Cystine/glutamate antiporter xCT deficiency reduces metastasis without impairing immune system function in breast cancer mouse models.

BACKGROUND: The upregulation of antioxidant mechanisms is a common occurrence in cancer cells, as they strive to maintain balanced redox state and prevent oxidative damage. This includes the upregulation of the cystine/glutamate antiporter xCT, which plays a crucial role in protecting cancer cells from oxidative stress. Consequently, targeting xCT has become an attractive strategy for cancer treatment. However, xCT is also expressed by several types of immune cells where it has a role in proliferation and effector functions. In light of these observations, a comprehensive understanding of the specific role of xCT in the initiation and progression of cancer, as well as its potential impact on the immune system within the tumor microenvironment and the anti-tumor response, require further investigation. METHODS: We generated xCTnull BALB/c mice to investigate the role of xCT in the immune system and xCTnull/Erbb2-transgenic BALB-neuT mice to study the role of xCT in a mammary cancer-prone model. We also used mammary cancer cells derived from BALB-neuT/xCTnull mice and xCTKO 4T1 cells to test the contribution of xCT to malignant properties in vitro and in vivo. RESULTS: xCT depletion in BALB-neuT/xCTnull mice does not alter autochthonous tumor initiation, but tumor cells isolated from these mice display proliferation and redox balance defects in vitro. Although xCT disruption sensitizes 4T1 cells to oxidative stress, it does not prevent transplantable tumor growth, but reduces cell migration in vitro and lung metastasis in vivo. This is accompanied by an altered immune cell recruitment in the pre-metastatic niche. Finally, systemic depletion of xCT in host mice does not affect transplantable tumor growth and metastasis nor impair the proper mounting of both humoral and cellular immune responses in vivo. CONCLUSIONS: xCT is dispensable for proper immune system function, thus supporting the safety of xCT targeting in oncology. Nevertheless, xCT is involved in several processes required for the metastatic seeding of mammary cancer cells, thus broadening the scope of xCT-targeting approaches.

Toll-like receptor 2 promotes breast cancer progression and resistance to chemotherapy.

Cancer stem cells (CSCs) are the main drivers of disease progression and chemotherapy resistance in breast cancer. Tumor progression and chemoresistance might then be prevented by CSC-targeted therapies. We previously demonstrated that Toll-like Receptor (TLR)2 is overexpressed in CSCs and fuels their self-renewal. Here, we show that high TLR2 expression is linked to poor prognosis in breast cancer patients, therefore representing a candidate target for breast cancer treatment. By using a novel mammary cancer-prone TLR2KO mouse model, we demonstrate that TLR2 is required for CSC pool maintenance and for regulatory T cell induction. Accordingly, cancer-prone TLR2KO mice display delayed tumor onset and increased survival. Transplantation of TLR2WT and TLR2KO cancer cells in either TLR2WT or TLR2KO hosts shows that tumor initiation is mostly sustained by TLR2 expression in cancer cells. TLR2 host deficiency partially impairs cancer cell growth, implying a pro-tumorigenic effect of TLR2 expression in immune cells. Finally, we demonstrate that doxorubicin-induced release of HMGB1 activates TLR2 signaling in cancer cells, leading to a chemotherapy-resistant phenotype. Unprecedented use of TLR2 inhibitors invivo reduces tumor growth and potentiates doxorubicin efficacy with no negative impact on the host immune system, opening new perspectives for the treatment of breast cancer patients.

Are Cancer Stem Cells a Suitable Target for Breast Cancer Immunotherapy?

There is substantial evidence to suggest that complete tumor eradication relies on the effective elimination of cancer stem cells (CSCs). CSCs have been widely described as mediators of resistance to conventional therapies, including chemo- and radiotherapy, as well as of tumor metastasization and relapse in different tumor types, including breast cancer. However, the resistant phenotype of CSCs makes their targeting a tough task, and immunotherapy may therefore be an interesting option. Nevertheless, although immunotherapeutic approaches to cancer treatment have generated great enthusiasm due to recent success in clinics, breast cancer treatment mostly relies on standard approaches. In this context, we review the existing literature on the immunological properties of breast CSC and immunotherapeutic approaches to them. We will thus attempt to clarify whether there is room for the immunotargeting of breast CSCs in the current landscape of breast cancer therapies. Finally, we will provide our opinion on the CSC-targeting immunotherapeutic strategies that could prospectively be attempted.

Intracellular Water Lifetime as a Tumor Biomarker to Monitor Doxorubicin Treatment via FFC-Relaxometry in a Breast Cancer Model.

This study aims to explore whether the water exchange rate constants in tumor cells can act as a hallmark of pathology status and a reporter of therapeutic outcomes. It has been shown, using 4T1 cell cultures and murine allografts, that an early assessment of the therapeutic effect of doxorubicin can be detected through changes in the cellular water efflux rate constant kio. The latter has been estimated by analyzing the magnetization recovery curve in standard NMR T1 measurements when there is a marked difference in the proton relaxation rate constants (R1) between the intra- and the extra-cellular compartments. In cellular studies, T1 measurements were carried out on a relaxometer working at 0.5 T, and the required difference in R1 between the two compartments was achieved via the addition of a paramagnetic agent into the extracellular compartment. For in-vivo experiments, the large difference in the R1 values of the two-compartments was achieved when the T1 measurements were carried out at low magnetic field strengths. This task was accomplished using a Fast Field Cycling (FFC) relaxometer that was properly modified to host a mouse in its probe head. The decrease in kio upon the administration of doxorubicin is the result of the decreased activity of Na+/K+-ATPase, as shown in an independent test on the cellular uptake of Rb ions. The results reported herein suggest that kio can be considered a non-invasive, early and predictive biomarker for the identification of responsive patients immediately from the first doxorubicin treatment.

Identification of TENM4 as a Novel Cancer Stem Cell-Associated Molecule and Potential Target in Triple Negative Breast Cancer.

Triple-negative breast cancer (TNBC) is insensitive to endocrine and Her2-directed therapies, making the development of TNBC-targeted therapies an unmet medical need. Since patients with TNBC frequently show a quicker relapse and metastatic progression compared to other breast cancer subtypes, we hypothesized that cancer stem cells (CSC) could have a role in TNBC. To identify putative TNBC CSC-associated targets, we compared the gene expression profiles of CSC-enriched tumorspheres and their parental cells grown as monolayer. Among the up-regulated genes coding for cell membrane-associated proteins, we selected Teneurin 4 (TENM4), involved in cell differentiation and deregulated in tumors of different histotypes, as the object for this study. Meta-analysis of breast cancer datasets shows that TENM4 mRNA is up-regulated in invasive carcinoma specimens compared to normal breast and that high expression of TENM4 correlates with a shorter relapse-free survival in TNBC patients. TENM4 silencing in mammary cancer cells significantly impaired tumorsphere-forming ability, migratory capacity and Focal Adhesion Kinase (FAK) phosphorylation. Moreover, we found higher levels of TENM4 in plasma from tumor-bearing mice and TNBC patients compared to the healthy controls. Overall, our results indicate that TENM4 may act as a novel biomarker and target for the treatment of TNBC.

Teneurins: Role in Cancer and Potential Role as Diagnostic Biomarkers and Targets for Therapy.

Teneurins have been identified in vertebrates as four different genes (TENM1-4), coding for membrane proteins that are mainly involved in embryonic and neuronal development. Genetic studies have correlated them with various diseases, including developmental problems, neurological disorders and congenital general anosmia. There is some evidence to suggest their possible involvement in cancer initiation and progression, and drug resistance. Indeed, mutations, chromosomal alterations and the deregulation of teneurins expression have been associated with several tumor types and patient survival. However, the role of teneurins in cancer-related regulatory networks is not fully understood, as both a tumor-suppressor role and pro-tumoral functions have been proposed, depending on tumor histotype. Here, we summarize and discuss the literature data on teneurins expression and their potential role in different tumor types, while highlighting the possibility of using teneurins as novel molecular diagnostic and prognostic biomarkers and as targets for cancer treatments, such as immunotherapy, in some tumors.

Tumor-Associated Antigen xCT and Mutant-p53 as Molecular Targets for New Combinatorial Antitumor Strategies.

The cystine/glutamate antiporter xCT is a tumor-associated antigen that has been newly identified in many cancer types. By participating in glutathione biosynthesis, xCT protects cancer cells from oxidative stress conditions and ferroptosis, and contributes to metabolic reprogramming, thus promoting tumor progression and chemoresistance. Moreover, xCT is overexpressed in cancer stem cells. These features render xCT a promising target for cancer therapy, as has been widely reported in the literature and in our work on its immunotargeting. Interestingly, studies on the TP53 gene have revealed that both wild-type and mutant p53 induce the post-transcriptional down modulation of xCT, contributing to ferroptosis. Moreover, APR-246, a small molecule drug that can restore wild-type p53 function in cancer cells, has been described as an indirect modulator of xCT expression in tumors with mutant p53 accumulation, and is thus a promising drug to use in combination with xCT inhibition. This review summarizes the current knowledge of xCT and its regulation by p53, with a focus on the crosstalk of these two molecules in ferroptosis, and also considers some possible combinatorial strategies that can make use of APR-246 treatment in combination with anti-xCT immunotargeting.

Immunotargeting of the xCT Cystine/Glutamate Antiporter Potentiates the Efficacy of HER2-Targeted Immunotherapies in Breast Cancer.

Despite HER2-targeted therapies improving the outcome of HER2+ breast cancer, many patients experience resistance and metastatic progression. Cancer stem cells (CSC) play a role in this resistance and progression, thus combining HER2 targeting with CSC inhibition could improve the management of HER2+ breast cancer. The cystine-glutamate antiporter, xCT, is overexpressed in mammary CSCs and is crucial for their redox balance, self-renewal, and resistance to therapies, representing a potential target for breast cancer immunotherapy. We developed a combined immunotherapy targeting HER2 and xCT using the Bovine Herpes virus-4 vector, a safe vaccine that can confer immunogenicity to tumor antigens. Mammary cancer-prone BALB-neuT mice, transgenic for rat Her2, were immunized with the single or combined vaccines. Anti-HER2 vaccination slowed primary tumor growth, whereas anti-xCT vaccination primarily prevented metastasis formation. The combination of the two vaccines exerted a complementary effect by mediating the induction of cytotoxic T cells and of HER2 and xCT antibodies that induce antibody-dependent cell-mediated cytotoxicity and hinder cancer cell proliferation. Antibodies targeting xCT, but not those targeting HER2, directly affected CSC viability, self-renewal, and migration, inducing the antimetastatic effect of xCT vaccination. Our findings present a new therapy for HER2+ breast cancer, demonstrating that CSC immunotargeting via anti-xCT vaccination synergizes with HER2-directed immunotherapy.

L-ferritin: A theranostic agent of natural origin for MRI visualization and treatment of breast cancer.

The altered regulation of iron uptake and metabolism in cancerous cells, along with the potential of this metal to cause oxidative stress and cell death, makes iron overload an attractive therapeutic strategy for cancer treatment. In this study, the selective uptake of native HoS-ferritin (Horse-Spleen Ferritin) was assessed in TS/A breast cancer cells and compared with benign cystadenoma NMuMG. The higher expression of L-ferritin receptor SCARA5 led to an enhanced uptake in TS/A that is detected by the generation of a negative contrast in the corresponding MR images. The toxicity of HoS-ferritin toward TS/A cells has been investigated in detail in vitro, showing that cellular vitality is inversely related to the amount of internalized iron content. Finally, biodistribution and therapeutic efficacy of HoS-ferritin have been shown for the first time in vivo on a orthotopic breast cancer mice model, suggesting that iron overdose delivered by the HoS-ferritin can trigger selective mechanisms of regulated cell death.

Cancer stem cell immunology and immunotherapy: Harnessing the immune system against cancer's source.

Despite recent advances in diagnosis and therapy having improved cancer outcome, many patients still do not respond to treatments, resulting in the progression or relapse of the disease, eventually impairing survival expectations. The limited efficacy of therapy is often attributable to its inability to affect cancer stem cells (CSCs), a small population of cells resistant to current radio- and chemo-therapies. CSCs are characterized by self-renewal and tumor-initiating capabilities, and function as a reservoir for the local and distant recurrence of the disease. Therefore, new therapeutic approaches able to effectively target and deplete CSCs are urgently needed. Immunotherapy is facing a renewed interest for its potential in cancer treatment, and the possibility of harnessing the immune system to target CSCs is being addressed by a new exciting research field. In this chapter, we discuss the cancer stem cell model and illustrate CSC biological and molecular properties, critically addressing theoretical and practical issues linked with their definition and study. We then review the existing literature regarding the immunological properties of CSCs and the complex interplay occurring between CSCs and immune cells. Finally, we present up-to-date studies on CSC immunotargeting and its potential future perspective. In conclusion, understanding the interplay between CSC biology and tumor immunology will provide a deeper understanding of the mechanisms that regulate CSC immunological properties. This will contribute to the design of new CSC-directed immunotherapeutic strategies with the potential of strongly improving cancer outcomes.

Fighting breast cancer stem cells through the immune-targeting of the xCT cystine-glutamate antiporter.

Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2+ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.

Bovine herpesvirus 4-based vector delivering the full length xCT DNA efficiently protects mice from mammary cancer metastases by targeting cancer stem cells.

Despite marked advancements in its treatment, breast cancer is still the second leading cause of cancer death in women, due to relapses and distal metastases. Breast cancer stem cells (CSCs), are a cellular reservoir for recurrence, metastatic evolution and disease progression, making the development of novel therapeutics that target CSCs, and thereby inhibit metastases, an urgent need. We have previously demonstrated that the cystine-glutamate antiporter xCT (SLC7A11), a protein that was shown to be overexpressed in mammary CSCs and that plays a key role in the maintenance of their redox balance, self-renewal and resistance to chemotherapy, is a potential target for mammary cancer immunotherapy. This paper reports on the development of an anti-xCT viral vaccine that is based on the bovine herpesvirus 4 (BoHV-4) vector, which we have previously showed to be a safe vaccine that can transduce cells in vivo and confer immunogenicity to tumor antigens. We show that the vaccination of BALB/c mice with BoHV-4 expressing xCT (BoHV-4-mxCT), impaired lung metastases induced by syngeneic mammary CSCs both in preventive and therapeutic settings. Vaccination induced T lymphocyte activation and the production of anti-xCT antibodies that can mediate antibody-dependent cell cytotoxicity (ADCC), and directly impair CSC phenotype, self-renewal and redox balance. Our findings pave the way for the potential future use of BoHV-4-based vector targeting xCT in metastatic breast cancer treatment.

Cripto-1 Plasmid DNA Vaccination Targets Metastasis and Cancer Stem Cells in Murine Mammary Carcinoma.

Metastatic breast cancer is a fatal disease that responds poorly to treatment. Cancer vaccines targeting antigens expressed by metastatic breast cancer cells and cancer stem cells could function as anticancer therapies. Cripto-1 is an oncofetal protein overexpressed in invasive breast cancer and cancer-initiating cells. In this study, we explored the potential of a Cripto-1-encoding DNA vaccine to target breast cancer in preclinical mouse models. BALB/c mice and BALB-neuT mice were treated with a DNA vaccine encoding mouse Cripto-1 (mCr-1). BALB/c mice were challenged with murine breast cancer 4T1 cells or TUBO spheres; BALB-neuT mice spontaneously developed breast cancer. Tumor growth was followed in all mouse models and lung metastases were evaluated. In vitro assays were performed to identify the immune response elicited by vaccination. Vaccination against mCr-1 reduced primary tumor growth in the 4T1 metastatic breast cancer model and reduced lung metastatic burden. In BALB-neuT mice, because the primary tumors are Cripto-1 negative, vaccination against mCr-1 did not affect primary tumors but did reduce lung metastatic burden. Spheroid-cultured TUBO cells, derived from a BALB/neuT primary tumor, develop a cancer stem cell-like phenotype and express mCr-1. We observed reduced tumor growth in vaccinated mice after challenge with TUBO spheres. Our data indicate that vaccination against Cripto-1 results in a protective immune response against mCr-1 expressing and metastasizing cells. Targeting Cripto-1 by vaccination holds promise as an immunotherapy for treatment of metastatic breast cancer. Cancer Immunol Res; 6(11); 1417-25. ©2018 AACR.

L-Ferritin targets breast cancer stem cells and delivers therapeutic and imaging agents.

A growing body of evidence suggests that cancer stem cells (CSC) have the unique biological properties necessary for tumor maintenance and spreading, and function as a reservoir for the relapse and metastatic evolution of the disease by virtue of their resistance to radio- and chemo-therapies. Thus, the efficacy of a therapeutic approach relies on its ability to effectively target and deplete CSC. In this study, we show that CSC-enriched tumorspheres from breast cancer cell lines display an increased L-Ferritin uptake capability compared to their monolayer counterparts as a consequence of the upregulation of the L-Ferritin receptor SCARA5. L-Ferritin internalization was exploited for the simultaneous delivery of Curcumin, a natural therapeutic molecule endowed with antineoplastic action, and the MRI contrast agent Gd-HPDO3A, both entrapped in the L-Ferritin cavity. This theranostic system was able to impair viability and self-renewal of tumorspheres in vitro and to induce the regression of established tumors in mice. In conclusion, here we show that Curcumin-loaded L-Ferritin has a strong therapeutic potential due to the specific targeting of CSC and the improved Curcumin bioavailability, opening up the possibility of its use in a clinical setting.

The non-inflammatory role of C1q during Her2/neu-driven mammary carcinogenesis.

There is an ever increasing amount of evidence to support the hypothesis that complement C1q, the first component of the classical complement pathway, is involved in the regulation of cancer growth, in addition to its role in fighting infections. It has been demonstrated that C1q is expressed in the microenvironment of various types of human tumors, including breast adenocarcinomas. This study compares carcinogenesis progression in C1q deficient (neuT-C1KO) and C1q competent neuT mice in order to investigate the role of C1q in mammary carcinogenesis. Significantly accelerated autochthonous neu+ carcinoma progression was paralleled by accelerated spontaneous lung metastases occurrence in C1q deficient mice. Surprisingly, this effect was not caused by differences in the tumor-infiltrating cells or in the activation of the complement classical pathway, since neuT-C1KO mice did not display a reduction in C3 fragment deposition at the tumor site. By contrast, a significant higher number of intratumor blood vessels and a decrease in the activation of the tumor suppressor WW domain containing oxidoreductase (WWOX) were observed in tumors from neuT-C1KO as compare with neuT mice. In parallel, an increase in Her2/neu expression was observed on the membrane of tumor cells. Taken together, our findings suggest that C1q plays a direct role both on halting tumor angiogenesis and on inducing apoptosis in mammary cancer cells by coordinating the signal transduction pathways linked to WWOX and, furthermore, highlight the role of C1q in mammary tumor immune surveillance regardless of complement system activation.

Immunotargeting of Antigen xCT Attenuates Stem-like Cell Behavior and Metastatic Progression in Breast Cancer.

Resistance to therapy and lack of curative treatments for metastatic breast cancer suggest that current therapies may be missing the subpopulation of chemoresistant and radioresistant cancer stem cells (CSC). The ultimate success of any treatment may well rest on CSC eradication, but specific anti-CSC therapies are still limited. A comparison of the transcriptional profiles of murine Her2(+) breast tumor TUBO cells and their derived CSC-enriched tumorspheres has identified xCT, the functional subunit of the cystine/glutamate antiporter system xc(-), as a surface protein that is upregulated specifically in tumorspheres. We validated this finding by cytofluorimetric analysis and immunofluorescence in TUBO-derived tumorspheres and in a panel of mouse and human triple negative breast cancer cell-derived tumorspheres. We further show that downregulation of xCT impaired tumorsphere generation and altered CSC intracellular redox balance in vitro, suggesting that xCT plays a functional role in CSC biology. DNA vaccination based immunotargeting of xCT in mice challenged with syngeneic tumorsphere-derived cells delayed established subcutaneous tumor growth and strongly impaired pulmonary metastasis formation by generating anti-xCT antibodies able to alter CSC self-renewal and redox balance. Finally, anti-xCT vaccination increased CSC chemosensitivity to doxorubicin in vivo, indicating that xCT immunotargeting may be an effective adjuvant to chemotherapy.

Microenvironment, oncoantigens, and antitumor vaccination: lessons learned from BALB-neuT mice.

The tyrosine kinase human epidermal growth factor receptor 2 (HER2) gene is amplified in approximately 20% of human breast cancers and is associated with an aggressive clinical course and the early development of metastasis. Its crucial role in tumor growth and progression makes HER2 a prototypic oncoantigen, the targeting of which may be critical for the development of effective anticancer therapies. The setup of anti-HER2 targeting strategies has revolutionized the clinical outcome of HER2(+) breast cancer. However, their initial success has been overshadowed by the onset of pharmacological resistance that renders them ineffective. Since the tumor microenvironment (TME) plays a crucial role in drug resistance, the design of more effective anticancer therapies should depend on the targeting of both cancer cells and their TME as a whole. In this review, starting from the successful know-how obtained with a HER2(+) mouse model of mammary carcinogenesis, the BALB-neuT mice, we discuss the role of TME in mammary tumor development. Indeed, a deeper knowledge of antigens critical for cancer outbreak and progression and of the mechanisms that regulate the interplay between cancer and stromal cell populations could advise promising ways for the development of the best anticancer strategy.