Activity Of Bcl-2 Family Members Research Articles

Abstract SoA2-02: Implications of therapy induced senescence in breast cancer

Abstract The goal of breast cancer treatment is to cause cellular stress sufficient to kill and eliminate the tumor cells. Unfortunately, in many cases cancer cells survive and are not removed by surgery and thus can cause relapse and eventual mortality. Surviving cells have necessarily avoided both intrinsic (e.g., apoptosis) and extrinsic (e.g., immune surveillance) mechanisms of death. Studies over the last 20 years have shown that chemotherapy-treated breast cancer cells can evade cell death by activating programs of cellular senescence mediated by the tumor suppressor p53. While p53 can induce apoptosis in response to stress in some cell types, in breast cancer, p53 preferentially induces cell cycle arrest and senescence in response to chemotherapy. Data show that patients with breast cancers that are wild type for TP53 have a much lower probability of being eradicated by chemotherapy, and have a much shorter survival, compared to patients with TP53 mutant tumors. Human and mouse mammary tumors induce many markers of senescence after chemotherapy treatment, including those associated with cell cycle arrest and the senescence associated secretory phenotype (SASP). These arrested tumors evade death from mitotic catastrophe and can block apoptotic programs via elevated activity of BCL2 family members. More recent studies have addressed how induction of senescence deters immune surveillance. Breast cancer cells exposed to chemotherapy reorganize chromatin structure that amplifies stromal derived signals that stimulate expression of many immune genes, including checkpoint ligands such as PD-L1 and Galectin-9. Further, p53 can directly transactivate other immune modulatory genes such as CD80. Collectively, these properties of senescent cells enable their persistence following treatment and contribute to their potential to drive relapse. These same properties, however, can also be exploited as vulnerabilities to therapeutics such as senolytic drugs that target reliance on BCL2 family members or immunotherapies that target PD-L1 expression. Citation Format: J. Jackson. Implications of therapy induced senescence in breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr SoA2-02.

Yeast Bax Inhibitor (Bxi1p/Ybh3p) Is Not Required for the Action of Bcl-2 Family Proteins on Cell Viability.

Permeabilization of mitochondrial membrane by proteins of the BCL-2 family is a key decisive event in the induction of apoptosis in mammalian cells. Although yeast does not have homologs of the BCL-2 family, when these are expressed in yeast, they modulate the survival of cells in a way that corresponds to their activity in mammalian cells. The yeast gene, alternatively referred to as BXI1 or YBH3, encodes for membrane protein in the endoplasmic reticulum that was, contradictorily, shown to either inhibit Bax or to be required for Bax activity. We have tested the effect of the deletion of this gene on the pro-apoptotic activity of Bax and Bak and the anti-apoptotic activity of Bcl-XL and Bcl-2, as well on survival after treatment with inducers of regulated cell death in yeast, hydrogen peroxide and acetic acid. While deletion resulted in increased sensitivity to acetic acid, it did not affect the sensitivity to hydrogen peroxide nor to BCL-2 family members. Thus, our results do not support any model in which the activity of BCL-2 family members is directly affected by BXI1 but rather indicate that it may participate in modulating survival in response to some specific forms of stress.

Highlights of This Issue

In the current study, Williams and colleagues develop a proximity ligation assay (PLA) to measure the activity of Bcl-2 family members in situ. The team assesses Bcl-2 family inhibition in estrogen receptor-positive (ER+) breast cancers, finding poor response to ABT-263, a dual Bcl-2/Bcl-xL inhibitor, due to acute upregulation of a related factor, Mcl-1. Breast cancers from the clinic were determined to express higher transcript levels of the MCL1 gene, and were more frequently amplified, than other Bcl-2 members. Mcl-1 knockdown increases tumor cell death alone and in combination with ABT-263. Importantly, MCL1 transcript levels predict sensitivity to Bcl-2/Bcl-xL blockade, a timely finding as inhibitors of the Bcl-2 family enter clinical trials in breast cancer.Cyclin-dependent kinase 4/6 inhibitors (CDKi's) block tumor proliferation by mimicking p16INK4a function and are the subject of ongoing clinical trials. Similar to prolonged endogenous p16INK4a expression, extended CDK4/6i treatment promotes senescence in normal, proliferating cell lines. Senescence is associated with decreased regenerative capacity, age-related disease and cytokine secretion (i.e., SASP). Here, Guan and colleagues establish that primary fibroblasts, coaxed into senescence by prolonged CDK4/6i treatment, produce a robust cytokine profile/SASP that limits the anti-tumor immune response and promotes melanoma growth in immunocompetent models. These data reveal that the effects of CDK4/6i treatment on primary cells hinders therapeutic efficacy and promotes long-term, gerontogenic consequences.Mutations in the PIK3CA gene, activating phosphoinositide-3 kinase (PI3K), are commonly observed in colorectal cancer. Unfortunately, attempts at targeting this signaling cascade in colon cancer have resulted in significant therapeutic resistance. To investigate therapeutic strategies for this subtype, APC and PIK3CA mutant genetically engineered mice and spheroid cultures were generated. Using these model systems, dual PI3K/mTOR inhibition was discovered to induce treatment responses in spheroid cultures and in vivo. These results identify dual PI3K/mTOR inhibitors as a potential treatment strategy for patients with PIK3CA mutant colorectal cancer and deserve further clinical investigation.Previously we have shown that HER2+ breast cancers are susceptible to PARP inhibition (PARPi) independent of a DNA repair defect, and express elevated levels of PARP1. Now, Wielgos and colleagues discover that HER2 regulates PARP1 expression through suppression of the let-7a miRNA. Mechanistically, let-7a regulates PARP1 at the PARP1 3′-UTR. In human breast tumors, HER2 status correlates with high PARP1 protein and low let-7a. Lastly, introducing let-7a into HER2+ breast cancer cells induces cytotoxicity to a similar extent as the PARP inhibitor veliparib. Collectively, these findings demonstrate the potential of let-7a as a therapeutic strategy to target PARP1, and that baseline levels of let-7a and PARP1 are prospective biomarkers for PARPi sensitivity.

Lifeguard Inhibits Fas Ligand-mediated Endoplasmic Reticulum-Calcium Release Mandatory for Apoptosis in Type II Apoptotic Cells

Death receptors are members of the tumor necrosis factor receptor superfamily involved in the extrinsic apoptotic pathway. Lifeguard (LFG) is a death receptor antagonist mainly expressed in the nervous system that specifically blocks Fas ligand (FasL)-induced apoptosis. To investigate its mechanism of action, we studied its subcellular localization and its interaction with members of the Bcl-2 family proteins. We performed an analysis of LFG subcellular localization in murine cortical neurons and found that LFG localizes mainly to the ER and Golgi. We confirmed these results with subcellular fractionation experiments. Moreover, we show by co-immunoprecipitation experiments that LFG interacts with Bcl-XL and Bcl-2, but not with Bax or Bak, and this interaction likely occurs in the endoplasmic reticulum. We further investigated the relationship between LFG and Bcl-XL in the inhibition of apoptosis and found that LFG protects only type II apoptotic cells from FasL-induced death in a Bcl-XL dependent manner. The observation that LFG itself is not located in mitochondria raises the question as to whether LFG in the ER participates in FasL-induced death. Indeed, we investigated the degree of calcium mobilization after FasL stimulation and found that LFG inhibits calcium release from the ER, a process that correlates with LFG blockage of cytochrome c release to the cytosol and caspase activation. On the basis of our observations, we propose that there is a required step in the induction of type II apoptotic cell death that involves calcium mobilization from the ER and that this step is modulated by LFG.

Synergistic effects of p53 activation via MDM2 inhibition in combination with inhibition of Bcl-2 or Bcr-Abl in CD34+ proliferating and quiescent chronic myeloid leukemia blast crisis cells.

The Bcr-Abl tyrosine kinase regulates several Bcl-2 family proteins that confer resistance to apoptosis in chronic myeloid leukemia (CML) cells. Given p53's ability to modulate the expression and activity of Bcl-2 family members, we hypothesized that targeting Bcr-Abl, Bcl-2, and p53 concomitantly could have therapeutic benefits in blast crisis (BC) CML and in quiescent CML CD34+ cells that are insensitive to tyrosine kinase inhibitors (TKI). We examined the effects of the MDM2 inhibitor nutlin3a and its combination with the dual Bcl-2 and Bcl-xL inhibitor ABT-737, and the Bcr-Abl inhibitor nilotinib on BC CML patient samples. We found that in quiescent CD34+ progenitors, p53 expression is significantly lower, and MDM2 is higher, compared to their proliferating counterparts. Treatment with nutlin3a induced apoptosis in bulk and CD34+CD38- cells, and in both proliferating and quiescent CD34+ progenitor CML cells. Nutlin3a synergized with ABT-737 and nilotinib, in part by inducing pro-apoptotic, and suppressing anti-apoptotic, Bcl-2 proteins. Nilotinib inhibited the expression of Bcl-xL and Mcl-1 in BC CML cells. These results demonstrate that p53 activation by MDM2 blockade can sensitize BC CML cells, including quiescent CD34+ cells, to Bcl-2 inhibitor- and TKI-induced apoptosis. This novel strategy could be useful in the therapy of BC CML.

Promises of Apoptosis-Inducing Peptides in Cancer Therapeutics

Until recently, most research efforts aimed at developing anti-cancer tools were focusing on small molecules. Alternative compounds are now being increasingly assessed for their potential anti-cancer properties, including peptides and their derivatives. One earlier limitation to the use of peptides was their limited capacity to cross membranes but this limitation was alleviated with the characterization of cell-permeable sequences. Additionally, means are designed to target peptides to their malignant targets. Most anti-cancer peptidic compounds induce apoptosis of tumor cells by modulating the activity of Bcl-2 family members that control the release of death factors from the mitochondria or by inhibiting negative regulators of caspases, the proteases that mediate the apoptotic response in cells. Some of these peptides have been shown to inhibit the growth of tumors in mouse models. Hopefully, pro-apoptotic anti-tumor peptides will soon be tested for their efficacy in patients with cancers.

Methods to measure membrane potential and permeability transition in the mitochondria during apoptosis.

Mitochondrial membrane permeabilization (MMP) constitutes an early event of the apoptotic process. MMP affects both mitochondrial membranes. Inner MMP leads to the dissipation of the inner transmembrane potential and outer MMP culminates in the efflux of apoptogenic factors. The exact molecular mechanisms of MMP are still controversial. A growing body of data suggests that the cell death regulatory activity of Bcl-2 family members depends, at least in some instances, on their ability to modulate the opening of the mitochondrial permeability transition pore complex. Here, we will detail some experimental protocols designed to measure mitochondrial membrane potential and permeability transition, either in intact cells or in isolated mitochondria.