Genetic aberrations that render cells incapable of executing apoptosis not only promote tumorigenesis, but also contribute to the observed resistance of human cancers to diverse anticancer agents. Unraveling mechanisms to unleash the apoptotic program in tumors that harbor such genetic changes could lead to effective therapeutic interventions against such malignancies.
Based on these insights, she is developing therapeutic strategies that are designed to activate death-receptor signaling in conjunction with a blockade of key molecular determinants of tumor-cell survival. These studies provide a foundation for collaborative translational projects aimed against a broad range of cancers. The frequent inactivation of the p53 tumor-suppressor gene in diverse human cancers contributes to their resistance to death signals triggered by aberrant cell-cycle progression, hypoxia, or DNA damage. Dr. Ravi's studies demonstrated that cells which are rendered resistant to chemotherapeutic agents or ionizing radiation by virtue of loss of p53 remain susceptible to induction of apoptosis by engagement of death receptors of the tumor necrosis factor receptor superfamily, such as CD95/Fas or death receptors for Apo2L/TRAIL (tumor necrosis factor-related apoptosis-inducing ligand). These findings demonstrated that death receptors and chemotherapeutic agents induce independent, yet synergistic, death-signaling pathways that converge at the mitochondrial activation of the apoptosome.
These insights provide a rationale for the development of Apo2L/TRAIL-based combination regimens for the treatment of p53-deficient cancers. Dr. Ravi discovered that death receptor-transduced signals can also overcome the protection conferred by overexpression of the Bcl-2 gene. She demonstrated that death receptor-induced caspase-mediated proteolytic cleavage of the loop domain of Bcl-2 inactivates its survival function. The carboxyl-terminal Bcl-2 cleavage product behaves as a BAX-like death effector and potentiates apoptosis.
These observations defined the precise molecular mechanism(s) by which death receptor-transduced signals destroy the survival function of Bcl-2 and provide a rationale for the use of death ligands, such as Apo2L/TRAIL, against Bcl-2-overexpressing tumor cells. Dr. Ravi's studies have provided important insights into the key molecular determinants of death receptor-induced apoptosis. Ligand-dependent oligomerization of death receptors results in activation of caspase-8, a member of a family of cysteine aspartate proteases that execute cell death. Caspase-8-induced cleavage of BID, a "BH-3 domain only" prodeath member of the Bcl-2 family, generates an active truncated form of BID (tBID), which in turn triggers the mitochondrial activation of caspase-9. Dr. Ravi demonstrated that tBID-mediated activation of caspase-9 in tumor cells in response to Apo2L/TRAIL requires BAX, a multidomain pro-apoptotic member of the Bcl-2 family. Her findings indicate that tumor cells are rendered profoundly resistant to Apo2L/TRAIL-induced death by inactivation of the BAX gene. Dr. Ravi's studies have provided critical insights into the regulation of death receptor-induced apoptosis by the NF-kappaB family of transcription factors.
Dr. Ravi demonstrated that that NF-kappaB protects cells from Apo2L/TRAIL-mediated apoptosis by inducing expression of survival factors, such as Bcl-xL, a Bcl-2 homolog that sequesters tBID and counteracts the activation of BAX or BAK. While activation of NF-kappaB inhibits death receptor-signaling, Dr. Ravi demonstrated that death receptors promote caspase-mediated cleavage of the RelA subunit of NF-kappaB. The truncation of the transactivation domain generates a transcriptionally inactive dominant-negative fragment of RelA that serves as an efficient pro-apoptotic feedback mechanism between caspase activation and NF-kappaB inactivation. Dr. Ravi's observations suggest that cell fate is determined by a dynamic balance between death receptor-transduced signals and NF-kappaB-induced survival proteins that counteract their ability to disrupt mitochondrial homeostasis and activate caspases.
Her findings indicate that the constitutive activation of NF-kappaB by diverse growth factors, cytokines, or genetic aberrations may protect tumor cells from Apo2L/TRAIL; conversely, such tumors may be sensitized to Apo2L/TRAIL by agents that inhibit NF-kappaB. Dr. Ravi's studies have demonstrated that tumor cells can be sensitized to Apo2L/TRAIL-induced apoptosis by the synergistic action of Interferon-?/? and NF-kappaB inhibition. Although Apo2L/TRAIL triggers apoptosis of cancer cells, including those that have lost or inactivated p53, it fails to activate caspase-9 or induce apoptosis in tumor cells that are deficient in BAX, a pro-apoptotic gene that is frequently mutated in cancers of the microsatellite mutator phenotype. However, she has found that BAX-deficient cancer cells can be sensitized to Apo2L/TRAIL-induced apoptosis by interferon or induced expression of caspase-8, BAK, and caspase-7.
Her studies show that interferon synergized with the inhibition of NF-kappaB-dependent expression of survival proteins (Bcl-xL and XIAP) to further augment Apo2L/TRAIL-induced death of p53+/+- or p53-/- BAX-proficient, as well as BAX-deficient, isogenic cancer cells. These results indicate that the combination of Apo2L/TRAIL with interferon and NF-kappaB inhibitors may be an effective regimen against both p53-deficient and BAX-deficient cancers. A majority of human cancers have lost or inactivated the p53 tumor-suppressor gene, a key determinant of cellular responses to DNA damage inflicted by chemotherapeutic agents. Using isogenic cancer cells that differ only in their p53 status, Dr. Ravi demonstrated that loss of p53 impairs DNA damage-induced expression of the pro-apoptotic proteins PUMA and Noxa and renders cancer cells relatively resistant to the topoisomerase I inhibitor irinotecan. However, p53-deficiency does not prevent irinotecan-induced expression of interferon regulatory factor-1 (IRF-1), an interferon-responsive transcription factor that sensitizes cancer cells to death receptor-mediated apoptosis. Irinotecan synergizes with the death-receptor ligand Apo2L ligand/tumor necrosis factor-related apoptosis-inducing ligand (Apo2L/TRAIL) to promote apoptosis of cancer cells independently of p53.
While xenografts of p53-deficient cancer cells are relatively resistant to irinotecan alone, combined treatment with irinotecan and Apo2L/TRAIL eliminates hepatic metastasis of both p53-proficient and p53-deficient cancer cells in vivo and markedly improves animal survival. These findings identify IRF-1 as a channel for p53-independent cross-talk between DNA damage- and death receptor-signaling and suggest that the addition of Apo2L/TRAIL can improve the therapeutic index of chemotherapeutic agents against p53-deficient cancers. Studies of how cancers evade elimination by the immune system have primarily focused on mechanisms by which tumor cells avoid recognition by the immune system.
Current efforts to improve immunotherapy rely on approaches to break antigen-specific tolerance and amplify immune responses to tumor antigens. While these endeavors may allow activation of immune responses against tumors, their ability to eliminate cancers remains contingent on the induction of tumor cell apoptosis by immune effector cells. Cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells induce apoptosis of their targets by two major mechanisms that operate through activation of a family of cysteine aspartate proteases (caspases). One mechanism involves engagement of death receptors on target cells by ligands expressed on CTLs or NK cells. The second mechanism by which CTLs and NK cells trigger apoptosis involves calcium-dependent exocytosis of granule proteins, perforin, and granzymes.
Both death receptors and granzyme B employ BID-BAX/BAK-dependent mitochondrial permeabilization to induce cell death. Dr. Ravi demonstrated that specific genetic alterations (inactivation of BAX or overexpression of Bcl-xL) that interfere with different points of this shared signaling pathway can protect tumor cells from immune effector cells in vitro and in vivo.
However, the relative resistance of such tumor cells may be overcome at high effector:target ratios via interferon-mediated potentiation of death-receptor signaling and by the granule-exocytosis pathway. Genetic impediments to death-signaling pathways used by immune effectors may be an important mechanism by which tumor cells could evade immune surveillance and immunotherapy.