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Purpose: Cancer cells possess traits reminiscent of those ascribed to normal stem cells. It is unclear whether these phenotypic similarities are the result of a common biological phenotype, such as regulatory pathways.Experimental Design: Lung cancer cell lines with corresponding gene expression data and genes associated with an embryonic stem cell identity were used to develop a signature of embryonic stemness (ES) activity specific to lung adenocarcinoma. Biological characteristics were elucidated as a function of cancer biology/oncogenic pathway dysregulation. The ES signature was applied to three independent early-stage (I-IIIa) lung adenocarcinoma data sets with clinically annotated gene expression data. The relationship between the ES phenotype and cisplatin (current standard of care) sensitivity was evaluated.Results: Pathway analysis identified specific regulatory networks [Ras (P = 0.0005), Myc (P = 0.0224), wound healing (P < 0.0001), chromosomal instability (P < 0.0001), and invasiveness (P < 0.0001)] associated with the ES phenotype. The prognostic relevance of the ES signature, as related to patient survival, was characterized in three cohorts [CALGB 9761 (n = 82; P = 0.0001), National Cancer Institute Director's Challenge Consortium (n = 442; P = 0.0002), and Duke (n = 45; P = 0.06)]. The ES signature was not prognostic in prostate, breast, or ovarian adenocarcinomas. Lung tumors (n = 569) and adenocarcinoma cell lines (n = 31) expressing the ES phenotype were more likely to be resistant to cisplatin (P < 0.0001 and P = 0.006, respectively).Conclusions: Lung adenocarcinomas that share a common gene expression pattern with normal human embryonic stem cells were associated with decreased survival, increased biological complexity, and increased likelihood of resistance to cisplatin. This indicates the aggressiveness of these tumors. (Clin Cancer Res 2009;15(24):7553–61)

Detailed and supplementary methods

List of significant gene symbols

Supplementary Data from Characterizing the Clinical Relevance of an Embryonic Stem Cell Phenotype in Lung Adenocarcinoma

SF1: IL-26 protein or mRNA expression in human cell lines and clinical samples; SF2: Single-cell profiling of CD4+ TH17 cells in TNBC patients; SF3: Confirmation of IL-26 knockdown in MDA-MB-231 cells; SF4: In vitro proliferation and in vivo metastasis of IL-26 knockdown MDA-MB-231 cells; SF5: Gene expression profiling of IL-26 knockdown MDA-MB-231 tumors; SF6: IL-26 knockdown in SUM159 and MDA-MB-468 cells and in vitro phenotyping; SF7: Western blot confirmation of IL-26 knock-in E0771 cells and IL-26 upregulation after growth in vivo; SF8: IL-26 knock-in E0771 tumors progress more rapidly than control in an immune competent setting; SF9: Stat3 activation by IL-26 is dependent on human IL10RB and IL20RA; SF10: DNA-bound IL-26 elicits inflammatory signaling in human and mouse immune cells; SF11: Confirmation of mouse neutrophil enrichment; SF12: Confirmation of Anti-Ly6G antibody depletion of mouse neutrophils; SF13: Inflammatory cytokine expression in MDA-MB-231 parental and MDA-MB-231 LM2 lung-metastatic subclone; SF14: CRISPR knockdown of IL-6, IL-8, and Cxcl1 in MDA-MB-231 LM2 CRISPR 3x cell lines/lung metastases and correlation of CRISPR 3x primary tumor volume with metastasis; SF15: IL-26 signature and Neutrophil enrichment score are significantly correlated in METABRIC dataset; SF16: T cell ELISPOT response to IL-26 vaccination in vivo.

Significant hits identified in CRISPR screen

CRISPR screen results

IL26 is a unique amphipathic member of the IL10 family of cytokines that participates in inflammatory signaling through a canonical receptor pathway. It also directly binds DNA to facilitate cellular transduction and intracellular inflammatory signaling. Although IL26 has almost no described role in cancer, our in vivo screen of inflammatory and cytokine pathway genes revealed IL26 to be one of the most significant inflammatory mediators of mammary engraftment and lung metastatic growth in triple-negative breast cancer (TNBC). Examination of human breast cancers demonstrated elevated IL26 transcripts in TNBC specimens, specifically in tumor cells as well as in Th17 CD4+ T cells within clinical TNBC specimens. IL26 did not have an autocrine effect on human TNBC cells, but rather its effect on engraftment and growth in vivo required neutrophils. IL26 enhanced mouse-derived DNA induction of inflammatory cytokines, which were collectively important for mammary and metastatic lung engraftment. To neutralize this effect, we developed a novel IL26 vaccine to stimulate antibody production and suppress IL26-enhanced engraftment in vivo, suggesting that targeting this inflammatory amplifier could be a unique means to control cancer-promoting inflammation in TNBC and other autoimmune diseases. Thus, we identified IL26 as a novel key modulator of TNBC metastasis and a potential therapeutic target in TNBC as well as other diseases reliant upon IL26-mediated inflammatory stimulation.Significance:These findings identify IL26 as a unique, clinically relevant, inflammatory amplifier that enhances TNBC engraftment and dissemination in association with neutrophils, which has potential as a therapeutic target.

Background: The majority of colorectal carcinomas (CRCs) are insensitive to anti-PD-1/PD-L1 immune checkpoint inhibitor (ICI) antibodies. While there are many causes for ICI insensitivity, recent studies suggest that the suppression of innate immunity through a loss of innate immune adaptor expression could be a root cause of this insensitivity and an important factor in the evolution of tumor immunosuppression. Methods: We interrogated mitochondrial antiviral signaling gene (MAVS) expression through bioinformatics analyses of multiple datasets to validate its suppression in clinical samples. We then engineered MAVS expressing tumor cells and tested its ability to elicit innate and adaptive anti-tumor immunity using both in vitro and in vivo approaches, which we then confirmed using MAVS expressing viral vectors. Finally, we observed that MAVS stimulated PD-L1expression in different types of tumor cells and then assessed the combination of PD-L1 ICI antibodies with MAVS tumor expression in vivo. Results: MAVS was significantly downregulated in CRCs, but its re-expression could stimulate broad cellular interferon-related responses, in both murine and patient-derived CRCs. In vivo, local MAVS expression elicited significant anti-tumor responses in both immune-sensitive and insensitive CRC models, through the stimulation of an interferon responsive axis that elicited tumor antigen-specific adaptive immunity. Critically, we found that tumor-intrinsic MAVS expression triggered systemic adaptive immune responses that enabled abscopal CD8+ T cell cytotoxicity against distant CRCs. As MAVS also induced PD-L1 expression, we further found synergistic anti-tumor responses in combination with anti-PD-L1 ICIs. Conclusion: These data demonstrate that intratumoral MAVS expression results in local and systemic tumor antigen-specific T cell responses, which could be combined with PD-L1 ICI to permit effective anti-tumor immunotherapy in ICI resistant cancers. We are further exploring to overcome the ICI insensitivity of other types of solid tumor, with MAVS-delivery strategies other than viral vectors. Citation Format: Bin-Jin Hwang, Li-Chung Tsao, Chaitanya Acharya, Timothy Trotter, Pankaj Agarwal, Tao Wang, Junping Wei, Xiao-Yi Yang, Gang-jun Lei, Takuya Osada, Herbert Kim Lyerly, Michael A. Morse, Zachary Hartman. Sensitizing immune unresponsive colorectal cancers to immune checkpoint inhibitors through MAVS overexpression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3531.

Supplementary text. This is a PDF document, which elaborates some methodological aspects of our paper. (PDF 200 kb)