Histologic Alterations Produced by Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) in SENCAR Mouse Skin: Relationship to Skin Tumor Promoting Activity

Histologic changes induced in SENCAR skin following a single treatment with chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) exhibited differences in time course from that observed with 12-O-tetradecanoylphorbol-13-acetate (TPA). Although not significantly different, maximum elevations in epidermal thickness, total number of nucleated epidermal cells, and dark basal keratinocytes (DCs) induced by 220nmol chrysarobin occurred at 96h after treatment, while those induced by 3.4nmol TPA occurred at 48h. Both compounds elicited comparable inflammatory responses. Twice-weekly applications of chrysarobin for 2.5 weeks induced a moderate hyperplasia, increase in total nucleated epidermal cells, and increased DCs at 48 and 96h after the last treatment, with a higher value for these parameters occurring at 48h. Interestingly, the magnitude of these changes was similar to that observed after a single application. In contrast, twice-weekly applications of TPA induced a dramatic, potentiated induction of epidermal hyperplasia and DCs. Once-weekly applications of chrysarobin led to a potentiated induction of both hyperplasia and DCs compared to the twice-weekly treatment regimen and also more effectively promoted epidermal papillomas in previously initiated SENCAR mice. Skin sections from mice treated with chrysarobin displayed overt signs of epidermal toxicity including altered basal cell morphology and a decreased number of basal cells per 125μm of basement membrane. Hyperplasia induced by multiple but not single treatments with chrysarobin and TPA correlated quantitatively with their papilloma promoting activity. In addition, the data suggest that epidermal toxicity may play a role in tumor promotion by anthrones

Protein Tyrosine Phosphatases, TC-PTP, SHP1, and SHP2, Cooperate in Rapid Dephosphorylation of Stat3 in Keratinocytes Following UVB Irradiation

Stat3 is initially dephosphorylated in murine keratinocytes in response to UVB irradiation. Treatment with Na3VO4 desensitized keratinocytes to UVB-induced apoptosis with the recovery of phosphorylated Stat3 protein levels, implying that a protein tyrosine phosphatase (PTP) is involved in this mechanism. In the current work, we report that three PTPs including TC45 (the nuclear form of TC-PTP), SHP1, and SHP2 are involved in this rapid dephosphorylation of Stat3 in keratinocytes induced by UVB irradiation. Dephosphorylation of Stat3 was increased rapidly after UVB irradiation of cultured keratinocytes. Knockdown of TC-PTP, SHP1, or SHP2 using RNAi showed that these PTPs are likely responsible for most of the rapid Stat3 dephosphorylation observed following UVB irradiation. The level of phosphorylated Stat3 was significantly higher in keratinocytes transfected with TC-PTP, SHP1, or SHP2 siRNA in the presence or absence of UVB compared with keratinocytes transfected with control siRNA. TC45 was mainly localized in the cytoplasm of keratinocytes and translocated from cytoplasm to nucleus upon UVB irradiation. Stat3 dephosphorylation was associated with nuclear translocation of TC45. Further studies revealed that knockdown of all three phosphatases, using RNAi, prevented the rapid dephosphorylation of Stat3 following UVB irradiation. In mouse epidermis, the level of phosphorylated Stat3 was initially decreased, followed by a significant increase at later time points after UVB exposure. The levels of Stat3 target genes, such as cyclin D1 and c-Myc, followed the changes in activated Stat3 in response to UVB irradiation. Collectively, these results suggest that three phosphatases, TC45, SHP1, and SHP2, are primarily responsible for UVB-mediated Stat3 dephosphorylation and may serve as part of an initial protective mechanism against UV skin carcinogenesis

Active Stat3 is required for survival of human squamous cell carcinoma cells in serum-free conditions

BACKGROUND: Squamous cell carcinoma (SCC) of the skin is the most aggressive form of non-melanoma skin cancer (NMSC), and is the single most commonly diagnosed cancer in the U.S., with over one million new cases reported each year. Recent studies have revealed an oncogenic role of activated signal transducer and activator of transcription 3 (Stat3) in many human tumors, especially in those of epithelial origin, including skin SCC. Stat3 is a mediator of numerous growth factor and cytokine signaling pathways, all of which activate it through phosphorylation of tyrosine 705. RESULTS: To further address the role of Stat3 in skin SCC tumorigenesis, we have analyzed a panel of human skin-derived cell lines ranging from normal human epidermal keratinocytes (NHEK), to non-tumorigenic transformed skin cells (HaCaT), to highly tumorigenic cells (SRB1-m7 and SRB12-p9) and observed a positive correlation between Stat3 phosphorylation and SCC malignancy. We next determined the role of Stat3 activity in cell proliferation and viability under serum-free culture conditions. This was accomplished by suppressing Stat3 activity in the SRB12-p9 cells through stable expression of a dominant negative acting form of Stat3β, which contains a tyrosine 705 to phenylalanine mutation (S3DN). The S3DN cells behaved similar to parental SRB12-p9 cells when cultured in optimal growth conditions, in the presence of 10% fetal calf serum. However, unlike the SRB12-p9 cells, S3DN cells underwent apoptotic cell death when cultured in serum-free medium (SFM). This was evidenced by multiple criteria, including accumulation of sub-G1 particles, induced PARP cleavage, and acquisition of the characteristic morphological changes associated with apoptosis. CONCLUSION: This study provides direct evidence for a role for Stat3 in maintaining cell survival in the conditions of exogenous growth factor deprivation produced by culture in SFM. We also propose that delivery of the S3DN gene or protein to tumor cells could induce apoptosis and/or sensitize those cells to the apoptotic effects of cancer therapeutic agents, raising the possibility of using S3DN as an adjunct for treatment of skin SCC

Contributors to the Summer Issue/Notes

Notes by Louis F. DiGiovanni, E. A. Steffen, Jr., John L. Globensky, Lenton G. Sculthorp, William V. Phelan, and George Ratterman

Contributors to the Summer Issue/Notes

Notes by Louis F. DiGiovanni, E. A. Steffen, Jr., John L. Globensky, Lenton G. Sculthorp, William V. Phelan, and George Ratterman

UVB-induced nuclear translocation of TC-PTP by AKT/14-3-3σ axis inhibits keratinocyte survival and proliferation

Understanding protein subcellular localization is important to determining the functional role of specific proteins. T-cell protein tyrosine phosphatase (TC-PTP) contains bipartite nuclear localization signals (NLSI and NLSII) in its C-terminus. We previously have demonstrated that the nuclear form of TC-PTP (TC45) is mainly localized to the cytoplasm in keratinocytes and it is translocated to the nucleus following UVB irradiation. Here, we report that TC45 is translocated by an AKT/14-3-3σ-mediated mechanism in response to UVB exposure, resulting in increased apoptosis and decreased keratinocyte proliferation. We demonstrate that UVB irradiation increased phosphorylation of AKT and induced nuclear translocation of 14-3-3σ and TC45. However, inhibition of AKT blocked nuclear translocation of TC45 and 14-3-3σ. Site-directed mutagenesis of 14-3-3σ binding sites within TC45 showed that a substitution at Threonine 179 (TC45/T179A) effectively blocked UVB-induced nuclear translocation of ectopic TC45 due to the disruption of the direct binding between TC45 and 14-3-3σ. Overexpression of TC45/T179A in keratinocytes resulted in a decrease of UVB-induced apoptosis which corresponded to an increase in nuclear phosphorylated STAT3, and cell proliferation was higher in TC45/T179A-overexpressing keratinocytes compared to control keratinocytes following UVB irradiation. Furthermore, deletion of TC45 NLSII blocked its UVB-induced nuclear translocation, indicating that both T179 and NLSII are required. Taken together, our findings suggest that AKT and 14-3-3σ cooperatively regulate TC45 nuclear translocation in a critical step of an early protective mechanism against UVB exposure that signals the deactivation of STAT3 in order to promote keratinocyte cell death and inhibit keratinocyte proliferation

The role of T-cell protein tyrosine phosphatase in epithelial carcinogenesis

T-cell protein tyrosine phosphatase (TC-PTP, encoded by PTPN2) is a non-receptor PTP that is mostly highly expressed in hematopoietic tissues. TC-PTP modulates a variety of physiological functions including cell cycle progression, cell survival and proliferation, and hematopoiesis through tyrosine dephosphorylation of its target substrates, such as EGFR, JAK1, JAK3, STAT1, and STAT3. Studies with whole or tissue-specific loss of TC-PTP function transgenic mice have shown that TC-PTP has crucial roles in the regulation of the immune response, insulin signaling, and oncogenic signaling. More recently, generation of epidermal-specific TC-PTP-deficient mice for use in multistage skin carcinogenesis bioassays demonstrated that TC-PTP suppresses skin tumor formation by negatively regulating STAT3 and AKT signaling. Further investigation showed that TC-PTP also minimizes UVB-induced epidermal cell damage by promoting apoptosis through the negative regulation of Flk-1/JNK signaling. These findings provide major evidence for a tumor suppressive function for TC-PTP against environment-induced skin cancer. Here, we will discuss TC-PTP, its substrates, and its functions with an emphasis on its role in skin carcinogenesis

Clinical Utilization of Patient Reported Outcome (PROMIS) Scores for Surgical Reconstruction of Posterior Tibialis Tendon Dysfunction

Introduction/Purpose: Previous studies have demonstrated that preoperative Patient Reported Outcome Instrumentation System (PROMIS) scores effectively predict improvement in foot and ankle surgery. Adult acquired flatfoot deformity (AAFD) and Posterior Tibialis Tendon Dysfunction (PTTD) are a common surgical problem, but it is unclear if the specific thresholds for the physical function (PF), pain interference (PI) and depression published previously for all foot and ankle surgeries apply to a specific diagnosis. Furthermore, the interplay of PROMIS scores and clinical variables has not been evaluated. The purpose of this study was: 1) to investigate the change in PROMIS scales and radiographic measurements from pre- to postoperative follow up in AAFD/PTTD patients, 2) to determine if preoperative PROMIS scales predict post-surgical improvement, 3) to determine if demographic, clinical variables combined with pre-operative PROMIS scales predict post-surgical improvement

Determining Success or Failure After Foot and Ankle Surgery Using Patient Acceptable Symptom State (PASS) and Patient Reported Outcome Information System (PROMIS)

Background: As the role of generic patient-reported outcomes (PROs) expands, important questions remain about their interpretation. In particular, how the Patient Reported Outcome Measurement Instrumentation System (PROMIS) t score values correlate with the patients’ perception of success or failure (S/F) of their surgery is unknown. The purposes of this study were to characterize the association of PROMIS t scores, the patients’ perception of their symptoms (patient acceptable symptom state [PASS]), and determination of S/F after surgery. Methods: This retrospective cohort study contacted patients after the 4 most common foot and ankle surgeries at a tertiary academic medical center (n = 88). Patient outcome as determined by phone interviews included PASS and patients’ judgment of whether their surgery was a S/F. Assessment also included PROMIS physical function (PF), pain interference (PI), and depression (D) scales. The association between S/F and PASS outcomes was evaluated by chi-square analysis. A 2-way analysis of variance (ANOVA) evaluated the ability of PROMIS to discriminate PASS and/or S/F outcomes. Receiver operator curve (ROC) analysis was used to evaluate the ability of pre- (n = 63) and postoperative (n = 88) PROMIS scores to predict patient outcomes (S/F and PASS). Finally, the proportion of individuals classified by the identified thresholds were evaluated using chi-square analysis. Results: There was a strong association between PASS and S/F after surgery (chi-square \u3c0.01). Two-way ANOVA demonstrated that PROMIS t scores discriminate whether patients experienced positive or negative outcome for PASS (P \u3c .001) and S/F (P \u3c .001). The ROC analysis showed significant accuracy (area under the curve \u3e 0.7) for postoperative but not preoperative PROMIS t scores in determining patient outcome for both PASS and S/F. The proportion of patients classified by applying the ROC analysis thresholds using PROMIS varied from 43.0% to 58.8 % for PASS and S/F. Conclusions: Patients who found their symptoms and activity at a satisfactory level (ie, PASS yes) also considered their surgery a success. However, patients who did not consider their symptoms and activity at a satisfactory level did not consistently consider their surgery a failure. PROMIS t scores for physical function and pain demonstrated the ability to discriminate and accurately predict patient outcome after foot and ankle surgery for 43.0% to 58.8% of participants. These data improve the clinical utility of PROMIS scales by suggesting thresholds for positive and negative patient outcomes independent of other factors. Level of Evidence: II, prospective comparative series