Identification of novel molecular regulators of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in breast cancer cells by RNAi screening.

INTRODUCTION: Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors, TRAIL-receptor 1 (TRAIL-R1) and TRAIL-receptor 2 (TRAIL-R2), leading to apoptosis by activation of caspase-8 and the downstream executioner caspases, caspase-3 and caspase-7 (caspase-3/7). Triple-negative breast cancer (TNBC) cell lines with a mesenchymal phenotype are sensitive to TRAIL, whereas other breast cancer cell lines are resistant. The underlying mechanisms that control TRAIL sensitivity in breast cancer cells are not well understood. Here, we performed small interfering RNA (siRNA) screens to identify molecular regulators of the TRAIL pathway in breast cancer cells. METHODS: We conducted siRNA screens of the human kinome (691 genes), phosphatome (320 genes), and about 300 additional genes in the mesenchymal TNBC cell line MB231. Forty-eight hours after transfection of siRNA, parallel screens measuring caspase-8 activity, caspase-3/7 activity, or cell viability were conducted in the absence or presence of TRAIL for each siRNA, relative to a negative control siRNA (siNeg). A subset of genes was screened in cell lines representing epithelial TNBC (MB468), HER2-amplified breast cancer (SKBR3), and estrogen receptor-positive breast cancer (T47D). Selected putative negative regulators of the TRAIL pathway were studied by using small-molecule inhibitors. RESULTS: The primary screens in MB231 identified 150 genes, including 83 kinases, 4 phosphatases, and 63 nonkinases, as potential negative regulators of TRAIL. The identified genes are involved in many critical cell processes, including apoptosis, growth factor-receptor signaling, cell-cycle regulation, transcriptional regulation, and DNA repair. Gene-network analysis identified four genes (PDPK1, IKBKB, SRC, and BCL2L1) that formed key nodes within the interaction network of negative regulators. A secondary screen of a subset of the genes identified in additional cell lines representing different breast cancer subtypes and sensitivities to TRAIL validated and extended these findings. Further, we confirmed that small-molecule inhibition of SRC or BCL2L1, in combination with TRAIL, sensitizes breast cancer cells to TRAIL-induced apoptosis, including cell lines resistant to TRAIL-induced cytotoxicity. CONCLUSIONS: These data identify novel molecular regulators of TRAIL-induced apoptosis in breast cancer cells and suggest strategies for the enhanced application of TRAIL as a therapy for breast cancer.

Role of specific CDKs in regulating DNA damage repair responses and replication stress.

Cyclins along with their catalytic units, Cyclin-dependent kinases (CDKs) regulate the cell cycle transition and transcription; and are essentially known as 'master regulators' in modulating DNA damage response (DDR) and replication stress. In addition to influencing DNA repair and damage signaling, CDKs also play a pivotal role in cell division fidelity and the maintenance of genomic integrity after DNA damage. In this review, we focus on the intricate ways by which specific CDKs mainly CDK7, CDK9, and CDK12/13, regulate the cell cycle progression and transcription and how their modulation can lead to lethal effects on the integrity of the genome. With a better knowledge of how these CDKs control the DDR and replication stress, it is now possible to combine CDK inhibitors with chemotherapeutic drugs that damage DNA in ways that can be applied in clinical settings as successful therapeutic strategies.

Nano-TRAIL: a promising path to cancer therapy.

Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand, also called apo-2 ligand (TRAIL/Apo-2L), is a cytokine that triggers apoptosis by binding to TRAIL-R1 (DR4) and TRAIL-R2 (DR5) death receptors. Apoptosis occurs through either the extrinsic or intrinsic pathway. The administration of recombinant human TRAIL (rhTRAIL) or TRAIL-receptor (TRAIL-R) agonists promotes apoptosis preferentially in cancerous cells over normal cells in vitro; this phenomenon has also been observed in clinical studies. The limited efficacy of rhTRAIL in clinical trials could be attributed to drug resistance, short half-life, targeted delivery issues, and off-target toxicities. Nanoparticles are excellent drug and gene delivery systems characterized by improved permeability and retention, increased stability and biocompatibility, and precision targeting. In this review, we discuss resistance mechanisms to TRAIL and methods to overcome TRAIL resistance by using nanoparticle-based formulations developed for the delivery of TRAIL peptides, TRAIL-R agonists, and TRAIL genes to cancer cells. We also discuss combinatorial approaches of chemotherapeutic drugs with TRAIL. These studies demonstrate TRAIL's potential as an anticancer agent.

Mitochondria in Cancer Stem Cells: From an Innocent Bystander to a Central Player in Therapy Resistance.

Cancer continues to rank among the world's leading causes of mortality despite advancements in treatment. Cancer stem cells, which can self-renew, are present in low abundance and contribute significantly to tumor recurrence, tumorigenicity, and drug resistance to various therapies. The drug resistance observed in cancer stem cells is attributed to several factors, such as cellular quiescence, dormancy, elevated aldehyde dehydrogenase activity, apoptosis evasion mechanisms, high expression of drug efflux pumps, protective vascular niche, enhanced DNA damage response, scavenging of reactive oxygen species, hypoxic stability, and stemness-related signaling pathways. Multiple studies have shown that mitochondria play a pivotal role in conferring drug resistance to cancer stem cells, through mitochondrial biogenesis, metabolism, and dynamics. A better understanding of how mitochondria contribute to tumorigenesis, heterogeneity, and drug resistance could lead to the development of innovative cancer treatments.

Identification of WEE1 as a potential molecular target in cancer cells by RNAi screening of the human tyrosine kinome.

Breast cancers can be classified into those that express the estrogen (ER) and progesterone (PR) receptors, those with ERBB2 (HER-2/Neu) amplification, and those without expression of ER, PR, or amplification of ERBB2 (referred to as triple-negative or basal-like breast cancer). In order to identify potential molecular targets in breast cancer, we performed a synthetic siRNA-mediated RNAi screen of the human tyrosine kinome. A primary RNAi screen conducted in the triple-negative/basal-like breast cancer cell line MDA-MB231 followed by secondary RNAi screens and further studies in this cell line and two additional triple-negative/basal-like breast cancer cell lines, BT20 and HCC1937, identified the G2/M checkpoint protein, WEE1, as a potential therapeutic target. Similar sensitivity to WEE1 inhibition was observed in cell lines from all subtypes of breast cancer. RNAi-mediated silencing or small compound inhibition of WEE1 in breast cancer cell lines resulted in an increase in gammaH2AX levels, arrest in the S-phase of the cell cycle, and a significant decrease in cell proliferation. WEE1-inhibited cells underwent apoptosis as demonstrated by positive Annexin V staining, increased sub-G1 DNA content, apoptotic morphology, caspase activation, and rescue by the pan-caspase inhibitor, Z-VAD-FMK. In contrast, the non-transformed mammary epithelial cell line, MCF10A, did not exhibit any of these downstream effects following WEE1 silencing or inhibition. These results identify WEE1 as a potential molecular target in breast cancer.

WEE1 inhibition sensitizes basal breast cancer cells to TRAIL-induced apoptosis.

TRAIL is a member of the TNF super family and has been shown to induce apoptosis in many cancer cell lines but not in normal cells. Breast cancers can be divided into different subgroups on the basis of the expression of estrogen and progesterone receptors, HER-2 amplification, or the lack of these three markers (known as triple-negative or basal-type breast cancer). Our group and others have shown previously that triple-negative breast cancer cell lines are sensitive to TRAIL whereas others are relatively resistant. In an earlier study, we reported that inhibition of WEE1, a cell-cycle checkpoint regulator, causes increased cell death in breast cancer cell lines. In this study, we tested the effects of WEE1 inhibition on TRAIL-mediated apoptosis in breast cancer cell lines. Pretreatment with WEE1 inhibitor or knockdown of WEE1 increased the toxicity of TRAIL in the basal/triple-negative breast cancer cell lines compared with WEE1 inhibitor or TRAIL treatment alone. The enhanced cell death is attributed to increased surface expression of death receptors, increased caspase activation which could be blocked by the pan-caspase inhibitor, Z-VAD-FMK, thereby rescuing cells from caspase-mediated apoptosis. The cell death was initiated primarily by caspase-8 because knockdown of caspase-8 and not of any other initiator caspases (i.e., caspase-2, -9, or -10) rescued cells from WEE1 inhibitor-sensitized TRAIL-induced cell death. Taken together, the data suggest that the combination of WEE1 inhibitor and TRAIL could provide a novel combination for the treatment of basal/triple-negative breast cancer.