Nuclear Factor-Kappa B Inhibition Can Enhance Apoptosis of Differentiated Thyroid Cancer Cells Induced by 131I

Objective: To evaluate changes of nuclear factor-kappa B (NF-kB) during radioiodine 131 ( 131 I) therapy and whether NF-kB inhibition could enhance 131 I-induced apoptosis in differentiated thyroid cancer (DTC) cells in a synergistic manner. Methods: Three human DTC cell lines were used. NF-kB inhibition was achieved by using a NF-kB inhibitor (Bay 11-7082) or by p65 siRNA transfection. Methyl-thiazolyl-tetrazolium assay was performed for cell viability assessment. DNA-binding assay, luciferase reporter assay, and Western blot were adopted to determine function and expression changes of NF-kB. Then NF-kB regulated anti-apoptotic factors XIAP, cIAP1, and Bcl-xL were measured. Apoptosis was analyzed by Western blot for caspase 3 and PARP, and by flow cytometry as well. An iodide uptake assay was performed to determine whether NF-kB inhibition could influence radioactive iodide uptake. Results: The methyl-thiazolyl-tetrazolium assay showed significant decrease of viable cells by combination therapy than by mono-therapies. The DNA-binding assay and luciferase reporter assay showed enhanced NF-kB function and reporter gene activities due to 131 I, yet significant suppression was achieved by NF-kB inhibition. Western blot proved 131 I could increase nuclear NF-kB concentration, while NF-kB inhibition reduced NF-kB concentration. Western blot also demonstrated significant up-regulation of XIAP, cIAP1, and Bcl-xL after 131 I therapy. And inhibition of NF-kB could significantly downregulate these factors. Finally, synergism induced by combined therapy was displayed by significant enhancements o

Down-regulation of ABCC11 protein (MRP8) in human breast cancer

Aim of this article is to investigate the expression of ABCC11 (MRP8) protein in normal breast tissue, and examine the difference in ABCC11 mRNA and protein expression between normal breast and breast cancer tissues taking into account ABCC11 genotype (a functional SNP, rs17822931) and estrogen receptor (ER) status.We are grateful to Dr. Vladimir Saenko, Nagasaki University, for his valuable and critical comments. This study was supported by Grant-in-Aid for Science Research (Category B) for N. Niikawa from the Ministry of Education, Culture, Sports, Science and technology of Japan and Research fund in Health Sciences University of Hokkaido

Inhibitor of the transcription factor NF-κB, DHMEQ, enhances the effect of paclitaxel on cells of anaplastic thyroid carcinoma in vitro and in vivo

Anticancer drug paclitaxel (Ptx) effect on biochemical mechanisms, regulating apoptosis in anaplastic thyroid carcinoma cells, was studied. It was shown that in addition to apoptotic cell death, Ptx induces signa­ling cascades that ensure cell survival. Paclitaxel-induced activation of nuclear factor kappa B (NF‑κВ) leads to an increase of some antiapoptotic proteins expression such as survivin, cIAP, XIAP. A novel NF‑κВ inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), was found to enhance cytotoxic effect of Ptx in anaplastic thyroid carcinoma cells. An enhancement of caspase-3 and -9 activation and PARP cleavage as well as the decreased levels of proteins-inhibitors of apoptosis were observed when cells were treated with a combination of both drugs. Mitochondria transmembrane potential (ΔΨm) loss was observed at higher concentrations of Ptx and DHMEQ. NF-κВ inhibition also potentiates paclitaxel effect at tumors formed by xenotransplantation of FRO cells into mice. Tumor mass reduction, significantly different from the effects of each of the compounds alone, was observed in animals, treated with paclitaxel and NF-κВ inhibitor. Thus, the combined use of paclitaxel and NF-κВ inhibitor inhibits biochemical processes that contribute to the resistance of anaplastic thyroid carcinoma cells to paclitaxel action

Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth

Abstract We recently reported that aberrant expression of Major Histocompatibility Class II (MHCII) on human triple negative breast cancer cells correlates with increased tumor infiltrating lymphocytes and prolonged progression free survival. This observation led us to hypothesize that expression of MHCII enhanced the intratumoral CD4+ T cell response, thereby bolstering the tumor-specific CD8+ T cell response, leading to impaired tumor growth. To test this, we transfected the murine breast cancer line TS/A with human class II transcriptional activator (hCIITA) or empty vector, creating MHCII-expressing and MHCII-negative cell lines, respectively. We found that MHCII-expressing tumors grew slower than controls in immunocompetent recipients, but that this difference was abrogated in CD4-depleted and nullified in SCID mice. CD4+ T cells within hCIITA-transfected tumors produced more IFNγ for longer times than those from control tumors. Similarly, CD8+ T cells in MHCII-expressing tumors displayed a more activated phenotype and produced more IFNγ and granzyme B for longer times. Nevertheless, both CD4+ and CD8+ T cells eventually became exhausted in both groups. In addition to boosted effector function, TCR repertoire analysis demonstrated an increase in both breadth and amplitude of T cell response to MHCII-expressing tumors. We next investigated the possibility of inducing MHCII on non-expressing tumors using the histone deacetylase inhibitor Entinostat. Indeed, Entinostat turned on MHCII expression, which correlated with significantly reduced tumor burden. Thus, epigenetic modifiers capable of inducing MHCII expression on tumor cells could avail this augmented T cell response to all patients for enhanced tumor control.</jats:p

Abstract 643: Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth

Abstract We recently reported that the aberrant expression of Major Histocompatibility Class II (MHCII) molecules on human triple negative breast cancer (TNBC) cells correlates with prolonged progression-free survival and increased tumor infiltrating lymphocytes. We hypothesized that the expression of MHCII enhances the intratumoral CD4+ T cell response, thereby bolstering the tumor-specific CD8+ T cell response, resulting in more effective tumor control. To test our hypothesis, we created both MHCII-expressing and MHCII-negative tumor cells by transfecting murine breast cancer (TS/A) cells with the human class II transcriptional activator (hCIITA) or empty vector, respectively. Transfected cells were then injected into BALB/c mice and the resulting immune response analyzed by flow cytometry at four time points. We found that hCIITA-expressing tumors grew slower than control tumors in immunocompetent recipients, but that this difference was nullified in immunocompromised and markedly reduced in CD4+ T cell depleted mice. CD4+ T cells isolated from hCIITA-transfected tumors produced more IFNγ, IL-17A, and surprisingly granzyme B for longer times than their counterparts in control tumors. Similarly, CD8+ T cells isolated from hCIITA-transfected tumors displayed a more activated phenotype and produced more IFNγ and granzyme B for longer times. Nevertheless, both CD4+ and CD8+ T cells eventually became exhausted in both groups. In addition to enhanced effector functions, TCR repertoire analysis demonstrated that both the breadth and magnitude of expansion of responding T cell clones were increased in hCIITA-transfected tumors. Interestingly, TS/A-hCIITA tumors harbored more regulatory T cells (Tregs) with a more suppressive phenotype than Tregs from control tumors. Finally, we show that the histone deacetylase inhibitor (HDACi) Entinostat is capable of robust and dose-dependent induction of MHCII on tumor cells in vivo, an effect that correlates with dramatic reduction in tumor size. These results suggest that the clinical benefit associated with MHCII expression on TNBC cells is mediated by a delay in T cell exhaustion and increased intratumoral CD4+ T cell activation, which enhances the cytotoxic capacity of CD8+ T cells. Entinostat, and potentially other epigenetic modifying agents, may enable induction of MHCII expression on TNBC cells clinically and allow more patients to benefit from an augmented T cell response. These effects may be magnified by combinatorial therapy with checkpoint inhibitors to promote durable anti-tumor immune responses. Citation Format: Tyler R. McCaw, Mei Li, Selene Meza-Perez, Donald J. Buchsbaum, Dmytro Starenki, Sara Cooper, Andres Forero, Troy D. Randall. Induced MHCII expression on breast cancer cells broadens the responding T cell repertoire, delays tumor-specific T cell exhaustion, and impairs tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 643. doi:10.1158/1538-7445.AM2017-643</jats:p

Precisely timed histone deacetylase inhibition creates a highly proliferative intratumoral CD8 T cell population and sensitizes tumors to checkpoint blockade

Abstract We previously showed that a class I histone deacetylase (HDAC) inhibitor, entinostat (ENT), dramatically improves CD8 T cell-mediated control of TS/A murine mammary tumors, but only when given in a precise window. If given too early ENT halts initial T cell activation and expansion and has no effect if given too late— once T cells start becoming dysfunctional. This led us to hypothesize that HDAC inhibition can maintain CD8 T cells in a less differentiated state that enhances effector and proliferative capacities. To test this, we first treated TS/A tumor-bearing mice with ENT and performed single cell RNA-sequencing on sorted tumor-infiltrating CD8 T cells. Relative to vehicle, ENT treated samples clustered separately and included a distinct, highly proliferative cluster of CD8 T cells, a finding corroborated by bulk RNA-sequencing and flow cytometry. Although TS/A tumors do not respond to anti-PD1 alone, ENT-induced changes in CD8 T cell phenotype sensitized tumors to PD1 blockade, leading to tumor rejection in most mice when both agents were timed appropriately. Patient tumors, however, have often passed this window to intervene; thus, we devised a tripartite strategy to “reset the clock”. Treating first with an oncolytic virus to liberate new antigens and stimulate new tumor-specific CD8 T cells, then with ENT and anti-PD1 at the right times significantly impaired growth of late-stage tumors and even led to rejection. Our findings show that precisely timed HDAC inhibition generates highly proliferative and strongly cytotoxic CD8 T cell populations whose functions can be further elaborated with correctly timed checkpoint blockade. Resetting the clock using a tumor-lytic strategy then renders this a potent immunotherapy platform.</jats:p

Circulating Tregs Accumulate in Omental Tumors and Acquire Adipose-Resident Features

Abstract Tumors that metastasize in the peritoneal cavity typically end up in the omental adipose tissue, a particularly immune-suppressive environment that includes specialized adipose-resident regulatory T cells (Treg). Tregs rapidly accumulate in the omentum after tumor implantation and potently suppress antitumor immunity. However, it is unclear whether these Tregs are recruited from the circulation or derived from preexisting adipose-resident Tregs by clonal expansion. Here we show that Tregs in tumor-bearing omenta predominantly have thymus-derived characteristics. Moreover, naïve tumor antigen-specific CD4+ T cells fail to differentiate into Tregs in tumor-bearing omenta. In fact, Tregs derived from the pretumor repertoire are sufficient to suppress antitumor immunity and promote tumor growth. However, tumor implantation in the omentum does not promote Treg clonal expansion, but instead leads to increased clonal diversity. Parabiosis experiments show that despite tissue-resident (noncirculating) characteristics of omental Tregs in naïve mice, tumor implantation promotes a rapid influx of circulating Tregs, many of which come from the spleen. Finally, we show that newly recruited Tregs rapidly acquire characteristics of adipose-resident Tregs in tumor-bearing omenta. These data demonstrate that most Tregs in omental tumors are recruited from the circulation and adapt to their environment by altering their homing, transcriptional, and metabolic properties. </jats:sec