Kaempferol-Iron Assembled Nanoparticles for Synergistic Photothermal and Chemodynamic Therapy of Breast Cancer.

The median survival rate for women with metastatic breast cancer is 1-2 years, and the 5-year survival rate is only 15 percent. Early detection of metastatic disease is often ineffective, since once the cancer recurs at a distant site, it may be refractory to conventional treatments. A promising strategy for targeting metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. Immunotherapeutic approaches are effective treatments for many types of cancer, such as melanoma. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. Moreover, immunotherapy can cause adverse side effects such as colitis. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to induce a tumor-specific immune response, which can eradicate residual cancer cells. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) that in turn activate tumor-specific cytotoxic T cells. While not well-understood, ICD may also sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs, such as anthracyclines, used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of mice with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT) which is essential for the folding of actin and tubulin and other critical proteins into their native forms. We found CCT to be overexpressed in breast cancer cells susceptible to the peptide. Inhibition of CCT activity by CT20p, indicated by decreased F-actin, disrupted the cytoskeleton, causing loss of cell motility and adhesion that led to breast cancer cell death. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that can generate the danger signals associate with ICD. To this end, we observed that breast cancer cells treated with CT20p displayed alterations in PERK (protein kinase RNA-like ER kinase), one of the key mediators of the unfolded protein response (UPR). As a result, CT20p-treated breast cancer cells were more readily phagocytosed. This also suggests that peptide treatment could potentially have an impact on NK cell cytotoxicity by inducing the expression of activating ligands on cancer cells. In our experiments normal breast epithelial cells, macrophages or NK cells were unaffected by CT20p treatment due reduced levels and activity of CCT. These studies indicate that CT20p, by targeting CCT, can be used to induce ICD and stimulate essential innate immune functions like NK cell cytotoxic activity and leukocyte phagocytosis that are necessary for promoting a robust anti-cancer adaptive immune response in order to mitigate the development of lethal metastatic disease. Citation Format: Arati Limaye, Rania Bassiouni, Jeremiah Oyer, Robert W. Igarashi, Orielyz Flores, J. Manuel Perez, Alijca Copik, Annette R. Khaled. Use of a cytotoxic peptide that induces immunogenic cell death to engage innate immunity in the treatment of metastatic breast cancer. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr A47.

Highlights from Recent Cancer Literature

BackgroundImmune evasion is a characteristic hallmark of cancer. Immunotherapies aim to activate and support the body's immune system to recognize and fight tumor cells. Induction of immunogenic cell death (ICD) and the associated activation of danger signaling pathways can increase the immunogenicity of tumor cells. Therapeutic ICD stimuli activate endoplasmic reticulum stress pathways and apoptosis leading to the cellular expression of damage-associated molecular patterns (DAMPs).The aim of our in vitro study was to investigate whether mistletoe extracts induce characteristics of immunogenic tumor cell death in cancer cell lines.MethodsThree human breast cancer cell lines and one murine melanoma cell line (SKBR3, MDA-MB-231, MCF-7, and B16F10) were treated with aqueous, fermented Viscum album extract (VAE: Iscador Qu spec.) and taxol or tunicamycin as positive controls, respectively. To investigate whether VAE induces ribotoxic stress, we measured the ER stress regulators p-eIF2a, ATF4, and CHOP by Western blot. Cell surface exposure of DAMPs (calreticulin, heat shock proteins hsp70 and hsp90), apoptosis and induction of mitochondrial reactive oxygen species (ROS) were assessed by flow cytometry. HMGB1 and ATP were quantified by ELISA and chemiluminescence assay, respectively.ResultsTreatment with VAE resulted in phosphorylation of eIF2α in all cancer cell lines tested and increased calreticulin (CRT) exposure on the surface of pre-apoptotic SKBR3 breast cancer and B16F10 mouse melanoma cells. VAE exerted a concentration-dependent effect in all cell lines, resulting in a significantly increased exposure of three DAMPs (CRT, hsp70 and hsp90) on the surface of early apoptotic cells. Furthermore, VAE elevated mitochondrial ROS production and the release of ATP. HMGB1 release was not induced by VAE.ConclusionsIn this in vitro study, we demonstrated for the first time the potential of a mistletoe extract to induce surrogate markers of immunogenic cancer cell death. This is a primary step in investigating the potential of VAEs to contribute to ICD-induced tumor-specific immune activation.

Chelidonine-induced inhibition of FBP1 disrupts M2 macrophage polarization and attenuates breast cancer.

Objective: ER+ breast cancer is the most common type of breast cancer, which seriously affects the physical and mental health of women. Recently, lncRNAs mediated tumor-associated macrophages (TAM) were identified to involve in tumorigenesis. Therefore, the present study aimed at demonstrating the regulatory network of GNAS-AS1 in TAM-mediated ER+ breast cancer progress.Methods: The expression levels of genes were evaluated using qRT-PCR. The proportions of polarized macrophages (M1, M2) were assessed by flow cytometry. Cell proliferation, migration and invasion were evaluated by CCK-8, wound healing and transwell assay, respectively. Double-luciferase reporter system was used to detect the interaction between molecules. Western blot was applied to test protein levels.Results: The expression of GNAS-AS1 was obviously increased in ER+ breast cancer tissues and cell lines, as well as M2 macrophages. GNAS-AS1 facilitated the capabilities of proliferation, migration and invasion of ER+ breast cancer cells by accelerating M2 macrophage polarization via directly sponging miR-433-3p. GATA3, as a target of miR-433-3p, could positively regulate by GNAS-AS1. Furthermore, either miR-433-3p overexpression or GATA3 knockdown impaired the effects of GNAS-AS1 on M2 macrophage polarization and ER+ breast cancer cells progression.Conclusion: GNAS-AS1/miR-433-3p/GATA3 axis promoted proliferation, metastasis of ER+ breast cancer cells by accelerating M2 macrophage polarization. The mechanism may provide a new strategy and target for ER+ breast cancer treatment.

Targeting Androgen Receptor in Estrogen Receptor-Negative Breast Cancer

Chemodynamic therapy (CDT) has received more and more attention as an emerging therapeutic strategy, especially transition metals with Fenton or Fenton-like activity have good effects in CDT research, manganese dioxide nanosheets (MnO2 NSs) and their complexes have become one of the most favored nanomaterials in CDT of tumors. CDT is mainly based on the role of reactive oxygen species (ROS) in tumor treatment, which have clear chemical properties and produce clear chemical reactions. However, their mechanism of interaction with cells has not been fully elucidated. Here, we performed CDT on mouse breast cancer cells (4T1) based on MnO2 NSs, extracted the metabolites from the 4T1 cells during the treatment, and analyzed the differences in metabolites by using high-resolution liquid chromatography-mass spectrometry (LC-MS). Untargeted metabolomics studies were conducted using the relevant data. This study mainly explored the changes in MnO2 NSs on the metabolite profile of 4T1 cells and their potential impact on tumor therapy, in order to determine the mechanism of action of MnO2 NSs in the treatment of breast cancer. The results of the study showed the presence of 11 different metabolites in MnO2 NSs CDT for 4T1 tumor cells, including phosphoserine, sphingine, phosphocholine, and stearoylcarnitine. These findings provide a deeper understanding of breast cancer treatment, and are beneficial for the further research and clinical application of CDT.

In Vivo Evidence for the Role of CD44s in Promoting Breast Cancer Metastasis to the Liver

SY03-01: The desirable death of the cancer cell: Immunogenic cell death for optimal chemotherapy

Immunogenic cell death (ICD) in cancer represents a functionally unique therapeutic response that can induce tumor-targeting immune responses. ICD is characterized by the exposure and release of numerous damage-associated molecular patterns (DAMPs), which confer adjuvanticity to dying cancer cells. The spatiotemporally defined emission of DAMPs during ICD has been well described, whereas the epigenetic mechanisms that regulate ICD hallmarks have not yet been deeply elucidated. Here, we aimed to examine the involvement of miRNAs and their putative targets using well-established in vitro models of ICD. To this end, B cell lymphoma (Mino) and breast cancer (MDA-MB-231) cell lines were exposed to two different ICD inducers, the combination of retinoic acid (RA) and interferon-alpha (IFN-α) and doxorubicin, and to non ICD inducers such as gamma irradiation. Then, miRNA and mRNA profiles were studied by next generation sequencing. Co-expression analysis identified 16 miRNAs differentially modulated in cells undergoing ICD. Integrated miRNA-mRNA functional analysis revealed candidate miRNAs, mRNAs, and modulated pathways associated with Immune System Process (GO Term). Specifically, ICD induced a distinctive transcriptional signature hallmarked by regulation of antigen presentation, a crucial step for proper activation of immune system antitumor response. Interestingly, the major histocompatibility complex class I (MHC-I) pathway was upregulated whereas class II (MHC-II) was downregulated. Analysis of MHC-II associated transcripts and HLA-DR surface expression confirmed inhibition of this pathway by ICD on lymphoma cells. miR-4284 and miR-212-3p were the strongest miRNAs upregulated by ICD associated with this event and miR-212-3p overexpression was able to downregulate surface expression of HLA-DR. It is well known that MHC-II expression on tumor cells facilitates the recruitment of CD4+ T cells. However, the interaction between tumor MHC-II and inhibitory coreceptors on tumor-associated lymphocytes could provide an immunosuppressive signal that directly represses effector cytotoxic activity. In this context, MHC-II downregulation by ICD could enhance antitumor immunity. Overall, we found that the miRNA profile was significantly altered during ICD. Several miRNAs are predicted to be involved in the regulation of MHC-I and II pathways, whose implication in ICD is demonstrated herein for the first time, which could eventually modulate tumor recognition and attack by the immune system.

Breast cancer is the most commonly diagnosed cancer and the leading cause of cancer death in women worldwide. Recent advances in the field of immuno-oncology demonstrate the beneficial immunostimulatory effects of the induction of immunogenic cell death (ICD). ICD increases tumor infiltration by T cells and is associated with improved prognosis in patients affected by triple negative breast cancer (TNBC) with residual disease. The aim of this study was to evaluate the antitumoral effect of PKHB1, a thrombospondin-1 peptide mimic, against breast cancer cells, and the immunogenicity of the cell death induced by PKHB1 in vitro, ex vivo, and in vivo. Our results showed that PKHB1 induces mitochondrial alterations, ROS production, intracellular Ca2+ accumulation, as well calcium-dependent cell death in breast cancer cells, including triple negative subtypes. PKHB1 has antitumor effect in vivo leading to a reduction of tumor volume and weight and promotes intratumoral CD8 + T cell infiltration. Furthermore, in vitro, PKHB1 induces calreticulin (CALR), HSP70, and HSP90 exposure and release of ATP and HMGB1. Additionally, the killed cells obtained after treatment with PKHB1 (PKHB1-KC) induced dendritic cell maturation, and T cell antitumor responses, ex vivo. Moreover, PKHB1-KC in vivo were able to induce an antitumor response against breast cancer cells in a prophylactic application, whereas in a therapeutic setting, PKHB1-KC induced tumor regression; both applications induced a long-term antitumor response. Altogether our data shows that PKHB1, a thrombospondin-1 peptide mimic, has in vivo antitumor effect and induce immune system activation through immunogenic cell death induction in breast cancer cells.

Bone is the most common site to which breast cancer cells metastasize. We found that osteoblast-like MG63 cells and human bone tissue contain the bile acid salt sodium deoxycholate (DC). MG63 cells take up and accumulate DC from the medium, suggesting that the bone-derived DC originates from serum. DC released from MG63 cells or bone tissue promotes cell survival and induces the migration of metastatic human breast cancer MDA-MB-231 cells. The bile acid receptor farnesoid X receptor (FXR) antagonist Z-guggulsterone prevents the migration of these cells and induces apoptosis. DC increases the gene expression of FXR and induces its translocation to the nucleus of MDA-MB-231 cells. Nuclear translocation of FXR is concurrent with the increase of urokinase-type plasminogen activator (uPA) and the formation of F-actin, two factors critical for the migration of breast cancer cells. Our results suggest a novel mechanism by which DC-induced increase of uPA and binding to the uPA receptor of the same breast cancer cell self-propel its migration and metastasis to the bone.

A promising strategy for treating metastatic breast cancer is engaging the immune system to destroy disseminated breast cancer cells. However, breast cancer is a challenge for immunotherapy because of its inherent genetic heterogeneity and decreased immunogenicity. There is an unmet need to develop new, targeted therapies for breast cancer that stimulate the immune system and can be used in combination with checkpoint blockade or adoptive cell transfer to fight metastatic disease. One potential approach is to generate dying breast cancer cells that operate like a vaccine to stimulate a tumor-specific immune response. This is termed immunogenic cell death (ICD) and is characterized by a unique molecular signature, involving the release of molecules that attract and stimulate phagocytes like dendritic cells (DCs) and that could sensitize tumor cells to killing by natural killer (NK) cells. With the discovery of new ICD-inducing agents, interest in this approach is increasing. However, the current doses of drugs used to induce ICD may be too high to translate into clinically relevant regimens. To address these problems, our group developed a novel cytotoxic peptide, CT20p, and a nanotechnology-based platform to deliver and concentrate CT20p in breast tumors. We found that treatment of a murine xenograft model with nanomolar amounts of CT20p, encapsulated in nanoparticles formed with a novel hyperbranched polyester polymer (HBPE-NPs), resulted in regression of breast cancer tumors, and that dying breast cancer cells expressed markers characteristic of ICD, such as the pre-mortem exposure of the "eat me" signal, calreticulin (Crt). The intracellular target of CT20p is a protein called chaperonin-containing T-complex (CCT), which is essential for the folding of actin and tubulin and other critical proteins into their native forms. Disruption of CCT could cause endoplasmic reticulum (ER) stress through the accumulation of misfolded proteins. ER stress in turn initiates intracellular pathways that generate the danger signals associate with ICD. To this end, we observed that cancer cells treated with CT20p displayed alterations in PERK, a key mediator of the unfolded protein response (UPR) and translocation of Crt. As a result, CT20p-treated cancer cells were more readily phagocytosed. More importantly, NK cells more effectively killed cancer cells pre-treated with CT20p. This suggests that CT20p may stimulate NK cell cytotoxicity by inducing activating signals on cancer cells, which would greatly augment the curative effects of adoptive transfer of NK cells. In our experiments normal breast epithelial cells, macrophages or NK cells, themselves, were unaffected by CT20p treatment due reduced CCT. These studies indicate that a single molecule, CT20p, can do both the killing of cancer cells and activation of immune cells and can be combined with cellular immunotherapy (e.g. expanded NK cells) to provide a more complete protection from breast cancer recurrence and metastasis. Citation Format: Khaled AR, Limaye A, Bassiouni R, Showalter A, Oyer J, Pandey V, Igarashi R, Altomare DA, Copik AJ. Enhancing the immunogenicity of breast cancer cells to stimulate innate immunity and augment the effects of cellular immunotherapy. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-04-12.

In the United States, a woman has a 12% chance of developing breast cancer, and current treatments offer little relief to patients diagnosed with metastatic disease. Tumorigenesis and successful establishment of metastases depend upon tumor cell interactions with the surrounding immune microenvironment. Elevated tumor infiltration of immunosuppressive (M2) macrophages correlates with poor prognosis of breast cancer patients. The tumor microenvironment remarkably orchestrates molecular mechanisms that program these macrophages toward the M2 phenotype. Also, metabolic programming is instrumental in orchestrating the polarization of macrophages to assume an M1 (tumor-eradicating) or an M2 (tumor-promoting) phenotype. Aberrant activation of Hedgehog (Hh) signaling in breast cancer cells enables them to survive, proliferate, and metastasize, thus making it a promising target for breast cancer treatment. Hh signaling also enables a crosstalk between breast cancer cells and cells in their milieu, thus contributing to M2 macrophage polarization. We used two immunocompetent orthotopic mouse models of mammary tumors to test the effect of inhibiting Hh signaling on tumor-associated macrophages, and discovered that treatment with the pharmacologic Hh inhibitor, Vismodegib, induced a significant shift in the profile of tumor-infiltrating macrophages. We hypothesized that Hh activity calibrates the metabolism in macrophages, leading to enhanced M2 phenotype and function within the tumor microenvironment. Using a mass spectrometry-enabled untargeted metabolomics approach, we identified that inhibiting Hh signaling reduces flux through the hexosamine biosynthetic pathway, resulting in reduced cellular O-GlcNAcylation in M2 macrophages. This impinges upon diminished STAT6 O-GlcNAcylation, which consequently decreases fatty acid oxidation and ultimately enacts a metabolic cascade including lipid utilization, cellular bioenergetics, and mitochondrial dynamics. As such, inhibiting Hh activity mitigates the metabolomic and bioenergetic underpinnings of the immunosuppressive program of M2 macrophages, resulting in macrophages that are functionally and phenotypically reminiscent of inflammatory, anti-tumor macrophages. In conclusion, we discovered a novel role for Hh signaling in promoting polarization of tumor-associated macrophages to the M2 type through recalibrating their metabolic circuitries, ultimately leading to diminished M2 phenotype and function within the tumor microenvironment. This is the first evidence highlighting the relevance of Hh signaling in controlling a complex metabolic network in immune cells. This knowledge will help us to better understand how to target and diminish the pro-tumorigenic functions of tumor-infiltrating macrophages. Citation Format: Dominique C. Hinshaw, Ann Hanna, Tshering Lama-Sherpa, Brandon Metge, Sarah C. Kammerud, Gloria A. Benavides, Atul Kumar, Heba A. Alsheikh, Mateus Mota, Dongquan Chen, Scott Ballinger, Jeffrey C. Rathmell, Selvarangan Ponnazhagan, Victor Darley-Usmar, Rajeev S. Samant, Lalita A. Shevde. Hedgehog signaling regulates metabolism and polarization of mammary tumor-associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2103.

Breast cancer, a malignancy originating from the epithelium or ductal epithelium of the breast, is not only highly prevalent in women but is also the leading cause of cancer-related deaths in women worldwide. Research has indicated that breast cancer incidence is increasing in younger women, prompting significant interest from scientists actively researching breast cancer treatment. Copper is highly accumulated in breast cancer cells, leading to the development of copper complexes that cause immunogenic cell death, apoptosis, oxidative stress, redox-mediated cell death, and autophagy by regulating the expression of key cell death proteins or assisting in the onset of cell death. However, they have not yet been applied to clinical therapy due to their solubility in physiological buffers and their different and unpredictable mechanisms of action. Herein, we review existing relevant studies, summarize the detailed mechanisms by which they exert anti-breast cancer effects, and propose a potential mechanism by which copper complexes may exert antitumor effects by causing copper death in breast cancer cells. Since copper death in breast cancer is closely related to prognosis and immune infiltration, further copper complex research may provide an opportunity to mitigate the high incidence and mortality rates associated with breast cancer.