Dara Ditsworth

Activation of poly(ADP)-ribose polymerase (PARP-1) induces release of the pro-inflammatory mediator HMGB1 from the nucleus.

Necrotic cells release inflammatory mediators that activate cytokine production from innate immune cells. One mediator of this activation is high mobility group box 1 protein (HMGB1). HMGB1 is normally a chromatin-associated protein and is sequestered at condensed chromatin during apoptosis. How it is released from chromatin during necrotic cell death is not known. Here we show that after DNA-alkylating damage, the activation of poly(ADP)-ribose polymerase (PARP) regulates the translocation of HMGB1 from the nucleus to the cytosol. This displaced HMGB1 is subject to release if the cell then loses plasma membrane integrity as a result of necrosis. Both full-length HMGB1 and a truncated form of HMGB1 lacking the highly conserved glutamate-rich C-terminal tail can induce macrophage activation and tumor necrosis factor-α production. However, displacement of HMGB1 from the nucleus following PARP activation requires the presence of the glutamate-rich C-terminal tail. Although the C-terminal tail is not the sole substrate for PARP modification of HMGB1, it appears to be required to destabilize HMGB1 association with chromatin following PARP-dependent chromatin modifications. These data suggest that PARP-dependent nuclear-to-cytosolic translocation of HMGB1 serves to establish the ability of cells to release this potent inflammatory mediator upon subsequent necrotic death.

α4 Is an Essential Regulator of PP2A Phosphatase Activity

The activity and specificity of serine/threonine phosphatases are governed largely by their associated proteins. alpha4 is an evolutionarily conserved noncatalytic subunit for PP2A-like phosphatases. Though alpha4 binds to only a minority of PP2A-related catalytic subunits, alpha4 deletion leads to progressive loss of all PP2A, PP4, and PP6 phosphatase complexes. In healthy cells, association with alpha4 renders catalytic (C) subunits enzymatically inactive while protecting them from proteasomal degradation until they are assembled into a functional phosphatase complex. During cellular stress, existing PP2A complexes can become unstable. Under such conditions, alpha4 sequesters released C subunits and is required for the adaptive increase in targeted PP2A activity that can dephosphorylate stress-induced phosphorylated substrates. Consistent with this, overexpression of alpha4 protects cells from a variety of stress stimuli, including DNA damage and nutrient limitation. These findings demonstrate that alpha4 plays a required role in regulating the assembly and maintenance of adaptive PP2A phosphatase complexes.

DNA Alkylating Therapy Induces Tumor Regression through an HMGB1-Mediated Activation of Innate Immunity

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. We have previously shown in tumor xenografts that DNA alkylating agents induce sporadic cell necrosis and regression of apoptosis-deficient tumors. Sporadic tumor cell necrosis is associated with extracellular release of cellular content such as the high mobility group box 1 (HMGB1) protein and subsequent recruitment of innate immune cells into the tumor tissue. It remained unclear whether HMGB1 and the activation of innate immunity played a role in tumor response to chemotherapy. In this study, we show that whereas DNA alkylating therapy leads to a complete tumor regression in an athymic mouse tumor xenograft model, it fails to do so in tumors deficient in HMGB1. The HMGB1-deficient tumors have an impaired ability to recruit innate immune cells including macrophages, neutrophils, and NK cells into the treated tumor tissue. Cytokine array analysis reveals that whereas DNA alkylating treatment leads to suppression of protumor cytokines such as IL-4, IL-10, and IL-13, loss of HMGB1 leads to elevated levels of these cytokines upon treatment. Suppression of innate immunity and HMGB1 using depleting Abs leads to a failure in tumor regression. Taken together, these results indicate that HMGB1 plays an essential role in activation of innate immunity and tumor clearance in response to DNA alkylating agents.

Chemotherapy Induces Tumor Clearance Independent of Apoptosis

Dysregulation of apoptosis is associated with the development of human cancer and resistance to anticancer therapy. The ultimate goal of cancer treatment is to selectively induce cancer cell death and overcome drug resistance. A deeper understanding of how a given chemotherapy affects tumor cell death is needed to develop strategically designed anticancer agents. Here, we use a xenograft mouse tumor system generated from genetically defined cells deficient in apoptosis to examine the involvement of multiple forms of cell death induced by cyclophosphamide (CP), a DNA alkylating agent commonly used in chemotherapy. We find that although apoptosis facilitates tumor regression, it is dispensable for complete tumor regression as other forms of cell death are activated. Sporadic necrosis is observed in both apoptosis-competent and deficient tumors evident by tumor cell morphology, extracellular release of high mobility group box 1 protein, and activation of innate immune cells in CP-treated tumors. Our findings indicate that in apoptosis-deficient tumors, necrosis may play a fundamental role in tumor clearance by stimulating the innate immune response.

The PP2A-associated Protein α4 Plays a Critical Role in the Regulation of Cell Spreading and Migration

Compared with kinases, the role of protein phosphatases in regulating biological functions is less well understood. Here we show that α4, a non-catalytic subunit of the protein phosphatase 2A, plays a major role in the control of cell spreading, migration, and cytoskeletal architecture. Fibroblasts lacking α4 were impaired in their ability to spread and migrate compared with wild-type cells, whereas enforced expression of α4 promoted cell spreading and migration. These effects were not restricted to fibroblasts. Using a T cell-specific α4 transgenic mouse model, increased α4 expression was found to increase lymphocyte motility and chemotaxis. Elevated α4 expression results in an increase in the GTP-bound state of Rac1, and GTP-bound Rac1 was dramatically reduced in α4-deficient cells. A constitutively active mutant of Rac1 rescued the defects of cell spreading and migration caused by α4 deletion, while inhibition of Rac1 blocked the ability of α4 to promote cell migration. Together, these data define a novel role for the protein phosphatase 2A regulatory subunit α4 in the regulation of cell spreading and migration.

Non-apoptotic routes to defeat cancer

The mechanism of tumor cell death after treatment with DNA alkylating agents in vivo previously remained largely unknown. We demonstrate that tumor regression after chemotherapy occurs via sporadic necrosis and relies on activation of innate immunity in a manner dependent on high mobility group box 1 protein (HMGB1).

Abstract 2513: Molecular dissection of cell death mechanisms following ionizing radiation in vitro and in vivo

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Purpose: A majority of human cancers have the ability to evade cell death with dysregulated cell death and survival mechanisms. This may have a direct impact on treatment resistance and tumor recurrence. This study aims to elucidate the roles of apoptosis, autophagy and necrosis induced by ionizing radiation (IR). Materials/Methods: Immortalized murine embryonic fibroblast (MEF) cell lines with various genetic backgrounds were used in the study. Xenograft tumors were generated by injecting tumor cells (engineered by retrovirally transfecting the above MEF cell lines with oncoprotein E1A and KRAS) to bilateral flanks of nude mice. Mice bearing xenograft tumors were treated with radiation by using an orthovoltage X-ray machine. Tissue cultures were irradiated by using a Gammacell 40 irradiator with a Cs137 source. Cell survival was analyzed with several commonly accepted methods. Morphology changes before and after treatments were analyzed by phase contrast microscopy as well as transmission electron microscopy (TEM). Western blotting and Immunohistochemistry (IHC) methods were carried out as described. Results: There was no significant difference in the level of tumor regression between xenografted tumors lacking Bax and Bak (Bax−/−Bak−/−) and WT tumors after radiation. While apoptotic cell death was observed in immortalized WT MEF cell line and xenograft tumors after irradiation, the intrinsic apoptosis pathway was not required Autophagy was found to be activated after radiation, and pharmacologic inhibition of autophagy by 3-methyladenine (3MA) resulted in higher survival fraction after radiation, suggesting a cytotoxic role of autophagy following radiation. However, there was no difference in radiation response when an essential autophagy gene ATG5 was stably knocked down, suggesting that autophagy activation may not be required for radiation induced cell death. We then investigated the role of poly(ADP)-ribose polymerase (PARP) in radiation response by using PARP-1−/− immortalized MEFs. These cells were more resistant to MAF (an active metabolite of cyclophosphamide) induced necrosis as was shown previously, but were more sensitive to radiation both in vitro and in vivo (as in xenograft). We also found that a specific PARP inhibitor ABT-888 can significantly sensitize xenograft tumors to radiation treatment. Conclusion: While apoptosis and autophagy occur after radiation, the intrinsic apoptosis and autophagic pathways is not required for tumor regression following IR. Conversely, PARP protein plays an important pro-survival role after IR, and deficiency of PARP renders cells more sensitive to radiation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2513. doi:10.1158/1538-7445.AM2011-2513