Nicholas Blazanin

Ligand activation of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) inhibits chemically induced skin tumorigenesis

Peroxisome proliferator-activated receptor (PPAR)beta/delta-null mice exhibit enhanced tumorigenesis in a two-stage chemical carcinogenesis model as compared with wild-type mice. Previous work showed that ligand activation of PPARbeta/delta induces terminal differentiation and inhibits proliferation of primary keratinocytes, and this effect does not occur in the absence of PPARbeta/delta expression. In the present studies, the effect of ligand activation of PPARbeta/delta on skin tumorigenesis was examined using both in vivo and ex vivo skin carcinogenesis models. Inhibition of chemically induced skin tumorigenesis was observed in wild-type mice administered GW0742, and this effect was likely the result of ligand-induced terminal differentiation and inhibition of replicative DNA synthesis. These effects were not found in similarly treated PPARbeta/delta-null mice. Ligand activation of PPARbeta/delta also inhibited cell proliferation and induced terminal differentiation in initiated/neoplastic keratinocyte cell lines representing different stages of skin carcinogenesis. These studies suggest that topical administration of PPARbeta/delta ligands may be useful as both a chemopreventive and/or a chemotherapeutic approach to inhibit skin cancer.

The Nuclear Receptor Peroxisome Proliferator-activated Receptor-β/δ (PPARβ/δ) Promotes Oncogene-induced Cellular Senescence through Repression of Endoplasmic Reticulum Stress

Background: It is unclear whether ER stress and associated unfolded protein response (UPR) can influence oncogene-induced senescence. Results: ER stress attenuated senescence by modulating kinases, and a positive feed forward loop was delineated where ER stress caused loss of senescence and promotion of tumorigenesis. Conclusion: A new role for ER stress and UPR that attenuates H-RAS-induced senescence was discovered. Significance: PPARβ/δ may suppress RAS-dependent tumorigenesis. Endoplasmic reticulum (ER) stress and ER stress-associated unfolded protein response (UPR) can promote cancer cell survival, but it remains unclear whether they can influence oncogene-induced senescence. The present study examined the role of ER stress in senescence using oncogene-dependent models. Increased ER stress attenuated senescence in part by up-regulating phosphorylated protein kinase B (p-AKT) and decreasing phosphorylated extracellular signal-regulated kinase (p-ERK). A positive feed forward loop between p-AKT, ER stress, and UPR was discovered whereby a transient increase of ER stress caused reduced senescence and promotion of tumorigenesis. Decreased ER stress was further correlated with increased senescence in both mouse and human tumors. Interestingly, H-RAS-expressing Pparβ/δ null cells and tumors having increased cell proliferation exhibited enhanced ER stress, decreased cellular senescence, and/or enhanced tumorigenicity. Collectively, these results demonstrate a new role for ER stress and UPR that attenuates H-RAS-induced senescence and suggest that PPARβ/δ can repress this oncogene-induced ER stress to promote senescence in accordance with its role as a tumor modifier that suppresses carcinogenesis.

TGFβ1-Induced Inflammation in Premalignant Epidermal Squamous Lesions Requires IL-17

Overexpression of transforming growth factor-beta1 (TGFbeta1) in the normal epidermis can provoke an inflammatory response, but whether this occurs within a developing tumor is not clear. To test this, we used an inducible transgenic mouse to overexpress TGFbeta1 in premalignant squamous lesions. Within 48 hours of TGFbeta1 induction, there was an increase in IL-17 production by both CD4(+) and gammadelta(+) T cells, together with increased expression of T-helper-17 (Th17)-polarizing cytokines. Induction of TGFbeta1 in premalignant primary keratinocytes elevated the expression of proinflammatory and Th17-polarizing cytokines, and the keratinocyte-conditioned media caused IL-17 production by naive T cells that was dependent on T-cell TGFbeta1 signaling. Microarray analysis showed significant upregulation of proinflammatory genes 2 days after TGFbeta1 induction, and this was followed by increased MPO(+), F4/80(+), and CD8(+) cells in tumors, increased CD8(+) effectors and IFNgamma(+) cells in skin-draining LNs, and tumor regression. In parallel, the percentage of tumor CD11b(+)Ly6G(+) neutrophils was reduced. Neutralization of IL-17 blocked TGFbeta1-induced CD11b(+) Ly6G(-) tumor infiltration but did not alter the reduction of neutrophils or tumor regression. Thus, TGFbeta1 overexpression causes IL-17-dependent and IL-17-independent changes in the premalignant tumor inflammatory microenvironment.

Tumor suppressor and oncogene actions of TGFβ1 occur early in skin carcinogenesis and are mediated by Smad3

Interactions between TGFβ1 and ras signaling pathways play an important role in cancer development. Here we show that in primary mouse keratinocytes, v‐rasHa does not block the early biochemical events of TGFβ1 signal transduction but does alter global TGFβ1 mediated gene expression in a gene specific manner. Expression of Smad3 dependent TGFβ1 early response genes and the TGFβ1 cytostatic gene expression response were not altered by v‐rasHa consistent with an intact TGFβ1 growth arrest. However, TGFβ1 and v‐rasHa cause significant alteration in genes regulating matrix remodeling as the TGFβ1 induction of extracellular matrix genes was blocked by v‐rasHa but specific matrix proteases associated with cancer progression were elevated. Smad3 deletion in keratinocytes repressed normal differentiation maker expression and caused expression of Keratin 8 a simple epithelial keratin and marker of malignant conversion. Smad3 was required for the TGFβ1 cytostatic response in v‐rasHa keratinocytes, but also for protease induction, keratinocyte attachment and migration. These results show that pro‐oncogenic activities of TGFβ1 can occur early in carcinogenesis before loss of its tumor suppressive function and that selective regulation rather than complete inactivation of Smad3 function may be crucial for tumor progression.

PPARβ/δ promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling.

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARβ/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARβ/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparβ/δ-null cells. Higher PPARβ/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARβ/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

Peroxisome Proliferator-Activated Receptor β/δ Cross Talks with E2F and Attenuates Mitosis in HRAS-Expressing Cells

ABSTRACT The role of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in Harvey sarcoma ras (Hras)-expressing cells was examined. Ligand activation of PPARβ/δ caused a negative selection with respect to cells expressing higher levels of the Hras oncogene by inducing a mitotic block. Mitosis-related genes that are predominantly regulated by E2F were induced to a higher level in HRAS-expressing Pparβ/δ-null keratinocytes compared to HRAS-expressing wild-type keratinocytes. Ligand-activated PPARβ/δ repressed expression of these genes by direct binding with p130/p107, facilitating nuclear translocation and increasing promoter recruitment of p130/p107. These results demonstrate a novel mechanism of PPARβ/δ cross talk with E2F signaling. Since cotreatment with a PPARβ/δ ligand and various mitosis inhibitors increases the efficacy of increasing G2/M arrest, targeting PPARβ/δ in conjunction with mitosis inhibitors could become a suitable option for development of new multitarget strategies for inhibiting RAS-dependent tumorigenesis.

Pharmacologic inhibition of ALK5 causes selective induction of terminal differentiation in mouse keratinocytes expressing oncogenic HRAS.

TGFβ has both tumor suppressive and oncogenic roles in cancer development. We previously showed that SB431542 (SB), a small molecule inhibitor of the TGFβ type I receptor (ALK5) kinase, suppressed benign epidermal tumor formation but enhanced malignant conversion. Here, we show that SB treatment of primary K5rTA/tetORASV12G bitransgenic keratinocytes did not alter HRASV12G-induced keratinocyte hyperproliferation. However, continuous SB treatment significantly enhanced HRASV12G-induced cornified envelope formation and cell death linked to increased expression of enzymes transglutaminase (TGM) 1 and TGM3 and constituents of the cornified envelope small proline-rich protein (SPR) 1A and SPR2H. In contrast, TGFβ1 suppressed cornified envelope formation in HRASV12G keratinocytes. Similar results were obtained in HRASV12G transgenic mice treated topically with SB or by coexpressing TGFβ1 and HRASV12G in the epidermis. Despite significant cell death, SB-resistant HRASV12G keratinocytes repopulated the primary culture that had overcome HRas-induced senescence. These cells expressed reduced levels of p16ink4a and were growth stimulated by SB but remained sensitive to a calcium-induced growth arrest. Together these results suggest that differential responsiveness to cornification may represent a mechanism by which pharmacologic blockade of TGFβ signaling can inhibit the outgrowth of preneoplastic lesions but may cause a more progressed phenotype in a separate keratinocyte population. Mol Cancer Res; 9(6); 746–56. ©2011 AACR.

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.

Abstract 2257: Divergent IRE1α endonuclease outputs dictate the senescence response of mouse keratinocytes to oncogenic HRAS

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oncogenic RAS plays a pivotal role in neoplastic transformation, however in primary cells it initially triggers a proliferative response followed by irreversible growth arrest and premature senescence, a potent barrier to tumor development. How premature senescence is bypassed to enable full transformation and tumor progression is not clear. Here we show that in primary mouse keratinocytes IRE1α, an endoplasmic reticulum (ER) transmembrane kinase/endoribonuclease (RNase) important in the unfolded protein response, regulates cell fate in response to oncogenic v-Ha-RAS (HRAS) through its two distinct RNase outputs. During the initial proliferation response, oncogenic HRAS activates IRE1α-mediated cleavage of XBP1 mRNA to produce the active transcription factor that is MEK-ERK dependent. Microarray analysis of keratinocytes with IRE1α knockdown using shRNA also showed that HRAS activates the regulated IRE1α-dependent mRNA decay (RIDD) function of IRE1α and this is responsible for downregulation of a subset of mRNAs by HRAS. Interestingly, IRE1α and XBP1 knockdown had distinct effects on premature senescence in HRAS keratinocytes. XBP1 knockdown induced premature senescence that was associated with extensive vacuolization whereas IRE1α knockdown did not despite similar inhibition of proliferation. Pharmacologic inhibition of IRE1α RNase activity inhibited premature senescence in XBP1-depleted HRAS keratinocytes and enhanced proliferation demonstrating a role for RIDD in this response. In addition, the potent cytostatic growth factor TGFβ1 which we previously showed induces rapid senescence in HRAS keratinocytes, inhibited IRE1α-mediated XBP1 splicing and enhanced RIDD possibly through alteration of IRE1α phosphorylation. Similarly, shRNA knockdown or pharmacologic inhibition of IRE1α bypassed TGFβ1-induced senescence, while XBP1 knockdown was susceptible to it. Using the RIDD gene signature obtained from microarray analysis of HRAS keratinocytes, we performed gene set enrichment analysis (GSEA) on previously identified gene expression profiles of benign and malignant mouse epidermal squamous tumors. Our results, validated by qPCR, show that genes in the RIDD signature are downregulated in benign tumors, paralleling the in vitro studies, but are upregulated in malignant tumors. In contrast, XBP1 splicing is similar in both tumor types. These results suggest that RIDD is linked senescence in vitro and benign tumor phenotype in vivo and that suppression of RIDD, but not XBP1 splicing, is a prerequisite for malignant progression. We propose that the balance between XBP1 splicing and RIDD determines whether a premalignant tumor cell undergoes proliferation and tumor progression or senescence and tumor suppression. Selective pharmacological agents that inhibit XBP1 splicing or enhance RIDD could be an important therapeutic modality to the eradication of cancer. Citation Format: Nicholas Blazanin, Christian John, Alayna Craig-Lucas, Adam Glick. Divergent IRE1α endonuclease outputs dictate the senescence response of mouse keratinocytes to oncogenic HRAS. [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 2257. doi:10.1158/1538-7445.AM2014-2257

Abstract 2172: Anti-oncogenic role of peroxisome proliferator-activated receptor-α/β (PPARδ/α) involves inhibition of mitosis and regulation of Hras1-induced senescence

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Ligand activation of PPARδ/α inhibits chemically-induced skin tumorigenesis and Pparα/α-null mice exhibit enhanced chemically-induced skin tumorigenesis compared to wild-type mice. The mechanism that mediates inhibition of skin carcinogenesis by PPARδ/α is still being unraveled and was examined in this study. Ligand activation of PPARδ/α caused a negative selection against cells expressing higher levels of HRAS by inducing a mitotic block. Mitosis-related genes that are predominantly regulated by E2F were induced to a higher level in HRAS-expressing Pparα/α-null keratinocytes as compared to HRAS-expressing wild-type keratinocytes. Ligand activated PPARδ/α repressed expression of these genes by direct binding with p130/p107, facilitating nuclear translocation, and increasing promoter recruitment of p130/p107. In addition, co-treatment with a PPARδ/α ligand and various mitosis inhibitors increases the efficacy of increasing G2/M arrest. PPARδ/α is also required for HRAS-induced senescence and suppression of malignant conversion of benign papillomas. HRAS-expressing Pparα/α-null keratinocytes evade senescence by utilizing the PI3K-AKT rather than the MEK-ERK pathway. PPARδ/α positively regulates MEK-ERK pathway by maintaining RASGRP1 levels and negatively regulating the PI3K-AKT pathway by repressing ILK and PDPK1 expression. Heightened AKT activity increases endoplasmic reticulum (ER) stress in Pparα/α-null keratinocytes, which in turn helps maintain higher AKT activity, leading to evasion of senescence. In addition, enhanced HRAS activity increases the synthesis of endogenous PPARδ/α ligands, thus providing a PPARδ/α-dependent cell-autonomous mechanism to suppress HRAS-induced carcinogenesis. (Supported by CA124533). Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2172. doi:1538-7445.AM2012-2172