Radiation-Induced Innate Neutrophil Response in Tumor Is Mediated by the CXCLs/CXCR2 Axis.

The early events that lead to the inflammatory and immune-modulatory effects of radiation therapy (RT) in the tumor microenvironment (TME) after its DNA damage response activating the innate DNA-sensing pathways are largely unknown. Neutrophilic infiltration into the TME in response to RT is an early innate inflammatory response that occurs within 24-48 h. Using two different syngeneic murine tumor models (RM-9 and MC-38), we demonstrated that CXCR2 blockade significantly reduced RT-induced neutrophilic infiltration. CXCR2 blockade showed the same effects on RT-induced tumor inhibition and host survival as direct neutrophil depletion. Neutrophils highly and preferentially expressed CXCR2 compared to other immune cells. Importantly, RT induced both gene and protein expression of CXCLs in the TME within 24 h, attracting neutrophils into the tumor. Expectedly, RT also upregulated the gene expression of both cGAS and AIM2 DNA-sensing pathways in cGAS-positive MC-38 tumors but not in cGAS-negative RM-9 tumors. Activation of these pathways resulted in increased IL-1ß, which is known to activate the CXCLs/CXCR2 axis. Gene ontology analysis of mRNA-Seq supported these findings. Taken together, the findings suggest that the CXCLs/CXCR2 axis mediates the RT-induced innate inflammatory response in the TME, likely translating the effects of innate DNA-sensing pathways that are activated in response to RT-induced DNA damage.

Outcome and Immune Correlates of a Phase II Trial of High-Dose Interleukin-2 and Stereotactic Ablative Radiotherapy for Metastatic Renal Cell Carcinoma.

PURPOSE: This phase II clinical trial evaluated whether the addition of stereotactic ablative radiotherapy (SAbR), which may promote tumor antigen presentation, improves the overall response rate (ORR) to high-dose IL2 (HD IL2) in metastatic renal cell carcinoma (mRCC). PATIENTS AND METHODS: Patients with pathologic evidence of clear cell renal cell carcinoma (RCC) and radiographic evidence of metastasis were enrolled in this single-arm trial and were treated with SAbR, followed by HD IL2. ORR was assessed based on nonirradiated metastases. Secondary endpoints included overall survival (OS), progression-free survival (PFS), toxicity, and treatment-related tumor-specific immune response. Correlative studies involved whole-exome and transcriptome sequencing, T-cell receptor sequencing, cytokine analysis, and mass cytometry on patient samples. RESULTS: Thirty ethnically diverse mRCC patients were enrolled. A median of two metastases were treated with SAbR. Among 25 patients evaluable by RECIST v1.1, ORR was 16% with 8% complete responses. Median OS was 37 months. Treatment-related adverse events (AE) included 22 grade ≥3 events that were not dissimilar from HD IL2 alone. There were no grade 5 AEs. A correlation was observed between SAbR to lung metastases and improved PFS (P = 0.0165). Clinical benefit correlated with frameshift mutational load, mast cell tumor infiltration, decreased circulating tumor-associated T-cell clones, and T-cell clonal expansion. Higher regulatory/CD8+ T-cell ratios at baseline in the tumor and periphery correlated with no clinical benefit. CONCLUSIONS: Adding SAbR did not improve the response rate to HD IL2 in patients with mRCC in this study. Tissue analyses suggest a possible correlation between frameshift mutation load as well as tumor immune infiltrates and clinical outcomes.

Neoadjuvant SABR for Renal Cell Carcinoma Inferior Vena Cava Tumor Thrombus-Safety Lead-in Results of a Phase 2 Trial.

PURPOSE: To evaluate the feasibility, safety, oncologic outcomes, and immune effect of neoadjuvant stereotactic radiation (Neo-SAbR) followed by radical nephrectomy and thrombectomy (RN-IVCT). METHODS AND MATERIALS: These are results from the safety lead-in portion of a single-arm phase 1 and 2 trial. Patients with kidney cancer (renal cell carcinoma [RCC]) and inferior vena cava (IVC) tumor thrombus (TT) underwent Neo-SAbR (40 Gy in 5 fractions) to the IVC-TT followed by open RN-IVCT. Absence of grade 4 to 5 adverse events (AEs) within 90 days of RN-IVCT was the primary endpoint. Exploratory studies included pathologic and immunologic alterations attributable to SAbR. RESULTS: Six patients were included in the final analysis. No grade 4 to 5 AEs were observed. A total of 81 AEs were reported within 90 days of surgery: 73% (59/81) were grade 1, 23% (19/81) were grade 2, and 4% (3/81) were grade 3. After a median follow-up of 24 months, all patients are alive. One patient developed de novo metastatic disease. Of 3 patients with metastasis at diagnosis, 1 had a complete and another had a partial abscopal response without the concurrent use of systemic therapy. Neo-SABR led to decreased Ki-67 and increased PD-L1 expression in the IVC-TT. Inflammatory cytokines and autoantibody titers reflecting better host immune status were observed in patients with nonprogressive disease. CONCLUSIONS: Neo-SAbR followed by RN-IVCT for RCC IVC-TT is feasible and safe. Favorable host immune environment correlated with abscopal response to SABR and RCC relapse-free survival, though direct causal relation to SABR has yet to be established.

Type I Interferon Response in Radiation-Induced Anti-Tumor Immunity.

The anti-tumor activity of interferons (IFNs) was first appreciated about half a century ago, and IFN-α2 was the first cancer immunotherapy approved by the US Food and Drug Administration. Radiation therapy (RT), one of the pillars of cancer treatment, directly causes DNA damage, which can lead to senescence and cell death in tumor cells. In recent years, however, RT-induced immunomodulatory effects have been recognized to play an indispensable role in achieving the optimum therapeutic effect of RT. Increasing evidence indicates that RT enhances adaptive anti-tumor immunity by augmenting the innate immune sensing of tumors in a type I IFN-dependent matter. This review briefly introduces the role of type I interferon in cancer and the available evidence on the overall effects of RT on tumor immunity mediated via type I IFN. Recent advances in deciphering the molecular mechanisms underlying the induction of type I IFNs triggered by RT, their clinical implications, and therapeutic opportunities will be highlighted.

Tumor neoantigenicity assessment with CSiN score incorporates clonality and immunogenicity to predict immunotherapy outcomes.

Lack of responsiveness to checkpoint inhibitors is a central problem in the modern era of cancer immunotherapy. Tumor neoantigens are critical targets of the host antitumor immune response, and their presence correlates with the efficacy of immunotherapy treatment. Many studies involving assessment of tumor neoantigens principally focus on total neoantigen load, which simplistically treats all neoantigens equally. Neoantigen load has been linked with treatment response and prognosis in some studies but not others. We developed a Cauchy-Schwarz index of Neoantigens (CSiN) score to better account for the degree of concentration of immunogenic neoantigens in truncal mutations. Unlike total neoantigen load determinations, CSiN incorporates the effect of both clonality and MHC binding affinity of neoantigens when characterizing tumor neoantigen profiles. By analyzing the clinical responses in 501 treated patients with cancer (with most receiving checkpoint inhibitors) and the overall survival of 1978 patients with cancer at baseline, we showed that CSiN scores predict treatment response to checkpoint inhibitors and prognosis in patients with melanoma, lung cancer, and kidney cancer. CSiN score substantially outperformed prior genetics-based prediction methods of responsiveness and fills an important gap in research involving assessment of tumor neoantigen burden.

ERRα regulates the growth of triple-negative breast cancer cells via S6K1-dependent mechanism.

Estrogen-related receptor alpha (ERRα) is an orphan nuclear factor that is a master regulator of cellular energy metabolism. ERRα is overexpressed in a variety of tumors, including ovarian, prostate, colorectal, cervical and breast, and is associated with a more aggressive tumor and a worse outcome. In breast cancer, specifically, high ERRα expression is associated with an increased rate of recurrence and a poor prognosis. Because of the common functions of ERRα and the mTORC1/S6K1 signaling pathway in regulation of cellular metabolism and breast cancer pathogenesis, we focused on investigating the biochemical relationship between ERRα and S6K1. We found that ERRα negatively regulates S6K1 expression by directly binding to its promoter. Downregulation of ERRα expression sensitized ERα-negative breast cancer cells to mTORC1/S6K1 inhibitors. Therefore, our results show that combinatorial inhibition of ERRα and mTORC1/S6K1 may have clinical utility in treatment of triple-negative breast cancer, and warrants further investigation.

Estrogen induces RAD51C expression and localization to sites of DNA damage.

Homologous recombination (HR) is a conserved process that maintains genome stability and cell survival by repairing DNA double-strand breaks (DSBs). The RAD51-related family of proteins is involved in repair of DSBs; consequently, deregulation of RAD51 causes chromosomal rearrangements and stimulates tumorigenesis. RAD51C has been identified as a potential tumor suppressor and a breast and ovarian cancer susceptibility gene. Recent studies have also implicated estrogen as a DNA-damaging agent that causes DSBs. We found that in ERα-positive breast cancer cells, estrogen transcriptionally regulates RAD51C expression in ERα-dependent mechanism. Moreover, estrogen induces RAD51C assembly into nuclear foci at DSBs, which is a precursor to RAD51 complex recruitment to the nucleus. Additionally, disruption of ERα signaling by either anti-estrogens or siRNA prevented estrogen induced upregulation of RAD51C. We have also found an association of a worse clinical outcome between RAD51C expression and ERα status of tumors. These findings provide insight into the mechanism of genomic instability in ERα-positive breast cancer and suggest that individuals with mutations in RAD51C that are exposed to estrogen would be more susceptible to accumulation of DNA damage, leading to cancer progression.

Tamoxifen Action in ER-Negative Breast Cancer.

Breast cancer is a highly heterogeneous disease. Tamoxifen is a selective estrogen receptor (ER) modulator and is mainly indicated for the treatment of breast cancer in postmenopausal women and postsurgery neoadjuvant therapy in ER-positive breast cancers. Interestingly, 5-10% of the ER-negative breast cancers have also shown sensitivity to tamoxifen treatment. The involvement of molecular markers and/or signaling pathways independent of ER signaling has been implicated in tamoxifen sensitivity in the ER-negative subgroup. Studies reveal that variation in the expression of estrogen-related receptor alpha, ER subtype beta, tumor microenvironment, and epigenetics affects tamoxifen sensitivity. This review discusses the background of the research on the action of tamoxifen that may inspire future studies to explore effective therapeutic strategies for the treatment of ER-negative and triple-negative breast cancers, the latter being an aggressive disease with worse clinical outcome.

ERRα Is a Marker of Tamoxifen Response and Survival in Triple-Negative Breast Cancer.

PURPOSE: Estrogen-related receptor alpha (ERRα) signaling has recently been implicated in breast cancer. We investigated the clinical value of ERRα in randomized cohorts of tamoxifen-treated and adjuvant-untreated patients. EXPERIMENTAL DESIGN: Cox proportional hazards regression was used to evaluate the significance of associations between ERRα gene expression levels and patient DMFS in a previously published microarray dataset representing 2,000 breast tumor cases derived from multiple medical centers worldwide. The 912 tumors used for immunostaining were from a tamoxifen-randomized primary breast cancer trial conducted in Stockholm, Sweden, during 1976-1990. Mouse model was used to study the effect of tamoxifen treatment on lung colonization of MDA-MB-231 control cells and MDA-MB-231 cells with stable knockdown of ERRα. The phenotypic effects associated with ERRα modulation were studied using immunoblotting analyses and wound-healing assay. RESULTS: We found that in ER-negative and triple-negative breast cancer (TNBC) adjuvant-untreated patients, ERRα expression indicated worse prognosis and correlated with poor outcome predictors. However, in tamoxifen-treated patients, an improved outcome was observed with high ERRα gene and protein expression. Reduced ERRα expression was oncogenic in the presence of tamoxifen, measured by in vitro proliferation and migration assays and in vivo metastasis studies. CONCLUSIONS: Taken together, these data show that ERRα expression predicts response to tamoxifen treatment, and ERRα could be a biomarker of tamoxifen sensitivity and a prognostic factor in TNBC.

Proteasome inhibition increases recruitment of IκB kinase ß (IKKß), S536P-p65, and transcription factor EGR1 to interleukin-8 (IL-8) promoter, resulting in increased IL-8 production in ovarian cancer cells.

Proinflammatory and pro-angiogenic chemokine interleukin-8 (IL-8, CXCL8) contributes to ovarian cancer progression through its induction of tumor cell proliferation, survival, angiogenesis, and metastasis. Proteasome inhibition by bortezomib, which has been used as a frontline therapy in multiple myeloma, has shown only limited effectiveness in ovarian cancer and other solid tumors. However, the responsible mechanisms remain elusive. Here, we show that proteasome inhibition dramatically increases the IL-8 expression and release in ovarian cancer cells. The responsible mechanism involves an increased nuclear accumulation of IκB kinase ß (IKKß) and an increased recruitment of the nuclear IKKß, p65-phosphorylated at Ser-536, and the transcription factor early growth response-1 (EGR-1) to the endogenous IL-8 promoter. Coimmunoprecipitation studies identified the nuclear EGR-1 associated with IKKß and with p65, with preferential binding to S536P-p65. Both IKKß activity and EGR-1 expression are required for the increased IL-8 expression induced by proteasome inhibition in ovarian cancer cells. Interestingly, in multiple myeloma cells the IL-8 release is not increased by bortezomib. Together, these data indicate that the increased IL-8 release may represent one of the underlying mechanisms responsible for the decreased effectiveness of proteasome inhibition in ovarian cancer treatment and identify IKKß and EGR-1 as potential new targets in ovarian cancer combination therapies.

Proteasome inhibition by bortezomib increases IL-8 expression in androgen-independent prostate cancer cells: the role of IKKα.

Expression of the proinflammatory and proangiogenic chemokine IL-8, which is regulated at the transcriptional level by NF-κB, is constitutively increased in androgen-independent metastatic prostate cancer and correlates with poor prognosis. Inhibition of NF-κB-dependent transcription was used as an anticancer strategy for the development of the first clinically approved 26S proteasome inhibitor, bortezomib (BZ). Even though BZ has shown remarkable antitumor activity in hematological malignancies, it has been less effective in prostate cancer and other solid tumors; however, the mechanisms have not been fully understood. In this article, we report that proteasome inhibition by BZ unexpectedly increases IL-8 expression in androgen-independent prostate cancer PC3 and DU145 cells, whereas expression of other NF-κB-regulated genes is inhibited or unchanged. The BZ-increased IL-8 expression is associated with increased in vitro p65 NF-κB DNA binding activity and p65 recruitment to the endogenous IL-8 promoter. In addition, proteasome inhibition induces a nuclear accumulation of IκB kinase (IKK)α, and inhibition of IKKα enzymatic activity significantly attenuates the BZ-induced p65 recruitment to IL-8 promoter and IL-8 expression, demonstrating that the induced IL-8 expression is mediated, at least partly, by IKKα. Together, these data provide the first evidence, to our knowledge, for the gene-specific increase of IL-8 expression by proteasome inhibition in prostate cancer cells and suggest that targeting both IKKα and the proteasome may increase BZ effectiveness in treatment of androgen-independent prostate cancer.

Electrophoretic mobility shift assay analysis of NFκB transcriptional regulation by nuclear IκBα.

Transcription factor NFκB is a key regulator of genes involved in immune and inflammatory responses, as well as genes regulating cell proliferation and survival. In addition to many inflammatory disorders, NFκB is constitutively activated in a variety of human cancers and leukemia. Thus, inhibition of NFκB DNA binding activity represents an important therapeutic approach for disorders characterized by high levels of constitutive NFκB activity. We have previously shown that NFκB DNA binding activity is suppressed by the nuclear translocation and accumulation of IκBα, which is induced by inhibition of the 26S proteasome. In this chapter, we describe a protocol that uses small inhibitory RNA (si RNA) interference followed by electrophoretic mobility shift assay (EMSA) to analyze the regulation of NFκB DNA binding by nuclear IκBα induced by the proteasome inhibitor MG132. Using this protocol, we show that in human leukemia Hut-78 cells that exhibit high levels of NFκB DNA binding activity, MG132 induces nuclear translocation and accumulation of IκBα, which then specifically inhibits NFκB DNA binding. This protocol uses human leukemia Hut-78 cells; however, it can be easily adapted for other cells exhibiting high levels of constitutive NFκB DNA binding.

Chromatin immunoprecipitation analysis of NFκB transcriptional regulation by nuclear IκBα in human macrophages.

Transcription factor NFκB comprises a family of proteins that serve as crucial regulators of genes involved in host immune and inflammatory responses, cell survival, proliferation, and differentiation. Since transcription of NFκB-dependent genes is increased in numerous inflammatory disorders as well as in many types of cancer and leukemia, inhibition of NFκB-dependent transcription thus represents an important therapeutic target. We have previously shown that in human leukocytes, transcription of NFκB-dependent genes is inhibited by the nuclear translocation and accumulation of IκBα, which can be induced by an inhibitor of CRM1-dependent nuclear export, leptomycin B (LMB). In this chapter, we describe a protocol that uses chromatin immunoprecipitation (ChIP) to analyze the regulation of NFκB recruitment to NFκB-dependent promoters by nuclear IκBα induced by LMB. We show that in lipopolysaccharide (LPS)-stimulated human U-937 macrophages, recruitment of NFκB p65 and p50 proteins to NFκB-dependent promoters of IκBα and cIAP2 genes is suppressed by the LMB-induced nuclear IκBα. Even though in this study we use U-937 macrophages, this protocol should be readily adaptable to analyze the regulation of NFκB recruitment by nuclear IκBα also in other cell types.

Bortezomib induces nuclear translocation of IκBα resulting in gene-specific suppression of NF-κB--dependent transcription and induction of apoptosis in CTCL.

Cutaneous T-cell lymphoma (CTCL) is characterized by constitutive activation of nuclear factor κB (NF-κB), which plays a crucial role in the survival of CTCL cells and their resistance to apoptosis. NF-κB activity in CTCL is inhibited by the proteasome inhibitor bortezomib; however, the mechanisms remained unknown. In this study, we investigated mechanisms by which bortezomib suppresses NF-κB activity in CTCL Hut-78 cells. We demonstrate that bortezomib and MG132 suppress NF-κB activity in Hut-78 cells by a novel mechanism that consists of inducing nuclear translocation and accumulation of IκBα (nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha), which then associates with NF-κB p65 and p50 in the nucleus and inhibits NF-κB DNA binding activity. Surprisingly, however, while expression of NF-κB-dependent antiapoptotic genes cIAP1 and cIAP2 is inhibited by bortezomib, expression of Bcl-2 is not suppressed. Chromatin immunoprecipitation indicated that cIAP1 and cIAP2 promoters are occupied by NF-κB p65/50 heterodimers, whereas Bcl-2 promoter is occupied predominantly by p50/50 homodimers. Collectively, our data reveal a novel mechanism of bortezomib function in CTCL and suggest that the inhibition of NF-κB-dependent gene expression by bortezomib is gene specific and depends on the subunit composition of NF-κB dimers recruited to NF-κB-responsive promoters.