Tapas Saha

Redox Regulation in Cancer: A Double-edged Sword with Therapeutic Potential

Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of reactive oxygen species (ROS) and cell’s own antioxidant defenses. ROS deregulate the redox homeostasis and promote tumor formation by initiating an aberrant induction of signaling networks that cause tumorigenesis. Ultraviolet (UV) exposures, γ-radiation and other environmental carcinogens generate ROS in the cells, which can exert apoptosis in the tumors, thereby killing the malignant cells or induce the progression of the cancer growth by blocking cellular defense system. Cancer stem cells take the advantage of the aberrant redox system and spontaneously proliferate. Oxidative stress and gene-environment interactions play a significant role in the development of breast, prostate, pancreatic and colon cancer. Prolonged lifetime exposure to estrogen is associated with several kinds of DNA damage. Oxidative stress and estrogen receptor-associated proliferative changes are suggested to play important roles in estrogen-induced breast carcinogenesis. BRCA1, a tumor suppressor against hormone responsive cancers such as breast and prostate cancer, plays a significant role in inhibiting ROS and estrogen mediated DNA damage; thereby regulate the redox homeostasis of the cells. Several transcription factors and tumor suppressors are involved during stress response such as Nrf2, NFκB and BRCA1. A promising strategy for targeting redox status of the cells is to use readily available natural substances from vegetables, fruits, herbs and spices. Many of the phytochemicals have already been identified to have chemopreventive potential, capable of intervening in carcinogenesis.

Tgf-Beta signaling in development.

The transforming growth factor–β (TGF-β) superfamily comprises nearly 30 growth and differentiation factors that include TGF-βs, activins, inhibins, and bone morphogenetic proteins (BMPs). Multiple members of the TGF-β superfamily serve key roles in stem cell fate commitment. The various members of the family can exhibit disparate roles in regulating the biology of embryonic stem (ES) cells and tumor suppression. For example, TGF-β inhibits proliferation of multipotent hematopoietic progenitors, promotes lineage commitment of neural precursors, and suppresses epithelial tumors. BMPs block neural differentiation of mouse and human ES cells, contribute to self-renewal of mouse ES cells, and also suppress tumorigenesis. ES cells and tumors may be exposed to multiple TGF-β members, and it is likely that the combination of growth factors and cross-talk among the intracellular signaling pathways is what precisely defines stem cell fate commitment. This Connections Map Pathway in the Database of Cell Signaling integrates signaling not only from TGF-β and BMP but also from the ligands nodal and activin, and describes the role of the signaling pathways activated by these ligands in mammalian development. Much of the evidence for the connections shown comes from studies on mouse and human ES cells or mouse knockouts. This pathway is important for understanding not only stem cell biology, but also the molecular effectors of TGF-β and BMP signaling that may contribute to cancer suppression or progression and thus are potential targets for therapeutic intervention.

Transcriptional Regulation of the Base Excision Repair Pathway by BRCA1

Inactivation of the breast cancer susceptibility gene BRCA1 plays a significant role in the development of a subset of breast cancers, although the major tumor suppressor function of this gene remains unclear. Previously, we showed that BRCA1 induces antioxidant-response gene expression and protects cells against oxidative stress. We now report that BRCA1 stimulates the base excision repair pathway, a major mechanism for the repair of oxidized DNA, by stimulating the activity of key base excision repair (BER) enzymes, including 8-oxoguanine DNA glycosylase (OGG1), the DNA glycosylase NTH1, and the apurinic endonuclease redox factor 1/apurinic endonuclease 1 (REF1/APE1), in human breast carcinoma cells. The increase in BER enzyme activity appears to be due, primarily, to an increase in enzyme expression. The ability of BRCA1 to stimulate the expression of the three BER enzymes and to enhance NTH1 promoter activity was dependent upon the octamer-binding transcription factor OCT1. Finally, we found that OGG1, NTH1, and REF1/APE1 each contribute to the BRCA1 protection against oxidative stress due to hydrogen peroxide and that hydrogen peroxide stimulates the expression of BRCA1 and the three BER enzymes. These findings identify a novel mechanism through which BRCA1 may regulate the repair of oxidative DNA damage.

BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage

BRCA1, a tumor suppressor, participates in DNA damage signaling and repair. Previously, we showed that BRCA1 overexpression caused inhibition of telomerase activity and telomere shortening in breast and prostate cancer cells. We now report that BRCA1 knockdown causes increased telomerase reverse transcriptase expression, telomerase activity, and telomere length; but studies utilizing a combination of BRCA1 and telomerase reverse transcriptase small interfering RNAs suggest that BRCA1 also regulates telomere length independently of telomerase. Using telomeric chromatin immunoprecipitation assays, we detected BRCA1 at the telomere and demonstrated time-dependent loss of BRCA1 from the telomere following DNA damage. Further studies suggest that BRCA1 interacts with TRF1 and TRF2 in a DNA-dependent manner and that some of the nuclear BRCA1 colocalizes with TRF1/2. Our findings further suggest that Rad50 is required to localize BRCA1 at the telomere and that the association of BRCA1 with Rad50 does not require DNA. Finally, we found that BRCA1 regulates the length of the 3′ G-rich overhang in a manner that is dependent upon Rad50. Our findings suggest that BRCA1 is recruited to the telomere in a Rad50-dependent manner and that BRCA1 may regulate telomere length and stability, in part through its presence at the telomere.

BRCA1 down-regulates cellular levels of reactive oxygen species

Previous studies have shown that the breast cancer suppressor BRCA1 stimulates antioxidant gene expression and protects cells against oxidative stress. To further examine this important function, we tested whether BRCA1 could modulate intracellular levels of reactive oxygen species (ROS). Wild‐type BRCA1 (but not a cancer‐associated mutant) significantly reduced ROS levels, determined by DCF fluorescence assays by flow cytometry and confocal microscopy. The BRCA1 and REF1 pathways for reduction of ROS levels appear to exhibit cross‐talk. BRCA1 also reduced the levels of protein nitration and H2O2‐induced oxidative damage to DNA. Thus, BRCA1 may protect cellular macromolecules by reducing intracellular ROS levels.

Chemopreventive properties of indole-3-carbinol, diindolylmethane and other constituents of cardamom against carcinogenesis.

Oxidative stress results from an imbalance in the production of reactive oxygen species (ROS) and cells own antioxidant defenses that in part lead to numerous carcinogenesis. Several phytochemicals, derived from vegetables, fruits, herbs and spices, have demonstrated excellent chemopreventive properties against carcinogenesis by regulating the redox status of the cells during oxidative stress. I3C (indole-3-carbinol) and DIM (diindolylmethane) are the phytochemicals that are found in all types of cruciferous vegetables and demonstrated exceptional anti-cancer effects against hormone responsive cancers like breast, prostate and ovarian cancers. Novel analogs of I3C were designed to enhance the overall efficacy, particularly with respect to the therapeutic activity and oral bioavailability and that results in several patent applications on symptoms associated with endometriosis, vaginal neoplasia, cervical dysplasia and mastalgia. Likewise, DIM and its derivatives are patented for treatment and prevention of leiomyomas, HPV infection, respiratory syncytial virus, angiogenesis, atherosclerosis and anti-proliferative actions. On the other hand, phytochemicals in cardamom have not been explored in great details but limonene and cineole demonstrate promising effects against carcinogenesis. Thus studies with selected phytochemicals of cardamom and bioavailability research might lead to many patent applications. This review is focused on the patents generated on the effects of I3C, DIM and selected phytochemicals of cardamom on carcinogenesis.

LAMP2A overexpression in breast tumors promotes cancer cell survival via chaperone-mediated autophagy.

Lysosome-associated membrane protein type 2A (LAMP2A) is a key protein in the chaperone-mediated autophagy (CMA) pathway. LAMP2A helps in lysosomal uptake of modified and oxidatively damaged proteins directly into the lumen of lysosomes for degradation and protein turnover. Elevated expression of LAMP2A was observed in breast tumor tissues of all patients under investigation, suggesting a survival mechanism via CMA and LAMP2A. Reduced expression of the CMA substrates, GAPDH and PKM, was observed in most of the breast tumor tissues when compared with the normal adjacent tissues. Reactive oxygen species (ROS) mediated oxidative stress damages regulatory cellular components such as DNA, proteins and/or lipids. Protein carbonyl content (PCC) is widely used as a measure of total protein oxidation in cells. Ectopic expression of LAMP2A reduces PCC and thereby promotes cell survival during oxidative stress. Furthermore, inhibition of LAMP2A stimulates accumulation of GAPDH, AKT1 phosphorylation, generation of ROS, and induction of cellular apoptosis in breast cancer cells. Doxorubicin, which is a chemotherapeutic drug, often becomes ineffective against tumor cells with time due to chemotherapeutic resistance. Breast cancer cells deficient of LAMP2A demonstrate increased sensitivity to the drug. Thus, inhibiting CMA activity in breast tumor cells can be exploited as a potential therapeutic application in the treatment of breast cancer.

BRCA1 regulation of base excision repair pathway

This feature item briefly reviews the relation of the tumor suppressor function of BRCA1 with DNA damage and repair. The main focus of this article is on the regulation of base excision repair (BER) pathway by BRCA1 and in that context we introduce poly (ADP-ribose) polymerase 1 (PARP-1), which is a key enzyme in BER pathway, and its possible regulation by BRCA1.

BRCA1-mediated signaling pathways in ovarian carcinogenesis

The link between loss or defect in functional BRCA1 and predisposition for development of ovarian and breast cancer is well established. Germ-line mutations in BRCA1 are responsible for both hereditary breast and ovarian cancer, which is around 5–10% for all breast and 10–15% of all ovarian cancer cases. However, majority of cases of ovarian cancer are sporadic in nature. The inactivation of cellular BRCA1 due to mutations or loss of heterozygosity is one of the most commonly observed events in such cases. Complement-resistant retroviral BRCA1 vector, MFG-BRCA1, is the only approved gene therapy for ovarian cancer patients by the Federal and Drug Administration. Given the limited available information, there is a need to evaluate the effects of BRCA1 on the global gene expression pattern for better understanding the etiology of the disease. Here, we use Ingenuity Pathway Knowledge Base to examine the differential pattern of global gene expression due to stable expression of BRCA1 in the ovarian cancer cell line, SKOV3. The functional analysis detected at least five major pathways that were significantly (p < 0.05) altered. These include: cell to cell signaling and interaction, cellular function and maintenance, cellular growth and proliferation, cell cycle and DNA replication, and recombination repair. In addition, we were able to detect several biologically relevant genes that are central for various signaling networks involved in cellular homeostasis; TGF-β1, TP53, c-MYC, NF-κB and TNF-α. This report provides a comprehensive rationale for tumor suppressor function(s) of BRCA1 in ovarian carcinogenesis.

Central Role of Ubiquitination in Genome Maintenance: DNA Replication and Damage Repair

Faithful transmission of genetic information through generations ensures genomic stability and integrity. However, genetic alterations occur every now and then during the course of genome duplication. In order to repair these genetic defects and lesions, nature has devised several repair pathways which function promptly to prevent the cell from accumulating permanent mutations. These repair mechanisms seem to be significantly impacted by posttranslational modifications of proteins like phosphorylation and ubiquitination. Protein ubiquitination is emerging as a critical regulatory mechanism of DNA damage response. Non-proteolytic, proteasome-independent functions of ubiquitin involving monoubiquitination and polyubiquitination of DNA repair proteins contribute significantly to the signaling of DNA repair pathways. In this paper, we will particularly highlight the work on ubiquitin-mediated signaling in the repair processes involving the Fanconi anemia pathway, translesional synthesis, nucleotide excision repair, and repair of double-strand breaks. We will also discuss the role of ubiquitin ligases in regulating checkpoint mechanisms, the role of deubiquitinating enzymes, and the growing possibilities of therapeutic intervention in this ubiquitin-conjugation system.