastasis, and a poor clinical prognosis of solid tumors. The discovery of HIF‐1 signaling has led to a rapidly increasing understanding of the complex mechanisms involved in tumor hypoxia and has helped greatly in screening novel anticancer agents. In this review, we will first introduce the cellular responses to hypoxia and HIF‐1 signaling pathway in hypoxia, and then summarize the multifaceted role of hypoxia in the hallmarks of human cancers. J. Cell. Biochem. 107: 1053–1062, 2009. © 2009 Wiley‐Liss, Inc.     

Phenotype‐rescue of cyclin‐dependent kinase inhibitor p16/INK4A defects in a spontaneous canine cell model of breast cancer

Mammary cancer is among the most frequently observed canine tumors in unspayed female dogs resulting in death due to metastatic disease. These tumors are excellent models of human breast cancer but until recently there was only anecdotal evidence regarding underlying genetic defects. We recently reported expression defects in the cyclin‐dependent kinase p21/Cip1 and p53 among three independent canine mammary tumor (CMT) cell lines derived from spontaneous canine mammary cancers. We investigated further defects in the same three cell lines focusing on additional tumor suppressor gene defects in cyclin‐dependent kinase inhibitors. p27/KIP1 appeared normally expressed and did not appear to encode inactivating mutations. In contrast, expression of p16/INK4A was defective/absent in two cell lines and normal/slightly induced in the third cell line. To determine if defects were causative in maintaining the transformed phenotype, a p16/INK4A transgene was permanently transfected followed by selection and single cell cloning. CMT/p16 clones were characterized for transgene expression, p16 protein content and phenotype including proliferation rate, cell cycle phase distribution, contact inhibition, substrate dependent cell growth and cell morphology. All cell lines appeared unique yet clear indications of phenotype rescue due to p16/INK4A transgene complementation were observed suggesting that defects in p16 expression were present in all three. In some cases cellular senescence also appeared to be induced. These data provide evidence supporting p16/INK4A mutations as causative defects promoting transformation in canine mammary cancer and further characterizes tumor suppressor gene defects with functional consequences in these cells supporting their application as spontaneous animal models of human disease. J. Cell. Biochem. 106: 491–505, 2009. © 2009 Wiley‐Liss, Inc.     

Prostate cancer regulatory networks

Although the timing with which common epithelial malignancies arise and become established remains a matter of debate, it is clear that by the time they are detected these tumors harbor hundreds of deregulated, aberrantly expressed or mutated genes. This enormous complexity poses formidable challenges to identify gene pathways that are drivers of tumorigenesis, potentially suitable for therapeutic intervention. An alternative approach is to consider cancer pathways as interconnected networks, and search for potential nodal proteins capable of connecting multiple signaling networks of tumor maintenance. We have modeled this approach in advanced prostate cancer, a condition with current limited therapeutic options. We propose that the integration of three signaling networks, including chaperone‐mediated mitochondrial homeostasis, integrin‐dependent cell signaling, and Runx2‐regulated gene expression in the metastatic bone microenvironment plays a critical role in prostate cancer maintenance, and offers novel options for molecular therapy. J. Cell. Biochem. 107: 845–852, 2009. © 2009 Wiley‐Liss, Inc.     

Select cyclopentenone prostaglandins trigger glutathione efflux and the role of ABCG2 transport

Electrophilic cyclopentenone prostaglandins (cyPGs), such as 15-deoxy-Δ^12,14-prostaglandin J₂ (15dPGJ₂), initiate redox-based cell signaling responses including increased intracellular glutathione (GSH) synthesis. We investigated whether cyPGs facilitated GSH efflux and if members of the ATP-binding cassette (ABC) protein family mediated the efflux. Four human cell lines were treated with 1–6 μM cyPGs for 48 h. Media and cells were harvested for GSH measurements using HPLC-EC. CyPG treatment increased extracellular GSH levels two- to threefold over controls in HN4 and C38 cells and five- to sixfold in SAEC and MDA 1586 cells and was dependent on increased GSH synthesis. Our studies show that prostaglandin D₂ and its metabolites, prostaglandin J₂ and 15dPGJ₂, specifically induce GSH efflux compared to other eicosanoids. These higher extracellular GSH levels were associated with protection from tert-butylhydroperoxide. Superarray analysis of ABC transporters suggested only ABCG2 expression had a positive relationship in the four cell types compared with extracellular GSH increases after cyPG treatment. The ABCG2 substrate Hoechst 33342 inhibited extracellular GSH increase after 15dPGJ₂ treatment. We report for the first time that ABCG2 may play a role in GSH efflux in response to cyPG treatment and may link inflammatory signaling with antioxidant adaptive responses.