Chemotherapy-induced Cancer Cell Death Research Articles

Tumor Microenvironment CD14+ Cells Correlate with Poor Overall Survival in Patients with Early-Stage Lung Adenocarcinoma.

Simple SummaryLung cancer is the leading cause of cancer-related deaths worldwide in part due to high rates of recurrence even with early-stage disease. This suggests that new biomarkers are needed to improve predictions for patient outcomes and better understand the underlying biology that drives poor outcomes. We believe the immune cells within the tumor microenvironment (TME) are a key differentiator between patients who are cured and patients who experience lung cancer relapse. In this study, we investigated the presence of CD14+ cells within the lung adenocarcinoma TME through immunohistochemistry and found that higher levels of CD14+ cells were correlated with shorter patient survival. In vitro studies revealed a bi-directional cross-talk between CD14+ cells and lung cancer that improved lung cancer cell recovery after chemotherapy. Our observations suggest that TME CD14+ infiltration is a prognostic marker in lung cancer and that further studies are needed to understand CD14+-cell-mediated mechanisms that influence patient outcomes.Patients with early-stage lung adenocarcinoma have a high risk of recurrent or metastatic disease despite undergoing curative intent therapy. We hypothesized that increased CD14+ cells within the tumor microenvironment (TME) could stratify patient outcomes. Immunohistochemistry for CD14 was performed on 189 specimens from patients with lung adenocarcinoma who underwent curative intent surgery. Outcomes and associations with clinical and pathologic variables were determined. In vitro studies utilized a coculture system to model the lung cancer TME containing CD14+ cells. Patients with high levels of TME CD14+ cells experienced a median overall survival of 5.5 years compared with 8.3 and 10.7 years for those with moderate or low CD14 levels, respectively (p < 0.001). Increased CD14+ cell tumor infiltration was associated with a higher stage at diagnosis and more positive lymph nodes at the time of surgery. This prognostic capacity remained even for patients with early-stage disease. Using an in vitro model system, we found that CD14+ cells reduced chemotherapy-induced cancer cell death. These data suggest that CD14+ cells are a biomarker for poor prognosis in early-stage lung adenocarcinoma and may promote tumor survival. CD14+ cell integration into the lung cancer TME can occur early in the disease and may be a promising new therapeutic avenue.

A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy-Induced Cancer Cell Death.

The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high-resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real-time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy-induced apoptosis in vivo in mouse models of breast cancer.

A Bivalent Activatable Fluorescent Probe for Screening and Intravital Imaging of Chemotherapy-Induced Cancer Cell Death.

The detection and quantification of apoptotic cells is a key process in cancer research, particularly during the screening of anticancer therapeutics and in mechanistic studies using preclinical models. Intravital optical imaging enables high‐resolution visualisation of cellular events in live organisms; however, there are few fluorescent probes that can reliably provide functional readouts in situ without interference from tissue autofluorescence. We report the design and optimisation of the fluorogenic probe Apotracker Red for real‐time detection of cancer cell death. The strong fluorogenic behaviour, high selectivity, and excellent stability of Apotracker Red make it a reliable optical reporter for the characterisation of the effects of anticancer drugs in cells in vitro and for direct imaging of chemotherapy‐induced apoptosis in vivo in mouse models of breast cancer.

ATAD3A stabilizes GRP78 to suppress ER stress for acquired chemoresistance in colorectal cancer.

AAA domain containing 3A (ATAD3A) is a nucleus-encoded mitochondrial protein with vital function in communication between endoplasmic reticulum (ER) and mitochondria which is participated in cancer metastasis. Here we show that elevated ATAD3A expression is clinically associated with poor 5-year disease-free survival in patients with colorectal cancer (CRC), especially high-risk CRC patients who received adjuvant chemotherapy. Our results indicated ATAD3A is significantly upregulated to reduce chemotherapy-induced cancer cell death. We found that knockdown of ATAD3A leads to dysregulation in protein processing for inducing ER stress by RNA sequencing (RNA-seq). In response to chemotherapy-induced ER stress, ATAD3A interacts with elevated GRP78 protein to assist protein folding and alleviate ER stress for cancer cell survival. This reduction of ER stress leads to reduce the surface exposure of calreticulin, which is the initiator of immunogenic cell death and antitumor immunity. However, silencing of ATAD3A enhances cell death, triggers the feasibility of chemotherapy-induced ER stress for antitumor immunity, increases infiltration of T lymphocytes and delays tumor regrowth in vitro and in vivo. Clinically, CRC patients with less ATAD3A have high density of CD45+ intratumoral infiltrating lymphocytes (TILs) and memory CD45RO+ TILs. Taken together, our results suggest that pharmacologic targeting to ATAD3A might be a potential therapeutic strategy to enhance antitumor immunity for CRC patients who received adjuvant chemotherapy.

Polarimetric Detection of Chemotherapy-Induced Cancer Cell Death

Imaging polarimetry is a focus of increasing interest in diagnostic medicine because of its non-destructive nature and its potential to distinguish normal from tumor tissue. However, handling and understanding polarimetric images is not an easy task, and different intermediate steps have been proposed in order to introduce helpful physical magnitudes. In this research, we look for a sensitive polarimetric parameter that allows us to detect cell death when cancer cells are treated with chemotherapy drugs. Experiments in two different myelomonocytic leukemia cell lines, U937 and THP1, are performed in triplicate, finding a highly-significant positive correlation between total diattenuation of samples in transmission configuration, D T , and chemotherapy-induced cell death. The location of the diattenuation enhancement gives some insight into the cell death process. The proposed method can be an objective complement to conventional methodologies based on pure observational microscopy and can be easily implemented in regular microscopes.

Inhibition of skin carcinogenesis by suppression of NF-\u03baB dependent ITGAV and TIMP-1 expression in IL-32\u03b3 overexpressed condition

BackgroundInterleukin-32 (IL-32) has been associated with various diseases. Previous studies have shown that IL-32 inhibited the development of several tumors. However, the role of IL-32γ, an isotype of IL-32, in skin carcinogenesis remains unknown.MethodsWe compared 7,12-Dimethylbenz[a]anthracene/12-O-Tetradecanoylphorbol-13-acetate (DMBA/TPA)-induced skin carcinogenesis in wild type (WT) and IL-32γ-overexpressing mice to evaluate the role of IL-32γ. We also analyzed cancer stemness and NF-κB signaling in skin cancer cell lines with or without IL-32γ expression by western blotting, quantitative real-time PCR and immunohistochemistry analysis.ResultsCarcinogen-induced tumor incidence in IL-32γ mice was significantly reduced in comparison to that in WT mice. Infiltration of inflammatory cells and the expression levels of pro-inflammatory mediators were decreased in the skin tumor tissues of IL-32γ mice compared with WT mice. Using a genome-wide association study analysis, we found that IL-32 was associated with integrin αV (ITGAV) and tissue inhibitor of metalloproteinase-1 (TIMP-1), which are critical factor for skin carcinogenesis. Reduced expression of ITGAV and TIMP-1 were identified in DMBA/TPA-induced skin tissues of IL-32γ mice compared to that in WT mice. NF-κB activity was also reduced in DMBA/TPA-induced skin tissues of IL-32γ mice. IL-32γ decreased cancer cell sphere formation and expression of stem cell markers, and increased chemotherapy-induced cancer cell death. IL-32γ also downregulated expression of ITGAV and TIMP-1, accompanied with the inhibition of NF-κB activity. In addition, IL-32γ expression with NF-κB inhibitor treatment further reduced skin inflammation, epidermal hyperplasia, and cancer cell sphere formation and downregulated expression levels of ITGAV and TIMP-1.ConclusionsThese findings indicated that IL-32γ suppressed skin carcinogenesis through the inhibition of both stemness and the inflammatory tumor microenvironment by the downregulation of TIMP-1 and ITGAV via inactivation of NF-κB signaling.

Abstract 69: Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages

Abstract One of the most common treatment options for cancer is a chemotherapy. After chemotherapy, however, unwanted host effects provoke tumor recurrence and aggressiveness of cancer cells, which often arise as a consequence of disruption in the patient's immune system. Lymphocytes, such as CD8+ cytotoxic T lymphocytes (CTLs), which have capability of suppressing cancer progression, are depleted following chemotherapy. Conventional chemotherapeutic agents kill cancer cells, which boosts recruitment of myeloid cells. Tumor-associated macrophages (TAMs), emerging as abundant tumor-infiltrating myeloid cells in tumor microenvironment, play an important role in immunosuppression caused by conventional chemotherapy. Chemotherapy-induced dying cancer cells could hijack accumulated TAMs, provoking tumor recurrence. Therefore, reprogramming of TAMs to maximize the chemotherapeutic efficacy is considered a promising novel anticancer strategy. In this study, we investigated whether dying cancer cells after chemotherapy could reduce therapeutic efficacy by modulating tumor-infiltrating macrophage activity. In a 4T1 syngeneic mouse breast cancer model, there was a marked decrease in the proportion of M1-like TAMs expressing CD86 with tumor-suppressive activity and reduced tumor infiltration of CTLs following treatment with paclitaxel. Paclitaxel treatment also resulted in an enhancement of heme oxygenase-1 (HO-1) mRNA levels in tumor-infiltrating myeloid cells engulfing dying cancer cells. Consistent with the in vivo profile of TAMs, bone marrow-derived macrophages (BMDMs) phagocytosing dying breast cancer cells exhibited significant upregulation of HO-1 expression. HO-1 induction in BMDMs engulfing dying breast cancer cells facilitated M2 polarization. In contrast, abrogation of HO-1 activity sustained M1 activation of BMDMs co-cultured with dying breast cancer cells. Pharmacologic inhibition of HO-1 activity with zinc protoporphyin IX augmented paclitaxel efficacy by stimulating tumor infiltration of CTLs in a 4T1 breast cancer model. Furthermore, blockade of HO-1 in breast tumor-bearing mice induced increased M1-like TAMs expressing CD86 that plays an important role in stimulating antitumor immune response. Taken together, HO-1 induction by the chemotherapy-derived dying cancer cells in TAMs dampens antitumor effects of chemotherapy by manipulating innate and adaptive immunity. Our results demonstrate that targeting overexpressed HO-1 in the breast tumor microenvironment can control the immune system to potentiate the efficacy of chemotherapy. Citation Format: Seung Hyeon Kim, Xiancai Zhong, Shin-Young Gwak, Ishrat Aklima Muna, Sin-Aye Park, Young-Nam Cha, Young-Joon Surh. Chemotherapy-induced cancer cell death diminishes therapeutic efficacy through heme oxygenase-1-mediated inactivation of M1-like tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 69.

Wnt Antagonist, Secreted Frizzled-Related Protein 4 (sFRP4), Increases Chemotherapeutic Response of Glioma Stem-Like Cells

Malignant gliomas have a highly tumorigenic subpopulation, termed cancer stem cells (CSCs), that drives tumor formation and proliferation. CSCs possess inherent resistance mechanisms against radiation- and chemotherapy-induced cancer cell death, enabling them to survive and initiate tumor recurrence. We examined the effect of secreted frizzled-related protein 4 (sFRP4), a Wnt signaling antagonist, in chemosensitizing the glioma cell line U138MG and glioma stem cells (GSCs) enriched from U138MG to chemotherapeutics. We found that sFRP4 alone and in combination with either doxorubicin or cisplatin induced apoptosis. Proliferation decreased substantially in GSC-enriched population as measured by MTT and BrdU assays. JC-1 and caspase-3 assays demonstrated that cell death was through the apoptotic pathway. sFRP4 treatment also decreased neurosphere formation and induced neuronal differentiation. Inhibition by sFRP4 was abolished by Wnt3a, indicating that sFRP4 acts through the frizzled receptor. Further indication that sFRP4 acts through the Wnt β-catenin pathway was provided by decrease in the β-catenin protein and decrease in the β-catenin-stimulated gene cyclin D1 upon sFRP4 induction. By real-time PCR, an increase in apoptotic markers Bax and p21, a decrease in pro-proliferative marker CycD1, and a decrease in the GSC marker CD133 were observed. These observations indicate that sFRP4 is able to sensitize glioma cells and stem cells to chemotherapeutics. We thus identified for the first time that sFRP4 could help to destroy cancer stem cells of glioma cell line, which would lead to effective treatment regimen to combat brain tumors.

Differentiation-Defective Class IV GCSF Receptor Promotes Resistance to Apoptosis Through Induction of Anti-Apoptotic Bcl-2 Family Proteins

Abstract 3819 INTRODUCTION.Granulocyte colony stimulating factor (GCSF) drives the production, proliferation and differentiation of myeloid progenitor and precursor cells via the GCSF Receptor (GCSFR). The Class I GCSFR constitutes the full-length, wild type receptor. Alternative splicing events create a truncated isoform, the Class IV GCSFR, which lacks 3 of the 4 distal tyrosine residues in the C-terminal domain, and results in impaired differentiation. A role for the Class IV receptor in human myeloid leukemia has been suggested from two clinical observations: 1) Bone marrow cells from patients with monosomy 7 overexpress the Class IV receptor and demonstrate increased proliferative response to GCSF; 2) Increased relapse rates were found in children and adolescents with AML whose blasts overexpress the Class IV receptor. Additionally, multiple splicing factors have recently been discovered to be mutated in myelodysplastic syndrome (MDS). We have discovered that the S34F mutation in the splicing factor U2AF1 is associated with exon skipping of the GCSFR. Yet, little is known about the biological properties of this Class IV isoform. METHODS.The IL-3 dependent murine Ba/F3 cell line was transfected with cDNA to express either human Class I (Ba/F3 GRI) or Class IV (Ba/F3 GRIV) GCSFR. Proliferation of Ba/F3 GRI and GRIV cells was measured using MTT assay after 48 hours of exposure to varying doses of GCSF. Cells were serum- and cytokine-starved for 0, 4, 8 and 16 hours. Cell count and viability were determined by trypan blue exclusion. Members of the mitochondrial apoptosis pathway were also evaluated by western blot analysis. Time-dependent changes in GCSF-mediated phosphoprotein signaling events were studied by western blotting. Samples were also stained with propidium iodide and used for cell cycle analysis by flow cytometry or used to perform AnnexinV apoptosis assay. RESULTS.Flow cytometry results demonstrated a G0/G1 cell cycle arrest at 8 hrs for both Ba/F3 GRI and GRIV cells with a similar magnitude of effect. However, longer starvation of 16 hrs resulted in increased cell death, more pronounced in the GRI than GRIV cells (46% v. 9% in the sub-G1 phase of the cell cycle, respectively). This pattern of apoptotic resistance in the GRIV cells was also seen in cell number and viability (viability of GRI and GRIV cells at 16 hrs was 67% ± 4.9% v. 90% ± 4%, respectively). These results were validated by flow cytometry using the AnnexinV apoptosis assay: proportion of GRI and GRIV cells staining positive for both AnnexinV and 7-AAD was 71% and 32%, respectively. To determine the molecular basis for this resistance to apoptosis, we performed western blotting to analyze Bcl-2, Mcl-1, Puma, GSK3ß, Bcl-xLand other Bcl-2 family members. Of these, the most prominent differences were seen in Bcl-2 and Mcl-1. The Class IV cells demonstrated increased levels of Bcl-2 throughout 16 hrs of starvation. The Class IV cells also displayed persistence of Mcl-1 for a longer duration than the GRI cells, correlating with the observed pattern of viability and cell cycle data. When these starved cells were stimulated with GCSF, there was a greater time dependent increase of phopho-p38 MAPK in the GRI cells. CONCLUSIONS.Here, we show that the Class IV GCSFR isoform confers both a proliferative and survival or anti-apoptotic advantage. Increased levels of both Mcl-1 and Bcl-2 may contribute to the apoptotic resistance seen in the Ba/F3 GRIV cells. Because there were no differences in activation state of GSK3ß, an alternate pathway for regulating Mcl-1 levels must be operative. The p38 MAPK stress response has been implicated in affecting downstream Mcl-1 and Bcl-2 regulation. p38 activation is important for chemotherapy induced cancer cell death, and chemotherapeutic resistance has been linked to overexpression of Bcl-2 or Mcl-1 in various cancer types. It is intriguing that Class IV expression has been linked to increased relapse rates in AML patients. Thus, we speculate that there may be a synergistic, protective effect of decreased p38 activation coincident with enhancement of the anti-apoptotic Bcl-2 and Mcl-1 proteins in the Class IV cells. Further elucidation of the mechanism for apoptotic resistance conferred by the Class IV GCSFR will help define its contribution to MDS transformation and provide candidate targets for novel therapeutics or prevention of disease progression in patients with primary or secondary MDS/AML. Disclosures:No relevant conflicts of interest to declare.

Abstract C59: The effects of combined treatment of erlotinib and cisplatin in resistant lung cancer and the role of autophagy in response to chemotherapy

Abstract Introduction: Non-small cell lung cancer (NSCLC) accounts for about 80% of all cases of lung cancer. The treatment of choice for NSCLC is complete surgical resection. However, in advance stages with metastasis and/or aggressive local infiltrate chemotherapy becomes the only option. At high therapeutic doses, chemotherapy can cause significant side effects, and the development of resistance may arise if initial treatment doses of therapy become ineffective. In order to prevent serious side effects and resistance, low-dose combination regimens that offer comparable results to current high-dose regimens are needed. The combination of cisplatin and erlotinib has been shown to inhibit tumor growth more effectively than a single drug in NSCLC cells. However, the mechanism underlying this finding remains unclear. Autophagy has been suggested to play a critical role in cancer and chemotherapy, but there is ongoing controversy among its role (promotion vs. prevention of cancer). In this study, we examined whether the combination of low-dose cisplatin and erlotinib is able to induce higher levels of cancer cell death than each drug alone in both sensitive and erlotinib-resistant lung adenocarcinoma. In addition, we looked into the role of autophagy in cancer cells and its association with chemotherapy. Methods: Alamar blue assay was used to construct dose response curves in which IC 50 values of cisplatin and erlotinib were determined. Erlotinib-resistant PC9 (PC9/ER) cells were developed by culturing PC9 (EGFR overexpression adenocarcinoma) cells in media containing an erlotinib dose 1000 times the IC50 value over a two week period; PC9/ER cells were verified to be 26-fold more resistant than PC9 sensitive cancer cells. Baseline levels of autophagy of normal human bronchial epithelial (NHBE) cells, PC9, and PC9/ER were examined by Western blot with antibodies against LC3I/II. The IC 25 of each drug was chosen as the low-dose treatments (3 uM cisplatin and 10 nM erlotinib). Cells were treated accordingly: control, cisplatin, erlotinib, and combination. Cell death and autophagy levels were measured. Next, cells were pretreated in one day in advance with rapamycin and cell death and changes in autophagy were assessed. Results: At baseline, there were higher levels of LC3II in PC9/ER compared to PC9 and NHBE. Combined treatment of cisplatin and erlotinib was able to induce significantly more cell death than individual drugs at the same dose (p&amp;lt;0.001). In fact, combination treatment provided synergistic killing effects in PC9 and PC9/ER cells. In PC9 cells, erlotinib alone had 79.9% survival, cisplatin 76.3%, and combination 45.0%. In PC9/ER, erlotinib alone had 96.6% survival, cisplatin 89.3%, and combination 61.1%. Levels of autophagy decreased in a similar pattern to killing-effects in both PC9 and PC9/ER cells. In addition, a significant increase in cell survival of about 20% was observed when pretreated with rapamycin (p&amp;lt;0.001). NHBE demonstrated no significant killing and no changes in autophagy levels. Conclusion: Our data is consistent for autophagy's role in promoting cancer cell survival, work as a protective mechanism for cancer-resistant cells, and a critical mechanism in chemotherapy-induced cancer cell death. The combination of erlotinib and cisplatin offers synergistic killing properties in vitro and may be a potential regimen against resistant lung cancer. In addition, the combination treatment does not induce significant cytotoxic effects and death in NHBE cells indicating its safe dosing. We plan to further characterize autophagy with 3-methyladenine pretreatment, changes in apoptosis, and invasiveness of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C59.

Autophagy as a mediator of chemotherapy-induced cell death in cancer

Since the 1940s, chemotherapy has been the treatment of choice for metastatic disease. Chemotherapeutic agents target proliferating cells, inducing cell death. For most of the history of chemotherapy, apoptosis was thought to be the only mechanism of drug-induced cell death. More recently, a second type of cell death pathway has emerged: autophagy, also called type II programmed cell death. Autophagy is a tightly regulated process by which selected components of a cell are degraded. It primarily functions as a cell survival adaptive mechanism during stress conditions. However, persistent stress can also promote extensive autophagy, leading to cell death, hence its name. Alterations in the autophagy pathway have been described in cancer cells that suggest a tumor-suppressive function in early tumorigenesis, but a tumor-promoting function in established tumors. Moreover, accumulating data indicate a role for autophagy in chemotherapy-induced cancer cell death. Here, we discuss some of the evidence showing autophagy-dependent cell death induced by anti-neoplastic agents in different cancer models. On the other hand, in some other examples, autophagy dampens treatment efficacy, hence providing a therapeutic target to enhance cancer cell killing. In this paper, we propose a putative mechanism that could reconcile these two opposite observations.

Survival of the Fittest: Cancer Stem Cells in Therapeutic Resistance and Angiogenesis

In an increasing number of cancers, tumor populations called cancer stem cells (CSCs), or tumor-initiating cells, have been defined in functional assays of self-renewal and tumor initiation. Moreover, recent work in several different cancers has suggested the CSC population as a source of chemotherapy and radiation-therapy resistance within tumors. Work in glioblastoma and breast cancers supports the idea that CSCs may possess innate resistance mechanisms against radiation- and chemotherapy-induced cancer cell death, allowing them to survive and initiate tumor recurrence. Several resistance mechanisms have been proposed, including amplified checkpoint activation and DNA damage repair as well as increased Wnt/beta-catenin and Notch signaling. Novel targeted therapies against the DNA damage checkpoint or stem-cell maintenance pathways may sensitize CSCs to radiation or other therapies. Another important category of cancer therapies are antiangiogenic and vascular targeting agents, which are also becoming integrated in the treatment paradigm of an increasing number of cancers. Recent results from our laboratory and others support a role for CSCs in the angiogenic drive as well as the mechanism of antiangiogenic agents. Identifying and targeting the molecular mechanisms responsible for CSC therapeutic resistance may improve the efficacy of current cancer therapies.