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Daniel Clayburgh, MD PhDOregon Health and Science UniversityEmail: clayburg@ohsu.eduPhone: 503-494-5674

Objectives1. To determine if Cancerous Inhibitor ofPhosphoprotein 2A (CIP2A), which increases c-myc activity by inhibiting the c-myc inhibitorPhosphoprotein 2A (PP2A), is overexpressed incutaneous squamous cell carcinoma of the headand neck (cSCCHN). 2. To determine if PP2A expression is altered incSCCHN.

MethodsSpecimens of normal skin, early stage cSCCHN,late stage primary cSCCHN, and metastaticcSCCHN were sectioned and subjected toimmunofluorescence staining for CIP2A andPP2A. CIP2A and PP2A expression wasquantitated using the validated open-sourcesoftware CellProfiler. Expression changes wereassessed with ANOVA and Bonferroni tests, withp<0.05 denoting significance.

ResultsA total of 38 specimens were examined (5 normalskin, 9 early stage cSCCHN, 12 late stagecSCCHN, and 12 metastatic cSCCHN). CIP2Awas significantly overexpressed in early stagecSCCHN relative to normal skin (30.0 vs 17.7% ofcells positive for CIP2A, p=0.049), whileexpression was even higher in late stage primarycSCCHN (46.3% positive) and metastaticcSCCHN (49.4% positive, p=0.004 vs normal,ANOVA among all 4 groups: p=0.002). However,PP2A expression was completely unchangedamong all four groups (p=0.9754 by ANOVA).

ConclusionWhile PP2A expression is unchanged, CIP2A isoverexpressed in cSCCHN, and increasingexpression of CIP2A is correlated with metastaticprogression of cSCCHN. Thus, CIP2A activitymay be important to cSCCHN tumorigenesis andserve both as a prognostic marker and as a noveltarget for new therapeutic agents.

Overexpression of CIP2A in head and neck cutaneous SCCJade Koide, BS1, Neil Gross, MD2, David Sauer, MD3, Melissa Wong, PhD4, and Daniel Clayburgh, MD PhD2

School of Medicine (1) and Departments of Otolaryngology-Head and Neck Surgery (2), Pathology (3) and Dermatology (4)Oregon Health and Science University

Figures 2-4 show representative images of CIP2A, PP2A,Ras, total Myc and T58 Myc expression. Cytokeratin isalso shown to define the cSCC cells within each image.On gross examination, both c-Myc and CIP2A expressionappear elevated in cSCC specimens relative to normalskin, while Ras and PP2A expression do not appearsignificantly altered. The relative expression of theseproteins across all specimens examined was thenquantified using CellProfiler software.

Figure 5 shows the results of CellProfiler quantificationCIP2A was significantly overexpressed in early stagecSCCHN relative to normal skin (Figure 4A). CIP2Aexpression in late stage primary cSCCHN and metastaticcSCCHN was also significantly higher than normal skin,and was also higher than early stage cSCC (p=0.01 vsnormal skin, p=0.02 vs early stage cSCC). In contrast,PP2A expression was unchanged among all four groups(Figure 4B, p=0.9754 by ANOVA). This increase inCIP2A expression had the expected effect of increasingc-Myc stabilization, as c-Myc expression was increased incSCC (Figure 4C). However, there was no significantchange in c-Myc t58 phosphorylation in cSCC (Figure4D).

• 36 Specimens from 30 patients with cSCCHN were identified and dividedinto early stage primaries ( T1-2 N0 M0; n=12), advanced stage primaries(any T N1-3 or M1; n=12), and metastatic lesions (n=12). Normal skinspecimens (n=6) were also obtained.

• Specimens were sectioned and immunofluorescent detection of CIP2A,PP2A, Ras, total c-Myc, and phospho-T58 c-Myc was performed.

•Images were captured using confocal microscopy with identical acquisitionsettings across all specimens.

• Expression was quantitated using the validated open-source softwareCellProfiler. Expression changes were assessed with ANOVA andBonferroni tests, with p<0.05 denoting significance.

CIP2A is overexpressed in cSCC, and increasing expression of thisprotein correlates with disease progression. This leads to increasedstabilization of c-Myc, thereby driving carcinogenesis. CIP2A may thusbe a future therapeutic target for the treatment of cSCC.

Cutaneous squamous cell carcinoma (cSCC) is the second mostcommon type of non-melanoma type skin cancer, with over 200,000new cases diagnosed each year in the United States1. While earlystage disease is easily treated by surgical excision, late stage diseaseinvolving metastatic cSCCHN carries a poor prognosis. This is in largepart due to the lack of effective therapies targeting metastatic cSCCHN;thus a better understanding of the mechanisms underlying metastaticspread of cSCCHN is crucial to developing new therapeutic strategiesfor this disease.

The proto-oncogene c-Myc is a transcription factor involved in a varietyof cellular processes, including cell cycle regulation, growth,differentiation, and metabolism. Its activity is tightly regulated by manyproteins2; a simplified schematic of this is shown in Figure 1. c-Mycstabilization and degradation is driven by phosphorylation at two sites,S62 and T58. This is in turn controlled by the action of several kinasesand phosphatases. Protein phosphatase 2 (PP2A) controls the crucialfinal dephosphorylation of S62 that leads to degradation of c-Myc, thusits activity functions as a tumor suppressor. Recently, the CellularInhibitor of PP2A (CIP2A) was discovered to inhibit PP2A activity3,thereby promoting c-Myc stabilzation and tumor progression. IncreasedCIP2A expression has been found in prostate, breast, gastric, and colonadenocarcinomas, and head and neck mucosal SCC4-8, but has not yetbeen investigated in cSCCHN.

INTRODUCTION

MATERIALS AND METHODS1. Alam M, Ratner D. Cutaneous squamous-cell carcinoma. N Engl J Med 2001;344:975-83.2. Junttila MR, Westermarck J. Mechanisms of MYC stabilization in human malignancies. Cell Cycle 2008;7:592-6.3. Junttila MR, Puustinen P, Niemela M, et al. CIP2A inhibits PP2A in human malignancies. Cell 2007;130:51-62.4. Come C, Laine A, Chanrion M, et al. CIP2A is associated with human breast cancer aggressivity. Clin Cancer

Res 2009;15:5092-100.5. Dong QZ, Wang Y, Dong XJ, et al. CIP2A is Overexpressed in Non-Small Cell Lung Cancer and Correlates with

Poor Prognosis. Ann Surg Oncol 2010.6. Katz J, Jakymiw A, Ducksworth MK, et al. CIP2A expression and localization in oral carcinoma and dysplasia.

Cancer Biol Ther 2010;10:694-9.7. Khanna A, Bockelman C, Hemmes A, et al. MYC-dependent regulation and prognostic role of CIP2A in gastric

cancer. J Natl Cancer Inst 2009;101:793-805.8. Vaarala MH, Vaisanen MR, Ristimaki A. CIP2A expression is increased in prostate cancer. J Exp Clin Cancer Res

2010;29:136.9. Qu W, Li W, Wei L, et al. CIP2A is overexpressed in esophageal squamous cell carcinoma. Med Oncol 2010.10. Lucas CM, Harris RJ, Giannoudis A, et al. Cancerous inhibitor of PP2A (CIP2A) at diagnosis of chronic myeloid

leukemia is a critical determinant of disease progression. Blood 2011;117:6660-8.11. Bockelman C, Hagstrom J, Makinen LK, et al. High CIP2A immunoreactivity is an independent prognostic

indicator in early-stage tongue cancer. Br J Cancer 2011;104:1890-

CONCLUSIONS

DISCUSSIONRESULTS

REFERENCES

Figure 2. Immunofluorescent detection of cytokeratin, PP2A,and CIP2A in normal, early primary, advanced primary, andmetastatic cSCC

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Figure 5. Quantification of protein expression using CellProfiler of (A) CIP2A, (B) PP2A, (C) c-Myc, and (D) T58 c-Myc. (* indicates p<0.05 vs normal skin)

Figure 3. Immunofluorescent detection of cytokeratin, total c-Myc, and Ras in normal, early primary, advanced primary, andmetastatic cSCC

Figure 4. Immunofluorescent detection ofcytokeratin and T58 Myc in normal, early primary,advanced primary, and metastatic cSCC

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CIP2A has recently been described to be a significant regulator of c-Mycactivity, and by extension an important mediator of tumorigenesis3.Overexpression of CIP2A has been implicated in a number of different typesof tumors4-6,8,9. However, the expression of CIP2A has not been defined incutaneous squamous cell carcinoma. In this study, we demonstrated that theexpression of CIP2A was significantly increased in cSCCHN. Moreover,CIP2A expression increases with higher stage disease, suggesting thatincreasing CIP2A expression may drive disease progression. High levels ofCIP2A have been shown to be a negative prognostic marker in othercancers10,11, and may have value as a pronostic marker in cSCC as well.

The observed increase in CIP2A in cSCC appeared to have the expectedeffect upon the c-Myc pathway. While PP2A expression was unchanged, c-Myc expression was increased, indicating that increased CIP2A expressioninhibited PP2A activity and led the c-Myc stabilization. Other c-Mycregulatory pathways, notably Ras, did not appear to play a role in thisincreased c-Myc stabilization. These results are similar to previous reports inother tumor types, which link increased CIP2A expression to c-Mycstabilization4,5,7.

Interestingly, no change in c-Myc phosphorylation at T58 was seen in cSCCcompared to normal skin. This may suggest that the phospho-S62 form of c-Myc accounts for much of the increased c-Myc expression. Alternatively, inblocking PP2A activity, CIP2A both decreases the amount of p-T58 c-Mycwhile increasing the amount of p-T58 p-S62 c-Myc (both forms would bedetected by the antibody used in this study), thereby leading to no netchange in overall p-T58 c-Myc. This question may be addressed by futureevaluation of the expression of p-S62 c-Myc.

Figure 1: c-Myc regulation