Michael A. Podolsky

CD8+ T Cells Mediate RAS-Induced Psoriasis-Like Skin Inflammation through IFN-γ

The RAS signaling pathway is constitutively activated in psoriatic keratinocytes. We expressed activated H-RASV12G in suprabasal keratinocytes of adult mice and observed rapid development of a psoriasis-like skin phenotype characterized by basal keratinocyte hyperproliferation, acanthosis, hyperkeratosis, intraepidermal neutrophil microabscesses and increased Th1/Th17 and Tc1/Tc17 skin infiltration. The majority of skin infiltrating CD8+ T cells co-expressed IFN-γ and IL-17A. When RAS was expressed on a Rag1−/− background, microabscess formation, iNOS expression and keratinocyte hyperproliferation were suppressed. Depletion of CD8+ but not CD4+ T cells reduced cutaneous and systemic inflammation, the RAS-induced increase in cutaneous Th17 and IL-17+ γΔ T cells, and epidermal hyperproliferation to levels similar to a Rag1−/− background. Reconstitution of Rag1−/− inducible RAS mice with purified CD8+ T cells restored microabscess formation and epidermal hyperproliferation. Neutralization of IFN-γ but not IL-17A in CD8+ T cell reconstituted Rag1−/− mice expressing RAS blocked CD8-mediated skin inflammation, iNOS expression and keratinocyte hyperproliferation. These results show for that CD8+ T cells can orchestrate skin inflammation with psoriasis-like pathology in response to constitutive RAS activation in keratinocytes, and this is primarily mediated through IFN-γ.

Genetic and Pharmacological Analysis Identifies a Physiological Role for the AHR in Epidermal Differentiation

Stimulation of the aryl hydrocarbon receptor (AHR) by xenobiotics is known to affect epidermal differentiation and skin barrier formation. The physiological role of endogenous AHR signaling in keratinocyte differentiation is not known. We used murine and human skin models to address the hypothesis that AHR activation is required for normal keratinocyte differentiation. Using transcriptome analysis of Ahr-/- and Ahr+/+ murine keratinocytes, we found significant enrichment of differentially expressed genes linked to epidermal differentiation. Primary Ahr-/- keratinocytes showed a significant reduction in terminal differentiation gene and protein expression, similar to Ahr+/+ keratinocytes treated with AHR antagonists GNF351 and CH223191, or the selective AHR modulator (SAhRM), SGA360. In vitro keratinocyte differentiation led to increased AHR levels and subsequent nuclear translocation, followed by induced CYP1A1 gene expression. Monolayer cultured primary human keratinocytes treated with AHR antagonists also showed an impaired terminal differentiation program. Inactivation of AHR activity during human skin equivalent development severely impaired epidermal stratification, terminal differentiation protein expression and stratum corneum formation. As disturbed epidermal differentiation is a main feature of many skin diseases, pharmacological agents targeting AHR signaling or future identification of endogenous keratinocyte-derived AHR ligands should be considered as potential new drugs in dermatology.

The TGFβ1 pathway is required for NFκB dependent gene expression in mouse keratinocytes

The transforming growth factor-beta 1 (TGFβ1) and NFκB pathways are important regulators of epidermal homeostasis, inflammatory responses and carcinogenesis. Previous studies have shown extensive crosstalk between these pathways that is cell type and context dependent, but this has not been well-characterized in epidermal keratinocytes. Here we show that in primary mouse keratinocytes, TGFβ1 induces NFκB-luciferase reporter activity that is dependent on both NFκB and Smad3. TGFβ1-induced NFκB-luciferase activity was blocked by the IκB inhibitor parthenolide, the IκB super-repressor, a dominant negative TGFβ1-activated kinase 1 (TAK1) and genetic deletion of NFκB1. Coexpression of NFκB p50 or p65 subunits enhanced NFκB-luciferase activity. Similarly, inhibition of the TGFβ1 type I receptor with SB431542 or genetic deletion of Smad3 blocked TGFβ1 induction of NFκB-luciferase. TGFβ1 rapidly induced IKK phosphorylation but did not cause a detectable decrease in cytoplasmic IκB levels or nuclear translocation of NFκB subunits, although EMSA showed rapid NFκB nuclear binding activity that could be blocked by SB431542 treatment. TNFα, a well characterized NFκB target gene was also induced by TGFβ1 and this was blocked in NFκB+/- and -/- keratinocytes and by the IκB super-repressor. To test the effects of the TGFβ1 pathway on a biologically relevant activator of NFκB, we exposed mice and primary keratinocytes in culture to UVB irradiation. In primary keratinocytes UVB caused a detectable increase in levels of Smad2 phosphorylation that was dependent on ALK5, but no significant increase in SBE-dependent gene expression. Inhibition of TGFβ1 signaling in primary keratinocytes with SB431542 or genetic deletion of Tgfb1 or Smad3 suppressed UVB induction of TNFα message. Similarly, UVB induction of TNFα mRNA was blocked in skin of Tgfb1+/- mice. These studies demonstrate that intact TGFβ1 signaling is required for NFκB-dependent gene expression in mouse keratinocytes and skin and suggest that a convergence of these pathways in the nucleus rather than the cytoplasm may be critical for regulation of inflammatory pathways in skin by TGFβ1.

Dietary broccoli impacts microbial community structure and attenuates chemically induced colitis in mice in an Ah receptor dependent manner

Consumption of broccoli mediates numerous chemo-protective benefits through the intake of phytochemicals, some of which modulate aryl hydrocarbon receptor (AHR) activity. Whether AHR activation is a critical aspect of the therapeutic potential of dietary broccoli is not known. Here we administered isocaloric diets, with or without supplementation of whole broccoli (15% w/w), to congenic mice expressing the high-affinity Ahrb/b or low-affinity Ahrd/d alleles, for 24 days and examined the effects on AHR activity, intestinal microbial community structure, inflammatory status, and response to chemically induced colitis. Cecal microbial community structure and metabolic potential were segregated according to host dietary and AHR status. Dietary broccoli associated with heightened intestinal AHR activity, decreased microbial abundance of the family Erysipelotrichaceae, and attenuation of colitis. In summary, broccoli consumption elicited an enhanced response in ligand-sensitive Ahrb/b mice, demonstrating that in part the beneficial aspects of dietary broccoli upon intestinal health are associated with heightened AHR activity.

ER stress and distinct outputs of the IRE1α RNase control proliferation and senescence in response to oncogenic Ras

Significance Inositol requiring enzyme 1α (IRE1α) is a mediator of the unfolded protein response that determines adaptation or cell death in response to endoplasmic reticulum (ER) stress through its distinct endoribonuclease (RNase) activities of Xbp1 splicing and mRNA decay, but its role in cancer is poorly understood. In normal epithelial cells, we find that Ras oncogene-induced proliferation and senescence are directly linked to IRE1α activation. Proliferation requires Xbp1 splicing and ER stress, while IRE1α-catalyzed degradation of Id1 mRNA drives senescence in conjunction with reduced ER stress. Thus, we propose that oncogene and ER stress regulation of the IRE1α RNase dictates tumor promotion or suppression in Ras-driven cancers. Oncogenic Ras causes proliferation followed by premature senescence in primary cells, an initial barrier to tumor development. The role of endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in regulating these two cellular outcomes is poorly understood. During ER stress, the inositol requiring enzyme 1α (IRE1α) endoribonuclease (RNase), a key mediator of the UPR, cleaves Xbp1 mRNA to generate a potent transcription factor adaptive toward ER stress. However, IRE1α also promotes cleavage and degradation of ER-localized mRNAs essential for cell death. Here, we show that oncogenic HRas induces ER stress and activation of IRE1α. Reduction of ER stress or Xbp1 splicing using pharmacological, genetic, and RNAi approaches demonstrates that this adaptive response is critical for HRas-induced proliferation. Paradoxically, reduced ER stress or Xbp1 splicing promotes growth arrest and premature senescence through hyperactivation of the IRE1α RNase. Microarray analysis of IRE1α- and XBP1-depleted cells, validation using RNA cleavage assays, and 5′ RACE identified the prooncogenic basic helix–loop–helix transcription factor ID1 as an IRE1α RNase target. Further, we demonstrate that Id1 degradation by IRE1α is essential for HRas-induced premature senescence. Together, our studies point to IRE1α as an important node for posttranscriptional regulation of the early Ras phenotype that is dependent on both oncogenic signaling as well as stress signals imparted by the tumor microenvironment and could be an important mechanism driving escape from Ras-induced senescence.

Ligand-mediated cytoplasmic retention of the Ah receptor inhibits macrophage-mediated acute inflammatory responses

The Ah receptor (AHR) has been shown to exhibit both inflammatory and anti-inflammatory activity in a context-specific manner. In vivo macrophage-driven acute inflammation models were utilized here to test whether the selective Ah receptor modulator 1-allyl-7-trifluoromethyl-1H–indazol-3-yl]-4-methoxyphenol (SGA360) would reduce inflammation. Exposure to SGA360 was capable of significantly inhibiting lipopolysaccharide (LPS)-mediated endotoxic shock in a mouse model, both in terms of lethality and attenuating inflammatory signaling in tissues. Topical exposure to SGA360 was also able to mitigate joint edema in a monosodium urate (MSU) crystal gout mouse model. Inhibition was dependent on the expression of the high-affinity allelic AHR variant in both acute inflammation models. Upon peritoneal MSU crystal exposure SGA360 pretreatment inhibited neutrophil and macrophage migration into the peritoneum. RNA-seq analysis revealed that SGA360 attenuated the expression of numerous inflammatory genes and genes known to be directly regulated by AHR in thioglycolate-elicited primary peritoneal macrophages treated with LPS. In addition, expression of the high-affinity allelic AHR variant in cultured macrophages was necessary for SGA360-mediated repression of inflammatory gene expression. Mechanistic studies revealed that SGA360 failed to induce nuclear translocation of the AHR and actually enhanced cytoplasmic localization. LPS treatment of macrophages enhanced the occupancy of the AHR and p65 to the Ptgs2 promoter, while SGA360 attenuated occupancy. AHR ligand activity was detected in peritoneal exudates isolated from MSU-treated mice, thus suggesting that the anti-inflammatory activity of SGA360 is mediated at least in part through AHR antagonism of endogenous agonist activity. These results underscore an important role of the AHR in participating in acute inflammatory signaling and warrants further investigations into possible clinical applications.

Differentiated state of initiating tumor cells is key to distinctive immune responses seen in H-RasG12V-induced squamous tumors

Immune therapies are usually successful in a fraction of patients. Squamous cell carcinomas originate in a stratified epithelium, and distinctly different immune responses were found to be driven by the state of differentiation of the tumor-initiating cell. Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. In two doxycycline-inducible models where oncogenic H-RasG12V is targeted either to the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), we observed strikingly distinct tumor immune responses. On threshold doxycycline levels yielding similar Ras expression, tumor latency, and numbers, tumors from K14Ras mice had an immunosuppressed microenvironment, whereas InvRas tumors had a proinflammatory microenvironment. On a Rag1−/− background, InvRas mice developed fewer and smaller tumors that regressed over time, whereas K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Adoptive transfer and depletion studies revealed that B-cell and CD4 T-cell cooperation was critical for tumor yield, lymphocyte polarization, and tumor immune phenotype in Rag1+/+ mice of both models. Coculture of tumor-conditioned B cells with CD4 T cells implicated direct contact for Th1 and regulatory T cell (Treg) polarization, and CD40-CD40L for Th1, Th2, and Treg generation, a response not observed from splenic B cells. Anti-CD40L caused regression of InvRas tumors but enhanced growth in K14Ras, whereas a CD40 agonist mAb had opposite effects in each tumor model. These data show that position of tumor-initiating cells within a stratified squamous epithelial tissue provokes distinct B- and CD4 T-cell interactions, which establish unique tumor microenvironments that regulate tumor development and response to immunotherapy. Cancer Immunol Res; 5(3); 198–210. ©2017 AACR.

Abstract 2673: Divergent therapeutic responses to CD40L blockade or CD40 activation in Ras-driven skin cancers determined by origin of tumor initiating cell

Heterogeneity in tumor immune responses is a poorly understood yet critical parameter for successful immunotherapy. We used doxycycline-inducible epidermal squamous cancer models driven by oncogenic Ras to test whether the origin of the tumor-initiating cell influenced the tumor immune response. Threshold doxycycline induced expression of H-RasG12V in either the basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or committed progenitor/suprabasal cells with an Involucrin-tTA transgene (InvRas), caused distinct immune responses despite similar levels of Ras, tumor latency, and tumor numbers. End stage K14Ras tumors had an immunosuppressed microenvironment with abundant Tregs and Bregs which evolved from an initially Th1 dominant environment in the earliest detectable lesions. In contrast InvRas tumors had a Th2 pro-inflammatory microenvironment, with significantly fewer Tregs or Bregs at any time point. This difference in immune microenvironment between the tumor models was also found in hyperplastic skin after 5 days of maximal Ras expression, indicating that it was not simply a property of the end stage tumor. Surprisingly, adaptive immunity had opposite roles in tumor development. InvRasRag1-/- mice developed fewer and smaller tumors that regressed while K14RasRag1-/- mice developed more tumors with shorter latency than Rag1+/+ controls. In both models adoptive transfer and depletion studies showed that cooperation between B and CD4 T cells drove the opposing tumor responses, lymphocyte polarization, and tumor immune phenotype. Co-culture of tumor-conditioned but not splenic B cells from each model with naive CD4 T cells showed that direct contact and CD40-CD40L ligation was required for opposite polarization towards either a Th2 or Treg phenotype. Importantly, we found that anti-CD40L mAb caused regression of preexisting InvRas tumors but enhanced growth of K14Ras tumors. In contrast, CD40 agonist mAb enhanced growth of preexisting InvRas tumors, and suppressed growth of K14Ras tumors. Thus in an in vivo setting the type of tumor immune microenvironment and opposing role of CD40-CD40L in tumor development generates distinct responses to therapeutic antibodies. Together these data show that the position of a tumor initiating cell within the stem cell hierarchy in a stratified squamous epithelia has important consequences for the type of tumor immune microenvironment and response to checkpoint therapy. Citation Format: Adam B. Glick, Jacob T. Bailey, Andrew Gunderson, Kyle Breech, Michael Podolsky. Divergent therapeutic responses to CD40L blockade or CD40 activation in Ras-driven skin cancers determined by origin of tumor initiating cell [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 2673. doi:10.1158/1538-7445.AM2017-2673

Abstract 530: Location of oncogene expression within a stratified squamous epithelium drives distinct B and CD4 T-cell crosstalk to dictate the tumor immune response

Tumors from stratified epithelium contain proliferating and differentiating compartments, but it is not clear how tumor cells in different layers alternatively engage the immune system. We have established two doxycycline inducible Ras models in which oncogenic RasV12G is targeted to either the epidermal basal/stem cell layer with a Keratin14-rtTA transgene (K14Ras), or suprabasal differentiating cells with an Involucrin-tTA transgene (InvRas). On threshold doxycycline levels yielding similar tumor numbers and Ras expression over 30 days, mice with basal cell targeted Ras developed focal squamous cell carcinoma while suprabasal targeting caused benign squamous papilloma formation. On a Rag1-/- background InvRas mice developed fewer tumors that regressed over time while K14Ras mice developed more tumors with shorter latency than Rag1+/+ controls. Depletion and adoptive transfer studies revealed that naive B and CD4 T cells together, but not alone, suppressed tumor formation in K14Ras mice but restored tumor numbers in InvRas mice. Tumors developing in K14Ras mice showed a loss of proinflammatory CD4 T and B cells and increased percentages of regulatory cells infiltrating the tumor tissue. In vivo cotransfers show that B and CD4 T cells reciprocally prime each other towards a regulatory phenotype in the K14Ras tumor microenvironment, but in the InvRas tumor microenvironment proinflammatory CD4 T and B cell phenotypes result. Coculture of tumor-conditioned B cells with stimulated naive CD4 T cells showed an importance of direct contact and CD40/CD40L interactions for the generation of regulatory T cells (Tregs) by B cells in K14Ras mice. In contrast, tumor-conditioned B cells from InvRas mice support generation of proinflammatory CD4 T cells, and antagonize Treg development. This function is restricted to tumor-conditioned B cells, as splenic B cells from tumor-bearing mice had no effect on CD4 T cell phenotype. In vivo blockade of CD40L in K14Ras mice resulted in significantly increased tumor counts as well as reduced B and Treg prevalence. Blockade of CD40L in InvRas mice significantly reduced tumor number, increasing Treg cell count and decreasing neutrophil infiltration into the tumor tissue. Time-course experiments suggest a protective role of Tregs in late stages of K14Ras tumors, and a tumor-promoting role of proinflammatory CD4 T cells in InvRas tumors. Thus, basal/stem cell expression of Ras provokes a regulatory cell inducing microenvironment, suppressing tumor-promoting inflammation in late-stage tumors. Ras expression in differentiating cells activates a tumor promoting proinflammatory phenotype in B and CD4 T cells without provoking immunosurveillance. Taken together, these data show that tumor cell position within a stratified epithelium differentiation hierarchy provokes distinct B and CD4 T cell interactions with opposing effects on tumor development. Citation Format: Michael A. Podolsky, Carrie J. Oakes, Andrew Gunderson, Kyle Breech, Jacob Bailey, Adam B. Glick. Location of oncogene expression within a stratified squamous epithelium drives distinct B and CD4 T-cell crosstalk to dictate the tumor immune response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 530.

Abstract 4083: Opposing roles of B cells in Ras-driven squamous tumor development dependent on tissue compartment of oncogene expression and tumor phenotype

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Studies in mouse models and human cancers show that B cells can both promote and inhibit cancer development, but the mechanisms generating these alternate outcomes are not well understood. Here we have developed a mouse model of Ras-driven epidermal tumor development in which opposite effects of B cells are manifest, dependent on the differentiation state of cells expressing oncogenic Ras. We used a doxycycline regulatable bitransgenic system to target oncogenic H-RasV12G to either the basal/stem cell (Keratin14-rTAxtetORasV12G, K14Ras) or differentiating (Involucrin-tTA x tetORasV12G, InvRas) compartment of the epidermis. K14Ras mice develop focal squamous cell carcinoma while InvRas mice develop squamous papillomas within 30 days on threshold levels of doxycycline resulting in ∼10 tumors/mouse. On a Rag1-/- background we observed opposite tumor responses, with InvRasRag1-/- mice developing fewer tumors and K14RasRag1-/- mice developing more tumors with shorter latency than Rag1+/+ controls. Depletion of CD4+ T cells caused an overall increase in tumor burden compared to isotype controls in both models, while adoptive transfer of naive CD4+ T cells (purity ≥ 93%) increased tumor burden in K14RasRag1-/- mice, but suppressed tumors in InvRasRag1-/- mice. Tumors from CD4 transferred K14RasRag1-/- mice had a predominant Th1 infiltrate with few Tregs and increased tumor-infiltrating neutrophils compared to mock transferred controls. Conversely, CD4+ T cells adoptively transferred to InvRasRag1-/- mice displayed a regulatory phenotype with low IFNγ and high Foxp3, which was associated with a decrease in neutrophilic infiltration. In contrast, B cell depletion with anti-CD20 caused a significant suppression of tumor outgrowth in InvRas mice but enhanced tumor burden in K14Ras mice. Furthermore, adoptive transfer of magnetically purified splenic B cells into K14RasRag1-/- mice suppressed tumor formation, and co-transfer of B cells with CD4+ T cells reduced tumor development to a greater extent than B cells alone. Tumor-infiltrating B cells from co-transferred mice had greater percentage of B regulatory cells, and increased costimulatory molecule expression compared to B cells alone, resembling K14RasRag1+/+ controls. B cell co-transfer significantly increased Foxp3+ Treg percentage to levels similar to K14RasRag1+/+ mice as measured by flow cytometry. Taken together these results show that B cells are the critical lymphocyte driving both tumor suppression or promotion in this model, that the differentiation state of the tumor initiating cell determines the B cell response, and that interactions between CD4+ T cells and B cells may be important for the development of a proper anti-tumor immune response. Citation Format: Michael A. Podolsky, Andrew Gunderson, Kyle Breech, Adam B. Glick. Opposing roles of B cells in Ras-driven squamous tumor development dependent on tissue compartment of oncogene expression and tumor phenotype. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4083. doi:10.1158/1538-7445.AM2014-4083