Grace Y. Wang

Basal Cell Carcinomas Arise From Hair Follicle Stem Cells in Ptch1+/− Mice

Basal cell carcinomas (BCCs) are hedgehog-driven tumors that resemble follicular and interfollicular epidermal basal keratinocytes and hence long have been thought to arise from these cells. However, the actual cell of origin is unknown. Using cell fate tracking of X-ray induced BCCs in Ptch1(+/-) mice, we found their essentially exclusive origin to be keratin 15-expressing stem cells of the follicular bulge. However, conditional loss of p53 not only enhanced BCC carcinogenesis from the bulge but also produced BCCs from the interfollicular epidermis, at least in part by enhancing Smo expression. This latter finding is consistent with the lack of visible tumors on ears and tail, sites lacking Smo expression, in Ptch1(+/-) mice.

Compromised Mitochondrial Fatty Acid Synthesis in Transgenic Mice Results in Defective Protein Lipoylation and Energy Disequilibrium

A mouse model with compromised mitochondrial fatty acid synthesis has been engineered in order to assess the role of this pathway in mitochondrial function and overall health. Reduction in the expression of mitochondrial malonyl CoA-acyl carrier protein transacylase, a key enzyme in the pathway encoded by the nuclear Mcat gene, was achieved to varying extents in all examined tissues employing tamoxifen-inducible Cre-lox technology. Although affected mice consumed more food than control animals, they failed to gain weight, were less physically active, suffered from loss of white adipose tissue, reduced muscle strength, kyphosis, alopecia, hypothermia and shortened lifespan. The Mcat-deficient phenotype is attributed primarily to reduced synthesis, in several tissues, of the octanoyl precursors required for the posttranslational lipoylation of pyruvate and α-ketoglutarate dehydrogenase complexes, resulting in diminished capacity of the citric acid cycle and disruption of energy metabolism. The presence of an alternative lipoylation pathway that utilizes exogenous free lipoate appears restricted to liver and alone is insufficient for preservation of normal energy metabolism. Thus, de novo synthesis of precursors for the protein lipoylation pathway plays a vital role in maintenance of mitochondrial function and overall vigor.

Establishment of murine basal cell carcinoma allografts – a potential model for preclinical drug testing and for molecular analysis

Dysregulated hedgehog (HH) signaling has been found in numerous cancers, suggesting that therapeutic targeting of this pathway may be useful vs. a wide range of cancers. Basal cell carcinoma (BCC) is an excellent model system for studying the influence of the HH pathway on carcinogenesis because aberrant activation of HH signaling not only is crucial for the development but also for the maintenance of BCC. Genetically engineered BCC mouse models provide one important tool for the study of the biology of human BCCs and for evaluating therapeutic interventions since these mice produce multiple genetically defined tumors within a relatively short period of time. However, these models remain expensive and cumbersome to use for large-scale pre-clinical drug testing. Here we report a method for growing allografts from murine BCC tumors in NOD/SCID mice. These allografts develop faster and reproduce the histology, immunophenotypes and response to at least one anti-BCC drug of the parental autochthonous tumors from which they arise. Therefore, the allograft model provides a practical model for (i) studying BCC carcinogenesis and (ii) initial pre-clinical screening for anti-HH pathway and other anti-BCC drugs.

Vitamin D3 Produced by Skin Exposure to UVR Inhibits Murine Basal Cell Carcinoma Carcinogenesis

The effect of UVR on human basal cell carcinoma (BCC) epidemiology is complex-the incidence rises until approximately 30,000 hours of lifetime sunlight exposure and then plateaus. We hypothesize that UVR has opposing effects on BCC carcinogenesis-stimulatory via mutagenesis and inhibitory via production of hedgehog-inhibiting vitamin D3 (D3). We find that UVR exposure of ionizing radiation-treated Ptch1+/- mice accelerates BCC carcinogenesis in male mice, in which UVR does not produce D3. By contrast, in female mice, in which UVR does produce D3, UVR fails to accelerate BCC carcinogenesis, thus mirroring the plateauing in humans. However, if D3 production is attenuated in female mice by deletion of keratinocyte lathosterol 5-desaturase, then UVR accelerates ionizing radiation-induced BCC carcinogenesis. Congruently, chronic topical application of D3 inhibits ionizing radiation-induced BCC tumorigenesis. These findings confirm that UVR-induced production of D3 in keratinocytes significantly restrains murine BCC tumorigenesis and demonstrate the counterintuitive conclusion that UVR has anti-BCC carcinogenic effects that can explain, at least in part, the complex relationship between exposure to UVR and BCC incidence.

PI3K-AKT Signaling Is a Downstream Effector of Retinoid Prevention of Murine Basal Cell Carcinogenesis

Basal cell carcinoma (BCC) is the most common human cancer. We have demonstrated previously that topical application of the retinoid prodrug tazarotene profoundly inhibits murine BCC carcinogenesis via retinoic acid receptor γ–mediated regulation of tumor cell transcription. Because topical retinoids can cause adverse cutaneous effects and because tumors can develop resistance to retinoids, we have investigated mechanisms downstream of tazarotenes antitumor effect in this model. Specifically we have used (i) global expression profiling to identify and (ii) functional cell-based assays to validate the phosphoinositide 3-kinase (PI3K)/AKT/mTOR pathway as a downstream target pathway of tazarotenes action. Crucially, we have demonstrated that pharmacologic inhibition of this downstream pathway profoundly reduces murine BCC cell proliferation and tumorigenesis both in vitro and in vivo. These data identify PI3K/AKT/mTOR signaling as a highly attractive target for BCC chemoprevention and indicate more generally that this pathway may be, in some contexts, an important mediator of retinoid anticancer effects. Cancer Prev Res; 7(4); 407–17. ©2014 AACR.

Targeting human glioma cells using HSV-1 amplicon peptide display vector

Targeting cell infection using herpes simplex virus type 1 (HSV-1) vectors is a complicated issue as the process involves multiple interactions of viral envelope glycoproteins and cellular host surface proteins. In this study, we have inserted a human glioma-specific peptide sequence (denoted as MG11) into a peptide display HSV-1 amplicon vector replacing the heparan sulfate-binding domain of glycoprotein C (gC). The modified MG11:gC envelope recombinant vectors were subsequently packaged into virions in the presence of helper virus deleted for gC. Our results showed that the tropism of these HSV-1 recombinant virions was increased for human glioma cells in culture as compared with wild-type virions. The binding of these recombinant virions could also be blocked effectively by pre-incubating the cells with the glioma-specific peptide, indicating that MG11 peptide and the recombinant virions competed for the same or similar receptor-binding sites on the cell surface of human glioma cells. Furthermore, preferential homing of these virions was shown in xenograft glioma mouse model following intravascular delivery. Taken together, these results validated the hypothesis that HSV-1 binding to cells can be redirected to human gliomas through the incorporation of MG11 peptide sequence to the virions.

Interleukin-13 receptor alpha 2 cooperates with EGFRvIII signaling to promote glioblastoma multiforme

The interleukin-13 receptor alpha2 (IL-13Rα2) is a cancer-associated receptor overexpressed in human glioblastoma multiforme (GBM). This receptor is undetectable in normal brain which makes it a highly suitable target for diagnostic and therapeutic purposes. However, the pathological role of this receptor in GBM remains to be established. Here we report that IL-13Rα2 alone induces invasiveness of human GBM cells without affecting their proliferation. In contrast, in the presence of the mutant EGFR (EGFRvIII), IL-13Rα2 promotes GBM cell proliferation in vitro and in vivo. Mechanistically, the cytoplasmic domain of IL-13Rα2 specifically binds to EGFRvIII, and this binding upregulates the tyrosine kinase activity of EGFRvIII and activates the RAS/RAF/MEK/ERK and STAT3 pathways. Our findings support the “To Go or To Grow” hypothesis whereby IL-13Rα2 serves as a molecular switch from invasion to proliferation, and suggest that targeting both receptors with STAT3 signaling inhibitor might be a therapeutic approach for the treatment of GBM.Interleukin-13 receptor alpha 2 is highly expressed in glioblastoma multiforme but its role in this malignancy is unclear. Here the authors show that this receptor interacts with mutant EGFR, stimulating its kinase activity, thus inducing proliferation.

Abstract 779: ARF activation is responsible for the tumor-suppressing function of p53 in basal cell carcinomas (BCCs) in Ptch1+/- mice.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Basal cell carcinomas (BCCs) are the most common cancer among people of European ancestry. Aberrant activation of hedgehog (HH) signaling plays a pivotal role in BCC carcinogenesis. Besides mutations in genes encoding HH pathway components, mutations in the p53 gene frequently are found in human BCCs. Consistently, our previous studies have found that the loss of p53 can dramatically accelerate BCC formation in ionizing radiation (IR)-treated Ptch1+/- mice. It remains elusive how p53 is activated in BCC development. DNA damage response (DDR) and oncogenic induced stress (OIS) are two essentially independent pathways leading to p53 activation although limited evidence suggests crosstalk between these pathways. In response to OIS, ARF is activated and leads to p53 activation, functioning as a tumor suppressor, in many cancers. However, the loss of ARF, unlike loss of p53, failed to affect the development of medulloblastoma in Ptch1+/- mice (Wetmore C., et al, 2001). In the present study, we investigated whether ARF affects BCC development in the same mice. To address this, we bred ARFGFP/GFP mice, in which ARF function is disabled and GFP is expressed under the control of the endogenous ARF promoter, with our Ptch1+/- mice to generate Ptch1+/- ARFGFP/GFP (designated as ARF mice) for comparison with Ptch1+/- K14CreER2 p53fl/fl (PF) mice. Following our standard protocol of tamoxifen injection at age 7.5 weeks to delete p53 specifically in K14-expressing keratinocytes (for PF mice) and treating both ARF and PF mice with ionizing radiation at age 8 weeks, we compared BCC development between the above 2 lines. We find that at age 5 months, ARF mice formed numbers of microscopic BCCs comparable to those in PF mice though the latter exhibited greater mouse-to-mouse variation. More importantly, both mouse lines developed initial visible BCCs at age 5 to 7 months, which is dramatically earlier than the age 9 to 18 months for the Ptch1+/- mice (ARF+/+ and p53+/+). In addition, ARFGFP/GFP mice responded to IR-induced DNA damage in a pattern essentially similar to ARF+/+ mice, indicating an intact DDR pathway as reported in the literature. In conclusion, our data demonstrate that ARF is involved in BCC carcinogenesis as a tumor suppressor, probably playing the major, perhaps the exclusive, role in activating p53 in this process. Citation Format: Grace Y. Wang, Carla Wood, Joy Wang, Lynn Wang, Eileen Libove, Ervin Epstein. ARF activation is responsible for the tumor-suppressing function of p53 in basal cell carcinomas (BCCs) in Ptch1+/- mice. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 779. doi:10.1158/1538-7445.AM2013-779

Abstract 208: The function of p53 is intact in murine basal cell carcinomas in IR-treated Ptch1+/- mice

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Basal cell carcinomas (BCC) are the most common cancer among people of European ancestry. Although mutational activation of Hedgehog (HH) signaling is pivotal for BCC carcinogenesis, p53 mutations also frequently are present in human and mouse BCCs. In humans, these p53 mutations are frequently of a “UV-signature” type, and human BCCs typically arise in sun-damaged skin containing epidermal clones of p53 mutant cells, implying that p53 is a crucial BCC tumor suppressor. In line with this notion, we found that deletion of p53 specifically in K14-expressing keratinocytes dramatically accelerates BCC carcinogenesis in ionizing radiation (IR)-treated Ptch1+/- mice. In IR-treated Ptch1+/- mice without p53 deletion, visible BCCs can also arise from but with longer tumor latency (9m vs. 5m) and a lower tumor incidence (10% vs. 100%) and mutiplicity. However it is not known whether loss of p53 signaling activity is required for the development of visible BCCs in p53+/+ mice. To address this question, we compared p53 signaling activity in IR-induced BCCs in Ptch1+/- p53+/+ mice vs. in BCCs in Ptch1+/- p53-/- mice after a second, DNA-damaging dose of IR. Using immunohistochemistry, we found the expected minimal IR-induced expression of p53, Puma, and Noxa as well as TUNEL-detected apoptosis in BCCs of p53-deleted mice. By contrast, IR induced robust expression of p53, Noxa, Puma, and apoptosis in BCCs from Ptch1+/- p53-intact mice. Without IR exposure, BCCs in p53+/+ mice had scattered p53+ cells and low level of expression of Puma and Noxa but no TUNEL+ cells. Our data thus suggest that IR-induced BCCs generated in Ptch1+/- p53+/+ mice retain their functional p53 signaling capacity and that these tumors are able to evade an intact DNA damage response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 208. doi:10.1158/1538-7445.AM2011-208

Ultraviolet Radiation Inhibits Mammary Carcinogenesis in an ER-Negative Murine Model by a Mechanism Independent of Vitamin D3

Three decades ago, the Garlands postulated that vitamin D3 produced in the skin by ultraviolet radiation (UVR)-induced conversion of 7-dehydrocholesterol to pre-D3 has anticancer effects, thus triggering more than 9,500 publications on D3 and cancer. Here, we report that UVR treatment of transgenic mice of the well-established C3(1)/SV40 Tag mammary cancer model significantly inhibits both autochthonous carcinogenesis and allograft tumor growth, but in contrast neither dietary nor topical D3 influences mammary carcinogenesis in this specific mouse model. Furthermore, UVRs inhibitory effects occur irrespective of whether or not the treatment increases circulating D3 in the mice. The inhibitory effect of UVR on autochthonous tumors occurs at or before the stage of ductal carcinoma in situ. Our studies indicate clearly that UVR can exert D3-independent anticancer effects in C3(1)/SV40 Tag mice. Therefore, supplemental D3 may not mimic all possible beneficial effects of UVR, and uncovering non–D3-mediated mechanisms of UVR tumor inhibition may lead to novel strategies for cancer prevention. Cancer Prev Res; 11(7); 383–92. ©2018 AACR.