DNA damage repair kinase DNA-PK and cGAS synergize to induce cancer-related inflammation in glioblastoma.

Cytosolic DNA promotes inflammatory responses upon detection by the cyclic GMP-AMP (cGAMP) synthase (cGAS). It has been suggested that cGAS downregulation is an immune escape strategy harnessed by tumor cells. Here, we used glioblastoma cells that show undetectable cGAS levels to address if alternative DNA detection pathways can promote pro-inflammatory signaling. We show that the DNA-PK DNA repair complex (i) drives cGAS-independent IRF3-mediated type I Interferon responses and (ii) that its catalytic activity is required for cGAS-dependent cGAMP production and optimal downstream signaling. We further show that the cooperation between DNA-PK and cGAS favors the expression of chemokines that promote macrophage recruitment in the tumor microenvironment in a glioblastoma model, a process that impairs early tumorigenesis but correlates with poor outcome in glioblastoma patients. Thus, our study supports that cGAS-dependent signaling is acquired during tumorigenesis and that cGAS and DNA-PK activities should be analyzed concertedly to predict the impact of strategies aiming to boost tumor immunogenicity.

Radiotherapy: An immune response modifier for immuno-oncology.

The ability of radiotherapy to enhance antigenicity and adjuvanticity of an irradiated tumor has stimulated the interest for its combination with immuno-oncology agents. However, radiotherapy often generates multiple layers of host responses which likely depends on the tumor biology, the immune cell infiltration and the induction of immunosuppressive signals post radiotherapy. Consequently, translation of preclinical findings to the clinic is more convoluted than anticipated which underscore the need to decipher molecular and cellular mechanisms elicited by radiotherapy. Here we review pro-inflammatory and immunosuppressive mechanisms triggered by radiotherapy that impact the outcome of antigen specific T cell killing and discuss how radiation-induced immunostimulatory mechanisms can be exploited to reactivate the host's immune system, especially in the context of immunotherapy.

Invasive micropapillary carcinoma of the breast overexpresses MUC4 and is associated with poor outcome to adjuvant trastuzumab in HER2-positive breast cancer.

BACKGROUND: Invasive micropapillary carcinoma of the breast (IMPC) is a histological tumor variant that occurs with low frequency characterized by an inside-out formation of tumor clusters with a pseudopapillary arrangement. IMPC is an aggressive tumor with poor clinical outcome. In addition, this histological subtype usually expresses human epidermal growth factor receptor 2 (HER2) which also correlates with a more aggressive tumor. In this work we studied the clinical significance of IMPC in HER2-positive breast cancer patients treated with adjuvant trastuzumab. We also analyzed mucin 4 (MUC4) expression as a novel biomarker to identify IMPC. METHODS: We retrospectively studied 86 HER2-positive breast cancer patients treated with trastuzumab and chemotherapy in the adjuvant setting. We explored the association of the IMPC component with clinicopathological parameters at diagnosis and its prognostic value. We compared MUC4 expression in IMPC with respect to other histological breast cancer subtypes by immunohistochemistry. RESULTS: IMPC, either as a pure entity or associated with invasive ductal carcinoma (IDC), was present in 18.6% of HER2-positive cases. It was positively correlated with estrogen receptor expression and tumor size and inversely correlated with patient's age. Disease-free survival was significantly lower in patients with IMPC (hazard ratio = 2.6; 95%, confidence interval 1.1-6.1, P = 0.0340). MUC4, a glycoprotein associated with metastasis, was strongly expressed in all IMPC cases tested. IMPC appeared as the histological breast cancer subtype with the highest MUC4 expression compared to IDC, lobular and mucinous carcinoma. CONCLUSION: In HER2-positive breast cancer, the presence of IMPC should be carefully examined. As it is often not informed, because it is relatively difficult to identify or altogether overlooked, we propose MUC4 expression as a useful biomarker to highlight IMPC presence. Patients with MUC4-positive tumors with IMPC component should be more frequently monitored and/or receive additional therapies.

Cancer cell metabolism and antitumour immunity.

Accumulating evidence suggests that metabolic rewiring in malignant cells supports tumour progression not only by providing cancer cells with increased proliferative potential and an improved ability to adapt to adverse microenvironmental conditions but also by favouring the evasion of natural and therapy-driven antitumour immune responses. Here, we review cancer cell-intrinsic and cancer cell-extrinsic mechanisms through which alterations of metabolism in malignant cells interfere with innate and adaptive immune functions in support of accelerated disease progression. Further, we discuss the potential of targeting such alterations to enhance anticancer immunity for therapeutic purposes.

Quantification of beta-galactosidase activity as a marker of radiation-driven cellular senescence.

Cellular senescence is a permanent state of cell cycle arrest that can be triggered by different stressors, including cancer treatments (the so-called "therapy-induced senescence"), such as radiation therapy (RT). Although senescent cells do not proliferate, they remain metabolically active and play a critical role in tumor progression, metastasis, and response to therapy. Therefore, investigating the induction of cellular senescence upon RT treatment is a critical read out for investigating RT efficacy or combinatorial strategies in cancer research. Senescent cells are characterized by a plethora of markers, including an increased content and activity of lysosomes, which can be detected by the activity of the lysosomal enzyme senescence-associated ß-galactosidase. In this chapter, we present a protocol for the gold standard cytochemical method for quantification of the activity of the senescence-associated ß-galactosidase in irradiated murine breast cancer cells in vitro.

Translating Science into Survival: Report on the Sixth International Cancer Immunotherapy Conference.

From September 28 to October 1, 2022, in New York City, the Sixth International Cancer Immunotherapy Conference was hosted jointly by the Cancer Research Institute (CRI), the American Association for Cancer Research, and the European Network for Cancer Immunotherapy. The four-day event covered the latest advances in cancer immunology and immunotherapy.

Fatty acid metabolism and radiation-induced anti-tumor immunity.

Fatty acid metabolic reprogramming has emerged as a major regulator of anti-tumor immune responses with large body of evidence that demonstrate its ability to impact the differentiation and function of immune cells. Therefore, depending on the metabolic cues that stem in the tumor microenvironment, the tumor fatty acid metabolism can tilt the balance of inflammatory signals to either promote or impair anti-tumor immune responses. Oxidative stressors such as reactive oxygen species generated from radiation therapy can rewire the tumor energy supply, suggesting that radiation therapy can further perturb the energy metabolism of a tumor by promoting fatty acid production. In this review, we critically discuss the network of fatty acid metabolism and how it regulates immune response especially in the context of radiation therapy.

Radiation therapy promotes unsaturated fatty acids to maintain survival of glioblastoma.

Radiation therapy (RT) is essential for the management of glioblastoma (GBM). However, GBM frequently relapses within the irradiated margins, thus suggesting that RT might stimulate mechanisms of resistance that limits its efficacy. GBM is recognized for its metabolic plasticity, but whether RT-induced resistance relies on metabolic adaptation remains unclear. Here, we show in vitro and in vivo that irradiated GBM tumors switch their metabolic program to accumulate lipids, especially unsaturated fatty acids. This resulted in an increased formation of lipid droplets to prevent endoplasmic reticulum (ER) stress. The reduction of lipid accumulation with genetic suppression and pharmacological inhibition of the fatty acid synthase (FASN), one of the main lipogenic enzymes, leads to mitochondrial dysfunction and increased apoptosis of irradiated GBM cells. Combination of FASN inhibition with focal RT improved the median survival of GBM-bearing mice. Supporting the translational value of these findings, retrospective analysis of the GLASS consortium dataset of matched GBM patients revealed an enrichment in lipid metabolism signature in recurrent GBM compared to primary. Overall, these results demonstrate that RT drives GBM resistance by generating a lipogenic environment permissive to GBM survival. Targeting lipid metabolism might be required to develop more effective anti-GBM strategies.

ELISA-based quantification of type I IFN secretion by irradiated cancer cells.

Cancer cell-intrinsic type I interferon (IFN-I) activation is required to initiate early innate immune responses and the subsequent radiation-induced anti-tumor immunity. Investigating the secretion of IFN-I cytokines in response to radiation therapy (RT) is therefore a critical readout for selecting the best immunogenic radiation dose-fractionation regimen. In this chapter, we present different ELISA-based quantification techniques that can be utilized to assess the secretion of tumor-derived IFN-I cytokines, namely IFN-α and IFN-ß.

Type I interferon induces cancer stem cells-mediated chemotherapy resistance.

In a recent study in Nature Immunology, Musella et al. demonstrate that suboptimal type I interferon (IFN-I) signaling in tumors undergoing immunogenic cell death (ICD) facilitates the accumulation of cancer stem cells (CSCs) by triggering the epigenetic regulator lysine demethylase 1B (KDM1B). KDM1B stands out as a promising target for the development of novel strategies to improve anti-cancer responses driven by ICD.

Halting ErbB-2 isoforms retrograde transport to the nucleus as a new theragnostic approach for triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is clinically defined by the absence of estrogen and progesterone receptors and the lack of membrane overexpression or gene amplification of receptor tyrosine kinase ErbB-2/HER2. Due to TNBC heterogeneity, clinical biomarkers and targeted therapies for this disease remain elusive. We demonstrated that ErbB-2 is localized in the nucleus (NErbB-2) of TNBC cells and primary tumors, from where it drives growth. We also discovered that TNBC expresses both wild-type ErbB-2 (WTErbB-2) and alternative ErbB-2 isoform c (ErbB-2c). Here, we revealed that the inhibitors of the retrograde transport Retro-2 and its cyclic derivative Retro-2.1 evict both WTErbB-2 and ErbB-2c from the nucleus of BC cells and tumors. Using BC cells from several molecular subtypes, as well as normal breast cells, we demonstrated that Retro-2 specifically blocks proliferation of BC cells expressing NErbB-2. Importantly, Retro-2 eviction of both ErbB-2 isoforms from the nucleus resulted in a striking growth abrogation in multiple TNBC preclinical models, including tumor explants and xenografts. Our mechanistic studies in TNBC cells revealed that Retro-2 induces a differential accumulation of WTErbB-2 at the early endosomes and the plasma membrane, and of ErbB-2c at the Golgi, shedding new light both on Retro-2 action on endogenous protein cargoes undergoing retrograde transport, and on the biology of ErbB-2 splicing variants. In addition, we revealed that the presence of a functional signal peptide and a nuclear export signal (NES), both located at the N-terminus of WTErbB-2, and absent in ErbB-2c, accounts for the differential subcellular distribution of ErbB-2 isoforms upon Retro-2 treatment. Our present discoveries provide evidence for the rational repurposing of Retro-2 as a novel therapeutic agent for TNBC.

Activin A backs-up TGF-ß to promote regulatory T cells.

The mechanisms accountable for the infiltration of regulatory T cells into an irradiated tumor remain elusive. In our recent study, we demonstrate that activin A promotes regulatory T cells in tumors, and impairs anti-tumor immune responses induced by radiotherapy and TGF-ß blockade. Dual blockade of activin A and TGF-ß may be necessary to reduce regulatory T cells mediated immunosuppression driven by radiation therapy.

Immunological barriers to immunotherapy in primary and metastatic breast cancer.

Patients with breast cancer obtain limited clinical benefits from immune checkpoint inhibitors (ICIs), pointing to the existence of multiple immunological alterations that cannot be simultaneously normalized with immunotherapy. Accumulating preclinical evidence suggests that radiation therapy (RT) can be harnessed to sensitize primary and metastatic mouse mammary carcinomas to ICIs. However, various clinical trials combining RT with ICIs in patients with breast cancer documented little cooperativity. Here, we discuss immunological barriers that may prevent RT from unlocking the therapeutic potential of ICIs in patients with breast cancer. These observations may inspire the development of combinatorial regimens that might benefit patients with diverse neoplastic conditions including brain tumors.

Exploiting Radiation Therapy to Restore Immune Reactivity of Glioblastoma.

Glioblastoma (GBM) is among the most aggressive of brain tumors and confers a dismal prognosis despite advances in surgical technique, radiation delivery methods, chemotherapy, and tumor-treating fields. While immunotherapy (IT) has improved the care of several adult cancers with previously dismal prognoses, monotherapy with IT in GBM has shown minimal response in first recurrence. Recent discoveries in lymphatics and evaluation of blood brain barrier offer insight to improve the use of ITs and determine the best combinations of therapies, including radiation. We highlight important features of the tumor immune microenvironment in GBM and potential for combining radiation and immunotherapy to improve prognosis in this devastating disease.

Activin A Promotes Regulatory T-cell-Mediated Immunosuppression in Irradiated Breast Cancer.

Increased regulatory T cells (Treg) after radiotherapy have been reported, but the mechanisms of their induction remain incompletely understood. TGFß is known to foster Treg differentiation within tumors and is activated following radiotherapy. Thus, we hypothesized that TGFß blockade would result in decreased Tregs within the irradiated tumor microenvironment. We found increased Tregs in the tumors of mice treated with focal radiotherapy and TGFß blockade. This increase was mediated by upregulation of another TGFß family member, activin A. In vitro, activin A secretion was increased following irradiation of mouse and human breast cancer cells, and its expression was further enhanced upon TGFß blockade. In vivo, dual blockade of activin A and TGFß was required to decrease intratumoral Tregs in the context of radiotherapy. This resulted in an increase in CD8+ T-cell priming and was associated with a reduced tumor recurrence rate. Combination of immune checkpoint inhibitors with the dual blockade of activin A and TGFß led to the development of tumor-specific memory responses in irradiated breast cancer. Supporting the translational value of activin A targeting to reduce Treg-mediated immunosuppression, retrospective analysis of a public dataset of patients with breast cancer revealed a positive correlation between activin A gene expression and Treg abundance. Overall, these results shed light on an immune escape mechanism driven by activin A and suggest that dual targeting of activin A and TGFß may be required to optimally unleash radiation-induced antitumor immunity against breast cancer.

Blockade of Stat3 oncogene addiction induces cellular senescence and reveals a cell-nonautonomous activity suitable for cancer immunotherapy.

Stat3 is constitutively activated in several tumor types and plays an essential role in maintaining their malignant phenotype and immunosupression. To take advantage of the promising antitumor activity of Stat3 targeting, it is vital to understand the mechanism by which Stat3 regulates both cell autonomous and non-autonomous processes. Here, we demonstrated that turning off Stat3 constitutive activation in different cancer cell types induces senescence, thus revealing their Stat3 addiction. Taking advantage of the senescence-associated secretory phenotype (SASP) induced by Stat3 silencing (SASP-siStat3), we designed an immunotherapy. The administration of SASP-siStat3 immunotherapy induced a strong inhibition of triple-negative breast cancer and melanoma growth associated with activation of CD4 + T and NK cells. Combining this immunotherapy with anti-PD-1 antibody resulted in survival improvement in mice bearing melanoma. The characterization of the SASP components revealed that type I IFN-related mediators, triggered by the activation of the cyclic GMP-AMP synthase DNA sensing pathway, are important for its immunosurveillance activity. Overall, our findings provided evidence that administration of SASP-siStat3 or low dose of Stat3-blocking agents would benefit patients with Stat3-addicted tumors to unleash an antitumor immune response and to improve the effectiveness of immune checkpoint inhibitors.

Canonical ErbB-2 isoform and ErbB-2 variant c located in the nucleus drive triple negative breast cancer growth.

Triple negative breast cancer (TNBC) refers to tumors that do not express clinically significant levels of estrogen and progesterone receptors, and lack membrane overexpression or gene amplification of ErbB-2/HER2, a receptor tyrosine kinase. Transcriptome and proteome heterogeneity of TNBC poses a major challenge to precision medicine. Clinical biomarkers and targeted therapies for this disease remain elusive, so chemotherapy has been the standard of care for early and metastatic TNBC. Our present findings placed ErbB-2 in an unanticipated scenario: the nucleus of TNBC (NErbB-2). Our study on ErbB-2 alternative splicing events, using a PCR-sequencing approach combined with an RNA interference strategy, revealed that TNBC cells express either the canonical (wild-type) ErbB-2, encoded by transcript variant 1, or the non-canonical ErbB-2 isoform c, encoded by alternative variant 3 (RefSeq), or both. These ErbB-2 isoforms function in the nucleus as transcription factors. Evicting both from the nucleus or silencing isoform c only, blocks TN cell and tumor growth. This reveals not only NErbB-2 canonical and alternative isoforms role as targets of therapy in TNBC, but also isoform c dominant oncogenic potential. Furthermore, we validated our findings in the clinic and observed that NErbB-2 correlates with poor prognosis in primary TN tumors, disclosing NErbB-2 as a novel biomarker for TNBC. Our discoveries challenge the present scenario of drug development for personalized BC medicine that focuses on wild-type RefSeq proteins, which conserve the canonical domains and are located in their classical cellular compartments.

Apoptotic Caspases: A Double-Edged Sword in Radiation-Induced Immunogenicity.

In a recent study in Oncoimmunology, Rodriguez-Ruiz et al. demonstrate that the apoptotic executioner caspase 3 (CASP3) attenuates antitumor immune responses induced by radiation therapy (RT) by inhibiting the release of cytosolic DNA. CASP3 stands out as a promising target for the development of novel strategies to improve anticancer responses driven by RT.