Kwon Ho Song

Nanog signaling in cancer promotes stem-like phenotype and immune evasion

Adaptation of tumor cells to the host is a major cause of cancer progression, failure of therapy, and ultimately death. Immune selection drives this adaptation in human cancer by enriching tumor cells with a cancer stem cell-like (CSC-like) phenotype that makes them resistant to CTL-mediated apoptosis; however, the mechanisms that mediate CSC maintenance and proliferation are largely unknown. Here, we report that CTL-mediated immune selection drives the evolution of tumor cells toward a CSC-like phenotype and that the CSC-like phenotype arises through the Akt signaling pathway via transcriptional induction of Tcl1a by Nanog. Furthermore, we found that hyperactivation of the Nanog/Tcl1a/Akt signaling axis was conserved across multiple types of human cancer. Inhibition of Nanog in a murine model of colon cancer rendered tumor cells susceptible to immune-mediated clearance and led to successful, long-term control of the disease. Our findings establish a firm link among immune selection, disease progression, and the development of a stem-like tumor phenotype in human cancer and implicate the Nanog/Tcl1a/Akt pathway as a central molecular target in this process.

API5 Confers Tumoral Immune Escape through FGF2-Dependent Cell Survival Pathway

Identifying immune escape mechanisms used by tumors may define strategies to sensitize them to immunotherapies to which they are otherwise resistant. In this study, we show that the antiapoptotic gene API5 acts as an immune escape gene in tumors by rendering them resistant to apoptosis triggered by tumor antigen-specific T cells. Its RNAi-mediated silencing in tumor cells expressing high levels of API5 restored antigen-specific immune sensitivity. Conversely, introducing API5 into API5(low) cells conferred immune resistance. Mechanistic investigations revealed that API5 mediated resistance by upregulating FGF2 signaling through a FGFR1/PKCδ/ERK effector pathway that triggered degradation of the proapoptotic molecule BIM. Blockade of FGF2, PKCδ, or ERK phenocopied the effect of API5 silencing in tumor cells expressing high levels of API5 to either murine or human antigen-specific T cells. Our results identify a novel mechanism of immune escape that can be inhibited to potentiate the efficacy of targeted active immunotherapies.

The siRNA cocktail targeting interleukin 10 receptor and transforming growth factor‐β receptor on dendritic cells potentiates tumour antigen‐specific CD8+ T cell immunity

Dendritic cells (DCs) are promising therapeutic agents in the field of cancer immunotherapy due to their intrinsic immune‐priming capacity. The potency of DCs, however, is readily attenuated immediately after their administration in patients as tumours and various immune cells, including DCs, produce various immunosuppressive factors such as interleukin (IL)‐10 and transforming growth factor (TGF)‐β that hamper the function of DCs. In this study, we used small interfering RNA (siRNA) to silence the expression of endogenous molecules in DCs, which can sense immunosuppressive factors. Among the siRNAs targeting various immunosuppressive molecules, we observed that DCs transfected with siRNA targeting IL‐10 receptor alpha (siIL‐10RA) initiated the strongest antigen‐specific CD8+ T cell immune responses. The potency of siIL‐10RA was enhanced further by combining it with siRNA targeting TGF‐β receptor (siTGF‐βR), which was the next best option during the screening of this study, or the previously selected immunoadjuvant siRNA targeting phosphatase and tensin homologue deleted on chromosome 10 (PTEN) or Bcl‐2‐like protein 11 (BIM). In the midst of sorting out the siRNA cocktails, the cocktail of siIL‐10RA and siTGF‐βR generated the strongest antigen‐specific CD8+ T cell immunity. Concordantly, the knock‐down of both IL‐10RA and TGF‐βR in DCs induced the strongest anti‐tumour effects in the TC‐1 P0 tumour model, a cervical cancer model expressing the human papillomavirus (HPV)‐16 E7 antigen, and even in the immune‐resistant TC‐1 (P3) tumour model that secretes more IL‐10 and TGF‐β than the parental tumour cells (TC‐1 P0). These results provide the groundwork for future clinical development of the siRNA cocktail‐mediated strategy by co‐targeting immunosuppressive molecules to enhance the potency of DC‐based vaccines.

Gain of HIF-1α under Normoxia in Cancer Mediates Immune Adaptation through the AKT/ERK and VEGFA Axes

Purpose: Adaptation to host immune surveillance is now recognized as a hallmark of cancer onset and progression, and represents an early, indispensable event in cancer evolution. This process of evolution is first instigated by an immune selection pressure imposed by natural host surveillance mechanisms and may then be propagated by vaccination or other types of immunotherapy. Experimental Design: We developed a system to simulate cancer evolution in a live host and to dissect the mechanisms responsible for adaptation to immune selection. Here, we show that the oxygen-sensitive α subunit of hypoxia-inducible factor 1 (HIF-1α) plays a central role in cancer immune adaptation under conditions of normal oxygen tension. Results: We found that tumor cells gain HIF-1α in the course of immune selection under normoxia and that HIF-1α renders tumor cells resistant to lysis by tumor-specific cytotoxic T lymphocytes (CTL) in culture and in mice. The effects of HIF-1α on immune adaptation were mediated through VEGFA-dependent activation of the AKT and ERK signaling pathways, which induced an antiapoptotic gene expression network in tumor cells. Conclusions: Our study therefore establishes a link between immune selection, overexpression of HIF-1α, and cancer immune adaptation under normoxia, providing new opportunities for molecular intervention in patients with cancer. Clin Cancer Res; 21(6); 1438–46. ©2015 AACR.

Apoptosis inhibitor-5 overexpression is associated with tumor progression and poor prognosis in patients with cervical cancer

BackgroundThe apoptosis inhibitor-5 (API5), anti-apoptosis protein, is considered a key molecule in the tumor progression and malignant phenotype of tumor cells. Here, we investigated API5 expression in cervical cancer, its clinical significance, and its relationship with phosphorylated extracellular signal-regulated kinase 1 and 2 (pERK1/2) in development and progression of cervical cancer.MethodsAPI5 effects on cell growth were assessed in cervical cancer cell lines. API5 and pERK1/2 immunohistochemical staining were performed on a cervical cancer tissue microarray consisting of 173 primary cervical cancers, 306 cervical intraepithelial neoplasias (CINs), and 429 matched normal tissues.ResultsAPI5 overexpression promoted cell proliferation and colony formation in CaSki cells, whereas API5 knockdown inhibited the both properties in HeLa cells. Immunohistochemical staining showed that API5 expression increased during the normal to tumor transition of cervical carcinoma (P < 0.001), and this increased expression was significantly associated with tumor stage (P = 0.004), tumor grade (P < 0.001), and chemo-radiation response (P = 0.004). API5 expression levels were positively associated with pERK1/2 in cervical cancer (P < 0.001) and high grade CIN (P = 0.031). In multivariate analysis, API5+ (P = 0.039) and combined API5+/pERK1/2+ (P = 0.032) were independent prognostic factors for overall survival.ConclusionsAPI5 expression is associated with pERK1/2 in a subset of cervical cancer patients and its expression predicts poor overall survival, supporting that API5 may be a promising novel target for therapeutic interventions.

Immune-mediated tumor evolution: Nanog links the emergence of a stem like cancer cell state and immune evasion

Tumor cells undergo molecular evolution under immune pressure. Using a murine metastatic lung cancer model, we recently reported that evolutionary pressure enforced through vaccination incites gain of Nanog, a master transcription factor that mediates both emergence of a stem-like cancer cell state and immune evasion. Thus, therapeutic strategies aiming to blunt NANOGs expression in patient tumors may improve the clinical management of cancer.

Apoptosis inhibitor 5 increases metastasis via Erk-mediated MMP expression

Apoptosis inhibitor 5 (API5) has recently been identified as a tumor metastasis-regulating gene in cervical cancer cells. However, the precise mechanism of action for API5 is poorly understood. Here, we show that API5 increases the metastatic capacity of cervical cancer cells in vitro and in vivo via up-regulation of MMP-9. Interestingly, API5-mediated metastasis was strongly dependent on the Erk signaling pathway. Conversely, knock-down of API5 via siRNA technology decreased the level of phospho-Erk, the activity of the MMPs, in vitro invasion, and in vivo pulmonary metastasis. Moreover, the Erk-mediated metastatic action was abolished by the mutation of leucine into arginine within the heptad leucine repeat region, which affects protein-protein interactions. Thus, API5 increases the metastatic capacity of tumor cells by up-regulating MMP levels via activation of the Erk signaling pathway. [BMB Reports 2015; 48(6): 330-335]

Targeting stemness is an effective strategy to control EML4-ALK+ non-small cell lung cancer cells.

The fusion between anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) is a causative factor in a unique subset of patients with non-small cell lung carcinoma (NSCLC). Although the inhibitor crizotinib, as it blocks the kinase activity of the resulting EML4-ALK fusion protein, displays remarkable initial responses, a fraction of NSCLC cases eventually become resistant to crizotinib by acquiring mutations in the ALK domain or activating bypass pathways via EGFR, KIT, or KRAS. Cancer stem cell (CSC) theory provides a plausible explanation for acquisition of tumorigenesis and resistance. However, the question as to whether EML4-ALK-driven tumorigenesis is linked with the stem-like property and whether the stemness is an effective target in controlling EML4-ALK+ NSCLC including crizotinib-resistant NSCLC cells has not been addressed. Here, we report that stem-like properties stem from ALK activity in EML4-ALK+ NSCLC cells. Notably, treatment with rapamycin, a CSC targeting agent, attenuates stem-like phenotypes of the EML4-ALK+ cells, which increased capability of tumor formation and higher expression of stemness-associated molecules such as ALDH, NANOG, and OCT4. Importantly, combinational treatment with rapamycin and crizotinib leads to synergistic anti-tumor effects on EML4-ALK+ NSCLC cells as well as on those resistant to crizotinib. Thus, we provide a proof of principle that targeting stemness would be a novel strategy to control intractable EML4-ALK+ NSCLC.

GM-CSF-loaded chitosan hydrogel as an immunoadjuvant enhances antigen-specific immune responses with reduced toxicity

BackgroundThe application of vaccine adjuvants has been vigorously studied for a diverse range of diseases in order to improve immune responses and reduce toxicity. However, most adjuvants have limited uses in clinical practice due to their toxicity.MethodsTherefore, to reduce health risks associated with the use of such adjuvants, we developed an advanced non-toxic adjuvant utilizing biodegradable chitosan hydrogel (CH-HG) containing ovalbumin (OVA) and granulocyte-macrophage colony-stimulating factor (GM-CSF) as a local antigen delivery system.ResultsAfter subcutaneous injection into mice, OVA/GM-CSF-loaded CH-HG demonstrated improved safety and enhanced OVA-specific antibody production compared to oil-based adjuvants such as Complete Freund’s adjuvant (CFA) or Incomplete Freund’s adjuvant (IFA). Moreover, CH-HG system-mediated immune responses was characterized by increased number of OVA-specific CD4+ and CD8+ INF-γ+ T cells, leading to enhanced humoral and cellular immunity.ConclusionsIn this study, the improved safety and enhanced immune response characteristics of our novel adjuvant system suggest the possibility of the extended use of adjuvants in clinical practice with reduced apprehension about toxic side effects.

A PEGylated hyaluronic acid conjugate for targeted cancer immunotherapy

ABSTRACT The cell‐free approach to foreignizing tumor cells with non‐self antigens has received increasing attention as a method to induce cytotoxic T lymphocyte (CTL)‐mediated immunological rejection of tumors, because the clinical translation of the conventional CTL‐based cancer immunotherapies has been limited by a complicated manufacturing process and autotransplantation. In this study, we prepared matrix metalloproteinase 9 (MMP9)‐responsive polymeric conjugates consisting of PEGylated hyaluronic acid (HA) as the targeting moiety and ovalbumin (OVA) as the model foreign antigen. The MMP9‐cleavable linker was introduced between PEG and the HA backbone to facilitate the detachment of the PEG corona from the conjugate at the tumor site. From the in vitro cellular uptake study, it was revealed that the conjugate was effectively taken up by the CD44‐expressing TC‐1 cancer cells in the presence of MMP9 via receptor‐mediated endocytosis. When the conjugate was systemically administered into the tumor‐bearing mice with endogenous OVA‐specific CTLs, the tumor growth was markedly inhibited, which was attributed to the significant antigen presentation on the tumor cells. Overall, the MMP9‐responsive conjugates bearing foreign antigens might have the potential as an alternative to CTL‐based cancer immunotherapeutics. Graphical abstract Figure. No caption available.