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Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma
YAJOT-01793; No of Pages 5 American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
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Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma Gino Marioni a,⁎, Elisabetta Zanoletti a, Antonio Mazzoni a, Andrea Gianatti b, Elisa Valentini c, Laura Girasoli a, Martina Guariento a, Luciano Giacomelli c, Alessandro Martini a, Stella Blandamura c a b c
Department of Neurosciences DNS, Otolaryngology Section, Padova University, Padova, Italy Anatomic Pathology Unit, Ospedali Riuniti Bergamo, Bergamo, Italy Department of Medicine DIMED, University of Padova, Italy
a r t i c l e
i n f o
Article history: Received 17 November 2016 Available online xxxx Keywords: Temporal bone squamous cell carcinoma Cortactin Phosphorylated Prognosis Target therapy
a b s t r a c t Purpose: Cortactin is a multidomain protein engaged in several cellular mechanisms involving actin assembly and cytoskeletal arrangement. Cortactin overexpression in several malignancies has been associated with increased cell migration, invasion, and metastatic potential. Cortactin needs to be activated by tyrosine or serine/threonine phosphorylation. The role of cortactin and phosphorylated cortactin (residue tyr466) was investigated in temporal bone squamous cell carcinoma (TBSCC). Materials and methods: Immunohistochemical expression of cortactin and phosphorylated cortactin (residue tyr466) was assessed in 27 consecutively-operated TBSCCs. Results: Several clinicopathological variables correlated with recurrence (pT stage, dura mater involvement), and disease-free survival (DFS) (cT stage, pT stage, pN status, dura mater involvement). Twenty-three of 24 immunohistochemically evaluable TBSCCs were cortactin-positive. Median cortactin expression was 75.0%. Cortactin reaction in the cytoplasm was more intense in carcinoma cells than in normal adjacent tissue. Recurrence and DFS rates did not correlate with cortactin and phosphorylated cortactin (residue tyr466) expression in TBSCC specimens. Conclusions: Cortactin upregulation in TBSCC supports the conviction that inhibiting cortactin functions could have selective effects on this malignancy. Multi-institutional studies should further investigate the role of cortactin and phosphorylated cortactin in TBSCC, and their potential clinical application in integrated treatment modalities. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Given the poor prognosis for patients with advanced temporal bone squamous cell carcinoma (TBSCC) [1], there is an undeniable need for novel, more effective diagnostic/therapeutic strategies to improve the outcomes of treatment. TBSCC primary therapies definitely need to be patient-tailored more rationally [1], also because managing TBSCC recurrences is particularly difficult, and any treatment carries a significant morbidity and mortality [2]. Extending surgery to ensure safe tissue margins is the premise for the adequate treatment of TBSCC. Molecular changes occurring before any morphological changes become apparent are responsible for the disease's biological behavior, prognosis, and response to primary therapy [3]. In clinical practice, the ideal molecular marker should have: (i) prognostic value; (ii) a significant capacity to predict the efficacy of specific treatments; and (iii) the
⁎ Corresponding author at: Dept of Neurosciences DNS, Otolaryngology Section, University of Padova, Via Giustiniani 2, 35128 Padova, Italy. E-mail address: [email protected] (G. Marioni).
features needed for it to become the target of integrated therapeutic approaches [4]. Cortactin is a multidomain protein engaged in several cellular mechanisms based on actin assembly and cytoskeletal arrangement, such as endocytosis, cell migration, neuronal morphogenesis and tumor invasion. Cortactin is an actin-binding protein that promotes the weak activation of the actin-related protein (ARP) 2/3 complex in actin nucleation, and it stabilizes the formation of newly-branching network of F-actin [5]. Cortactin occurs in the cytoplasm and in actin-rich peripheral structures such as lamellipodia and podosomes. The cortactin locus CTTN is located in the 11q13 region, the amplification of which is one of the alterations most often seen in head and neck squamous cell carcinoma (HNSCC). Studies have shown that 11q13 amplification correlates with a poor prognosis in HNSCC patients, and cortactin has been accused of being the gene responsible for tumor progression because of its role in cell motility and carcinoma invasion. Cortactin overexpression promotes tumor aggressiveness, even without the 11q13 amplicon, suggesting a post-translational activation rather than a gene amplification in HNSCC [6,7]. Cortactin needs to be activated by tyrosine or serine/threonine phosphorylation. Tyrosine phosphorylation stems from
http://dx.doi.org/10.1016/j.amjoto.2017.01.012 0196-0709/© 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
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G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
activation of the epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases, and it occurs at residues tyr421, tyr466 and tyr482, in the proline-rich domain of the carboxy terminus. Phospho-cortactin interacts with the adaptor protein NCK1, which binds the neuronal Wiskott Aldrich Syndrome protein, resulting in ARP 2/3 activation. The role of phosphorylation in cortactin function is still not entirely clear, but it correlates positively with the ability to induce cell migration through matrix metalloproteinase recruitment and secretion in invadopodia [8,9]. Cancer has the characteristic ability to invade surrounding tissues and metastasize to regional and distant sites. The events attendant on local invasion and metastasis by epithelial tumors such as TBSCC include: (i) loss of adhesion to surrounding tumor cells and basement membrane; (ii) production of enzymes and mediators that facilitate the incursion of malignant cells into the subjacent connective tissue; (iii) attachment to extracellular membrane molecules; (iv) neovascularization; (v) passage into and out of the circulation via attachment to endothelial cell ligands; and (vi) a repeat of this cascade at a metastatic site [10]. The details of these mechanisms are not fully known in the case of TBSCC. The present study is the first to have investigated the immunohistochemical expression of cortactin and phosphorylated cortactin (residue tyr466) in patients with primary TBSCC. The aim of the study was to conduct a preliminary assessment of the role of cortactin in this malignancy. 2. Methods 2.1. Patients (see Table 1) The present investigation was approved by our Otolaryngology Section's in-house ethical committee. The study was carried out in accordance with the principles of the Helsinki Declaration. The study was conducted on specimens from 27 patients with primary TBSCC operated by the same surgeon (A.Mz.) at a tertiary referral center. The patients included 15 women and 12 men with a mean age of 54.0±12.7 years, who were part of a larger series investigated for other purposes in a previous study [11]. Preoperatively, all patients underwent micro-otoscopy with biopsy, temporal bone computerized
tomography and/or contrast-enhanced magnetic resonance imaging, and neck ultrasonography (with or without fine-needle aspiration cytology). Positron emission tomography was performed in advanced cases to rule out distant metastasis. Based on the revised Pittsburgh staging system [12,13], the primary TBSCCs were classified as cT1 in 5 cases, cT2 in 6, cT3 in 9, and cT4 in 7. En-bloc lateral temporal bone resection was performed in 19 cases (two of them partial), and en-bloc subtotal temporal bone resection in 8. The surgical techniques involved have been described in detail elsewhere [14]. The facial nerve was sacrificed in 9 of the 27 patients, i.e. in all the cases of subtotal temporal bone resection to enable en-bloc radical excision of the carcinoma, and in one case intraoperatively showing clinical signs of nerve involvement by the malignancy. Dura mater specimens were sent for frozen section during the surgical procedure to check for histological clearance: the carcinoma involved the dura mater in 4 of the 27 cases. Twenty-four patients underwent cervical lymph node dissection, and 25 had ipsilateral parotidectomy. On pathological T staging, 5 cases were pT1, 5 were pT2, 6 were pT3, and 11 were pT4. The pathological grade of the primary TBSCC was G1 in 17 of the 27 cases and G2 in 10. The surgical margins were negative on definitive histopathological examination in all cases. Regional lymph node status was classified as N0 in 20 cases (3 cN0 and 17 pN0), and as N+ in 7 (3 pN1, 3 pN2a, and 1 pN2b). Postoperative radiotherapy (RT) was performed in 15 cases (conventional external once-daily fractions of 2 Gy for a total dose ranging from 50 to 70 Gy, median 60 Gy). No distant metastases (M) were detected at diagnosis. The mean follow-up for the cohort was 82.9 ± 67.1 months (median 76 months). Survivors were followed up for at least 5 years. 2.2. Immunohistochemistry Sections were obtained from each of the 27 tissue blocks for immunohistochemical examination, performed on 4 μm-thick formalin-fixed and paraffin-embedded sections from each tissue sample. Staining was done automatically (BondmaX, Menarini, Florence, Italy), as described elsewhere, using the Bond Polymer Refine Detection kit (Leica Microsystem, Wetzlar, Germany), with rabbit anti-cortactin antibody (monoclonal EP1922Y; Abcam, Cambridge, UK; working dilution
Table 1 Main clinical, pathological, and immunohistochemical characteristics (cortactin and phosphorylated cortactin [residue tyr466]) of the patients with TBSCC. Patient no.
Sex
cT
pT
G
N-status (0 vs +)
Dura mater involvement (0 vs 1)
Cortactin immunoreactivity (0 vs 1)
Cortactin %
Phosphorylated cortactin Y466 immunoreactivity (0 vs 1)
TBSCC recurrence (0 vs 1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
F M F F F M M F M M F F M F M F M F F M M M M F F F F
2 2 1 4 1 2 3 4 3 1 3 3 1 2 3 4 2 3 3 4 2 4 4 1 3 4 3
2 2 1 4 1 2 3 4 4 1 3 3 1 2 4 4 2 3 3 4 4 4 4 1 3 4 4
1 2 1 2 1 1 1 1 2 2 2 1 1 1 1 1 1 2 1 1 2 1 1 1 2 2 2
0 0 0 0 0 0 0 0 0 0 0 + 0 + 0 + 0 0 0 0 0 0 + 0 + + +
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 0
1 1 1 1 1 1 1 1 1 1 1 1 1 Not evaluable 1 1 0 Not evaluable Not evaluable 1 1 1 1 1 1 1 1
80% 90% 50% 60% 70% 90% 70% 90% 70% 95% 10% 80% 90% Not evaluable 60% 8% 0% Not evaluable Not evaluable 30% 80% 80% 100% 10% 40% 80% 80%
1 0 0 1 1 1 1 0 0 1 0 0 0 Not evaluable Not evaluable 0 0 1 Not evaluable 0 1 0 0 0 0 1 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
1:200, 30 min, citrate buffer), and rabbit anti-phospho-Y466 cortactin antibody (polyclonal; Abcam, Cambridge, UK; working dilution 1:200, 30 min, citrate buffer). Sections were then slightly counterstained with hematoxylin. Appropriate positive and negative controls were run concurrently. Cortactin and p-Y466-cortactin expressions were jointly scored by two pathologists (S.B. and E.V.) who were given no clinical information. Cortactin staining in the cytoplasm was scored semi-quantitatively on a two-tiered scale: 0 = negative, 1 = positive. In positive cases, the proportion of positive cells was calculated as a percentage. The same above-mentioned two-tiered scale was used to assess phosphorylated cortactin expression (residue tyr466). 2.3. Statistical analysis The statistical tests applied were Fisher's exact test and the MannWhitney U test, as appropriate. The odds ratios (ORs) - TBSCC recurrence (1/0) versus variable - with 95% confidence intervals (95% CIs) were also calculated. The log-rank test was used to compare diseasefree survival (DFS) (in months), stratified by the different parameters considered. As a cutoff for the immunohistochemical expression of cortactin in the TBSCC specimens, we chose the median value for the cohort of patients, considered as the analytically best-fitting value, while phosphorylated cortactin (residue tyr466) expression was dichotomized (0 = negative, 1 = positive staining). A p-value b 0.05 was considered significant. The STATA™ 8.1 (Stata Corp, College Station, TX, USA) statistical package was used for all analyses.
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whereas there was no staining in bone matrix or cartilage matrix. Cortactin expression in the neoplastic cells was always cytoplasmic (Fig. 1C), while the expression of phosphorylated cortactin (residue tyr466) was diffuse, in the cytoplasm (Fig. 1D) and membrane (Fig. 1E). The cortactin reaction was more intense in the cytoplasm of carcinoma cells than in the adjacent normal tissue (Fig. 1F). Twenty-three of the 27 TBSCCs were immunohistochemically cortactin-positive. One specimen showed no cortactin immunoreactivity, while 3 were not evaluable due to fixation artifacts. The mean cortactin expression was 63.1% ± 30.0% (median 75.0%). Of the 27 TBSCCs, 15 specimens showed no phosphorylated cortactin (residue tyr466) immunoreactivity, while 3 could not be assessed due to fixation artifacts. Of the remainder, 4 specimens showed only cytoplasmic expression (range 10%–35%), 4 showed only membranous expression (range 3–30%), and one showed both. The Mann-Whitney U test ruled out any significant differences in cortactin expression between the positive TBSCC cases stratified by pT (p = 0.81), N status (p = 0.81), grade (p = 0.69), dura mater involvement (p = 0.43), or disease relapse after treatment (p = 0.97). Our statistical analysis found no significant differences in DFS (in months) when patients were stratified by the cortactin expression in their TBSCC, using a cutoff of 75%, which was the median cortactin expression level for our cohort (log-rank test, p = 0.39). Fisher's exact test also identified no significant association between phosphorylated cortactin (residue tyr466) immunoreactivity (positive vs negative) and pT (p = 0.67), N status (p = 0.35), grade (p = 0.40), dura mater involvement (p = 0.61), or carcinoma relapse (p = 0.67). The log-rank test found no significant association between phosphorylated cortactin (residue tyr466) immunoreactivity and DFS (p = 0.41).
3. Results 4. Discussion 3.1. Patients' clinical outcomes Eight patients developed local recurrences of their TBSCC after a mean 4.9 ± 4.3 months. Seven of the 27 patients died of the disease after their treatment; and 9 died of other causes. When patients were divided into two sub-cohorts according to whether or not their disease recurred locally after treatment, Fisher's exact test found a difference in the distributions for pT stage (pT1-T2 vs pT3-T4: p = 0.03, OR 8.4, 95% CI 1.0–102.0), but not for cT (cT1-T2 vs cT3-T4: p = 0.14), lymph node status (N0 vs N+: p = 0.14), or pathological grade (G1 vs G2: p = 0.41). Dura mater involvement (Fisher's exact test, p = 0.004; OR 3.83) and sacrifice of the facial nerve during surgery (Fisher's exact test, p = 0.006; OR 16, 95% CI 1.6–207.0) correlated significantly with a higher TBSCC recurrence rate. The log-rank test showed a significant difference in DFS (in months) when patients were stratified by cT (p = 0.03), pT (p = 0.0025), or lymph node status (p = 0.028), but not if they were stratified by pathological grade (p = 0.17). A significant difference in DFS emerged when patients were stratified by dura mater involvement (log-rank test, p = 0.0003). Facial nerve sacrifice correlated significantly with DFS too (log-rank test, p = 0.0001). When the approach to treatment was considered, parotidectomy, neck dissection, and postoperative radiotherapy did not correlate significantly with either recurrence rate (Fisher's exact test, p = 0.51, p = 1 and p = 1, respectively), or DFS (log-rank test, p = 0.55, p = 0.28, and p = 0.78, respectively). 3.2. Associations between cortactin, phosphorylated cortactin (residue tyr466) immunoreactivities, clinicopathological features, and prognosis for TBSCC (see Table 1) The normal tissue examined consisted of skin, sometimes with glands. The skin of the external auditory canal showed diffuse, moderate, cytoplasmic staining for cortactin and phospho-Y466 cortactin, as did the adnexal cutaneous glands. Cortactin and its phosphorylated form stained some chondrocytes and osteocytes in the cartilage and bone tissue underlying and remote from the tumor (Fig. 1A and B),
One of the most relevant issues relating to TBSCC is the lack of generally-accepted prognostic factors. This is probably due to: (i) the small number of cases reported, even in the largest available series; (ii) the histological heterogeneity of several case series (because some authors included squamous cell carcinoma arising from the skin of the pinna, or non-epithelial tumors, which have a very different prognosis); (iii) failure to report information on the pathological status of surgical margins; and (iv) the heterogeneity of the treatments adopted at different centers [15,16]. The main strength of the present study lies in the homogeneity of the series of patients considered because: (i) the histological diagnosis was SCC in all cases; (ii) all tumors originated in the temporal bone (SCCs in the periauricular area and adjoining sites were not considered); (iii) all patients underwent primary en-bloc temporal bone surgery; (iv) all patients were treated consecutively by the same surgical team; (v) on final histopathological examination, the surgical margins were negative in all cases; and (vi) only surgical specimens (not biopsies) of TBSCC were considered for immunohistochemical examination. The main weaknesses of our investigation concern: (i) the retrospective setting; and (ii) the limited number of primary SCC cases considered. Although the prognostic value of the available clinicopathological systems for staging TBSCC is often debated [17], and temporal bone cancer has no recognized American Joint Committee on Cancer or International Union Against Cancer staging system of its own, the revised Pittsburgh staging system [12,13] proved quite consistent when applied to the patients considered in the present series: cT stage correlated with DFS (p = 0.03); and pT stage correlated with recurrence rate (p = 0.03), and DFS (p = 0.0025). Lymph node status (N0 vs N+) was also associated with a shorter DFS in our series (p = 0.028). It is worth adding that, although dura mater involvement was diagnosed on histopathological examination in a limited number of cases in our series, univariate analysis found this feature significantly associated with a higher recurrence rate (p = 0.004), and a shorter DFS (p = 0.0003). In recent years, oncological research has focused on tumor biology and discovered biomarkers, the expression of which may be associated
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
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G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
Fig. 1. Cortactin (A) and phospho-Y466 cortactin (B) only stain the chondrocytes and osteocytes in cartilage and bone tissue, while the cartilage and bone matrices remain unstained; C) strong, diffuse cytoplasmic staining for cortactin in TBSCC; D) moderate cytoplasmic staining for phospho-Y466 cortactin in TBSCC; E) moderate membranous staining for phospho-Y466 cortactin in TBSCC; F) strong cytoplasmic staining for cortactin in TBSCC, and no staining in non-neoplastic cartilage.
with the behavior of several malignancies, or predict their response to therapy. Unlike the case of most other human malignancies, a recent detailed review by our group showed that only a handful of such studies have dealt with this topic in TBSCC [4]. Cortactin is a cytoskeletal actin-binding protein and src kinase substrate [18]. It accumulates in peripheral, actin-rich cell structures, giving the impression that it facilitates actin network formation. Recent evidence points to cortactin regulating various aspects of cell dynamics. It is overexpressed in a number of epithelial carcinomas, including breast cancer and head and neck cancer. It has been claimed that this overexpression in human tumors results in an increased cell migration, invasion (with a crucial role in invadopodia [19]), and metastatic potential [20]. Cortactin is post-translationally modified by many different kinases: the best-characterized sites are in the C-terminus and include Y421, Y466, and Y482, which are phosphorylated by src family kinases. Protein phosphorylation serves as an important switch for many cell signaling events. It could act as a switch to regulate the conformation of a protein, thus modifying its cellular and biochemical properties and/or serving as a signature for the recruitment of other proteins in close proximity. The influence of cortactin phosphorylation on cell functions remains largely unknown, but its importance clearly emerges from
the strong positive correlation between high level of tyrosine phosphorylation with enhanced cell migration and tumor metastasis [8]. In the present cohort of TBSCCs, 23 of the 24 immunohistochemically evaluable cases were cortactin-positive, revealing a median 75.0% cortactin expression. Cortactin reaction was more intense in the cytoplasm of carcinoma cells than in the adjacent normal tissue. Our statistical analysis ruled out any significant differences in cortactin expression between TBSCC cases stratified by pT, N status, grade, or dura mater involvement. Cortactin expression was also found not significantly related to TBSCC prognosis (in terms of recurrence rate or DFS). When phosphorylated cortactin (residue tyr466) immunoreactivity was considered in the same series of TBSCC, 4 specimens were positive only in the cytoplasm, 4 only in the membrane, and one in both. Statistical analysis revealed no significant associations between phosphorylated cortactin (residue tyr466) immunoreactivity and TBSCC recurrence rate or DFS. In the present study, cortactin phosphorylation was only investigated in residue tyr466, so we cannot exclude a role in TBSCC for phosphorylated cortactin in tyr421 or tyr482 because they have yet to be analyzed. Further studies should focus on these issues to further clarify whether phosphorylated cortactin has a relevant role in this malignancy's behavior.
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
Considering the available literature, significant progress has been made in understanding how cortactin regulates cell migration, invadopodia formation and function, and cancer cell invasion and metastasis [21]. In 2006, investigating cortactin's role in HNSCC, Rothschild et al. [22] reported that down-regulating cortactin expression in CTTNamplified HNSCC cells by means of small interfering RNA impaired HNSCC motility and invasion. They found that treating HNSCC cells with the EGFR inhibitor gefitinib limited HNSCC motility and down-regulated cortactin tyrosine phosphorylation [22]. In 2010, Yamada et al. [23] successfully applied RNA silencing to inhibit cortactin expression, thereby curtailing the invasive potential of oral squamous cell carcinoma. In 2015, Ni et al. [24] conducted immunohistochemical analyses and found cortactin expression higher in colon cancer (70.0%) than in control tissues (30.0%). They concluded that cortactin promoted human colon cancer cell growth and tumorigenicity, and that it might serve as an effective target for gene therapy. Finally, in 2016, Long et al. [25] reported that transfection with miR-542-3p mimic reduced cortactin expression in colorectal cancer cells: it regulated CTTN in a targeted manner to modulate the growth and invasion of colorectal cancer cells. In our cohort of cortactin-positive TBSCCs, cortactin immunoreactivity (median 75.0%) was definitely higher in the cytoplasm of carcinoma cells than in the normal adjacent tissue. Cortactin upregulation in TBSCC supports the conviction that inhibiting cortactin functions could have selective effects on this malignancy. Prospective, multi-institutional studies — preferably on an international scale (given the small numbers of cases treated at single institutions) and adopting shared diagnostic/therapeutic approaches — should be designed to further investigate the role of cortactin and phosphorylated cortactin in the biology of TBSCC, and their potential clinical application in integrated treatment modalities. Funding This study was partly supported by grant No. 60A07-0199/14 (G. Marioni) from the University of Padova, Italy. Conflict of interest declared None. Acknowledgments The authors are indebted to Vincenza Guzzardo, DIMED, University of Padova, Italy for her technical contribution to the histopathological analyses. They also thank Frances Coburn for correcting the English version of this paper.
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[2] Zanoletti E, Marioni G, Franchella S, et al. Recurrent squamous cell carcinoma of the temporal bone: critical analysis of cases with a poor prognosis. Am J Otolaryngol 2015;36:352–5. [3] Marioni G, Blandamura S, Lionello M, et al. Indications for postoperative radiotherapy in laryngeal carcinoma: a panel of tumor tissue markers for predicting locoregional recurrence in surgically treated carcinoma. A pilot study. Head Neck 2014;36:1534–40. [4] Marioni G, Martini A, Favaretto N, et al. Temporal bone carcinoma: a first glance beyond the conventional clinical and pathological prognostic factors. Eur Arch Otorhinolaryngol 2016;273:2903–10. [5] Ammer AG, Weed SA. Cortactin branches out: roles in regulating protrusive actin dynamics. Cell Motil Cytoskeleton 2008;65:687–707. [6] Rodrigo JP, García-Carracedo D, García LA, et al. Distinctive clinicopathological associations of amplification of the cortactin gene at 11q13 in head and neck squamous cell carcinomas. J Pathol 2009;217:516–23. [7] Clark ES, Brown B, Whigham AS, et al. Aggressiveness of HNSCC tumors depends on expression levels of cortactin, a gene in the 11q13 amplicon. Oncogene 2009;28: 431–44. [8] Lua BL, Low BC. Cortactin phosphorylation as a switch for actin cytoskeletal network and cell dynamics control. FEBS Lett 2005;579:577–85. [9] Clark ES, Whigham AS, Yarbrough WG, et al. Cortactin is an essential regulator of matrix metalloproteinase secretion and extracellular matrix degradation in invadopodia. Cancer Res 2007;67:4227–35. [10] Petruzzelli GJ. The biology of tumor invasion, angiogenesis and lymph node metastasis. ORL J Otorhinolaryngol Relat Spec 2000;62:178–85. [11] Zanoletti E, Marioni G, Stritoni P, et al. Temporal bone squamous cell carcinoma: analyzing prognosis with univariate and multivariate models. Laryngoscope 2014;124: 1192–8. [12] Moody SA, Hirsch BE, Myers EN. Squamous cell carcinoma of the external auditory canal: an evaluation of a staging system. Am J Otol 2000;21:582–8. [13] Hirsch BE. Staging system revision. Arch Otolaryngol Head Neck Surg 2002;128: 93–4. [14] Mazzoni A, Zanoletti E, Marioni G, et al. En bloc temporal bone resections in squamous cell carcinoma of the ear. Technique, principles, and limits. Acta Otolaryngol 2016;136:425–32. [15] Lionello M, Stritoni P, Facciolo MC, et al. Temporal bone carcinoma. Current diagnostic, therapeutic, and prognostic concepts. J Surg Oncol 2014;110:383–92. [16] Marioni G, Zanoletti E, Giacomelli L, et al. Clinical and pathological parameters prognostic for increased risk of recurrence after postoperative radiotherapy for temporal bone carcinoma. Head Neck 2016;38:894–8. [17] Mazzoni A, Danesi G, Zanoletti E. Primary squamous cell carcinoma of the external auditory canal: surgical treatment and long-term outcomes. Acta Otorhinolaryngol Ital 2014;34:129–37. [18] Weaver AM. Cortactin in tumor invasiveness. Cancer Lett 2008;265:157–66. [19] Clark ES, Weaver AM. A new role for cortactin in invadopodia: regulation of protease secretion. Eur J Cell Biol 2008;87(8-9):81–590. [20] Buday L, Downward J. Roles of cortactin in tumor pathogenesis. Biochim Biophys Acta 1775;2007:263–73. [21] MacGrath SM, Koleske AJ. Cortactin in cell migration and cancer at a glance. J Cell Sci 2012;125(Pt 7):1621–6. [22] Rothschild BL, Shim AH, Ammer AG, et al. Cortactin overexpression regulates actinrelated protein 2/3 complex activity, motility, and invasion in carcinomas with chromosome 11q13 amplification. Cancer Res 2006;66:8017–25. [23] Yamada S, Yanamoto S, Kawasaki G, et al. Overexpression of cortactin increases invasion potential in oral squamous cell carcinoma. Pathol Oncol Res 2010;16:523–31. [24] Ni QF, Yu JW, Qian F, et al. Cortactin promotes colon cancer progression by regulating ERK pathway. Int J Oncol 2015;47:1034–42. [25] Long HC, Gao X, Lei CJ, et al. miR-542-3p inhibits the growth and invasion of colorectal cancer cells through targeted regulation of cortactin. Int J Mol Med 2016;37: 1112–8.
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Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
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American Journal of Otolaryngology–Head and Neck Medicine and Surgery journal homepage: www.elsevier.com/locate/amjoto
Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma Gino Marioni a,⁎, Elisabetta Zanoletti a, Antonio Mazzoni a, Andrea Gianatti b, Elisa Valentini c, Laura Girasoli a, Martina Guariento a, Luciano Giacomelli c, Alessandro Martini a, Stella Blandamura c a b c
Department of Neurosciences DNS, Otolaryngology Section, Padova University, Padova, Italy Anatomic Pathology Unit, Ospedali Riuniti Bergamo, Bergamo, Italy Department of Medicine DIMED, University of Padova, Italy
a r t i c l e
i n f o
Article history: Received 17 November 2016 Available online xxxx Keywords: Temporal bone squamous cell carcinoma Cortactin Phosphorylated Prognosis Target therapy
a b s t r a c t Purpose: Cortactin is a multidomain protein engaged in several cellular mechanisms involving actin assembly and cytoskeletal arrangement. Cortactin overexpression in several malignancies has been associated with increased cell migration, invasion, and metastatic potential. Cortactin needs to be activated by tyrosine or serine/threonine phosphorylation. The role of cortactin and phosphorylated cortactin (residue tyr466) was investigated in temporal bone squamous cell carcinoma (TBSCC). Materials and methods: Immunohistochemical expression of cortactin and phosphorylated cortactin (residue tyr466) was assessed in 27 consecutively-operated TBSCCs. Results: Several clinicopathological variables correlated with recurrence (pT stage, dura mater involvement), and disease-free survival (DFS) (cT stage, pT stage, pN status, dura mater involvement). Twenty-three of 24 immunohistochemically evaluable TBSCCs were cortactin-positive. Median cortactin expression was 75.0%. Cortactin reaction in the cytoplasm was more intense in carcinoma cells than in normal adjacent tissue. Recurrence and DFS rates did not correlate with cortactin and phosphorylated cortactin (residue tyr466) expression in TBSCC specimens. Conclusions: Cortactin upregulation in TBSCC supports the conviction that inhibiting cortactin functions could have selective effects on this malignancy. Multi-institutional studies should further investigate the role of cortactin and phosphorylated cortactin in TBSCC, and their potential clinical application in integrated treatment modalities. © 2017 Elsevier Inc. All rights reserved.
1. Introduction Given the poor prognosis for patients with advanced temporal bone squamous cell carcinoma (TBSCC) [1], there is an undeniable need for novel, more effective diagnostic/therapeutic strategies to improve the outcomes of treatment. TBSCC primary therapies definitely need to be patient-tailored more rationally [1], also because managing TBSCC recurrences is particularly difficult, and any treatment carries a significant morbidity and mortality [2]. Extending surgery to ensure safe tissue margins is the premise for the adequate treatment of TBSCC. Molecular changes occurring before any morphological changes become apparent are responsible for the disease's biological behavior, prognosis, and response to primary therapy [3]. In clinical practice, the ideal molecular marker should have: (i) prognostic value; (ii) a significant capacity to predict the efficacy of specific treatments; and (iii) the
⁎ Corresponding author at: Dept of Neurosciences DNS, Otolaryngology Section, University of Padova, Via Giustiniani 2, 35128 Padova, Italy. E-mail address: [email protected] (G. Marioni).
features needed for it to become the target of integrated therapeutic approaches [4]. Cortactin is a multidomain protein engaged in several cellular mechanisms based on actin assembly and cytoskeletal arrangement, such as endocytosis, cell migration, neuronal morphogenesis and tumor invasion. Cortactin is an actin-binding protein that promotes the weak activation of the actin-related protein (ARP) 2/3 complex in actin nucleation, and it stabilizes the formation of newly-branching network of F-actin [5]. Cortactin occurs in the cytoplasm and in actin-rich peripheral structures such as lamellipodia and podosomes. The cortactin locus CTTN is located in the 11q13 region, the amplification of which is one of the alterations most often seen in head and neck squamous cell carcinoma (HNSCC). Studies have shown that 11q13 amplification correlates with a poor prognosis in HNSCC patients, and cortactin has been accused of being the gene responsible for tumor progression because of its role in cell motility and carcinoma invasion. Cortactin overexpression promotes tumor aggressiveness, even without the 11q13 amplicon, suggesting a post-translational activation rather than a gene amplification in HNSCC [6,7]. Cortactin needs to be activated by tyrosine or serine/threonine phosphorylation. Tyrosine phosphorylation stems from
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Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
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G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
activation of the epidermal growth factor receptor (EGFR) and other receptor tyrosine kinases, and it occurs at residues tyr421, tyr466 and tyr482, in the proline-rich domain of the carboxy terminus. Phospho-cortactin interacts with the adaptor protein NCK1, which binds the neuronal Wiskott Aldrich Syndrome protein, resulting in ARP 2/3 activation. The role of phosphorylation in cortactin function is still not entirely clear, but it correlates positively with the ability to induce cell migration through matrix metalloproteinase recruitment and secretion in invadopodia [8,9]. Cancer has the characteristic ability to invade surrounding tissues and metastasize to regional and distant sites. The events attendant on local invasion and metastasis by epithelial tumors such as TBSCC include: (i) loss of adhesion to surrounding tumor cells and basement membrane; (ii) production of enzymes and mediators that facilitate the incursion of malignant cells into the subjacent connective tissue; (iii) attachment to extracellular membrane molecules; (iv) neovascularization; (v) passage into and out of the circulation via attachment to endothelial cell ligands; and (vi) a repeat of this cascade at a metastatic site [10]. The details of these mechanisms are not fully known in the case of TBSCC. The present study is the first to have investigated the immunohistochemical expression of cortactin and phosphorylated cortactin (residue tyr466) in patients with primary TBSCC. The aim of the study was to conduct a preliminary assessment of the role of cortactin in this malignancy. 2. Methods 2.1. Patients (see Table 1) The present investigation was approved by our Otolaryngology Section's in-house ethical committee. The study was carried out in accordance with the principles of the Helsinki Declaration. The study was conducted on specimens from 27 patients with primary TBSCC operated by the same surgeon (A.Mz.) at a tertiary referral center. The patients included 15 women and 12 men with a mean age of 54.0±12.7 years, who were part of a larger series investigated for other purposes in a previous study [11]. Preoperatively, all patients underwent micro-otoscopy with biopsy, temporal bone computerized
tomography and/or contrast-enhanced magnetic resonance imaging, and neck ultrasonography (with or without fine-needle aspiration cytology). Positron emission tomography was performed in advanced cases to rule out distant metastasis. Based on the revised Pittsburgh staging system [12,13], the primary TBSCCs were classified as cT1 in 5 cases, cT2 in 6, cT3 in 9, and cT4 in 7. En-bloc lateral temporal bone resection was performed in 19 cases (two of them partial), and en-bloc subtotal temporal bone resection in 8. The surgical techniques involved have been described in detail elsewhere [14]. The facial nerve was sacrificed in 9 of the 27 patients, i.e. in all the cases of subtotal temporal bone resection to enable en-bloc radical excision of the carcinoma, and in one case intraoperatively showing clinical signs of nerve involvement by the malignancy. Dura mater specimens were sent for frozen section during the surgical procedure to check for histological clearance: the carcinoma involved the dura mater in 4 of the 27 cases. Twenty-four patients underwent cervical lymph node dissection, and 25 had ipsilateral parotidectomy. On pathological T staging, 5 cases were pT1, 5 were pT2, 6 were pT3, and 11 were pT4. The pathological grade of the primary TBSCC was G1 in 17 of the 27 cases and G2 in 10. The surgical margins were negative on definitive histopathological examination in all cases. Regional lymph node status was classified as N0 in 20 cases (3 cN0 and 17 pN0), and as N+ in 7 (3 pN1, 3 pN2a, and 1 pN2b). Postoperative radiotherapy (RT) was performed in 15 cases (conventional external once-daily fractions of 2 Gy for a total dose ranging from 50 to 70 Gy, median 60 Gy). No distant metastases (M) were detected at diagnosis. The mean follow-up for the cohort was 82.9 ± 67.1 months (median 76 months). Survivors were followed up for at least 5 years. 2.2. Immunohistochemistry Sections were obtained from each of the 27 tissue blocks for immunohistochemical examination, performed on 4 μm-thick formalin-fixed and paraffin-embedded sections from each tissue sample. Staining was done automatically (BondmaX, Menarini, Florence, Italy), as described elsewhere, using the Bond Polymer Refine Detection kit (Leica Microsystem, Wetzlar, Germany), with rabbit anti-cortactin antibody (monoclonal EP1922Y; Abcam, Cambridge, UK; working dilution
Table 1 Main clinical, pathological, and immunohistochemical characteristics (cortactin and phosphorylated cortactin [residue tyr466]) of the patients with TBSCC. Patient no.
Sex
cT
pT
G
N-status (0 vs +)
Dura mater involvement (0 vs 1)
Cortactin immunoreactivity (0 vs 1)
Cortactin %
Phosphorylated cortactin Y466 immunoreactivity (0 vs 1)
TBSCC recurrence (0 vs 1)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27
F M F F F M M F M M F F M F M F M F F M M M M F F F F
2 2 1 4 1 2 3 4 3 1 3 3 1 2 3 4 2 3 3 4 2 4 4 1 3 4 3
2 2 1 4 1 2 3 4 4 1 3 3 1 2 4 4 2 3 3 4 4 4 4 1 3 4 4
1 2 1 2 1 1 1 1 2 2 2 1 1 1 1 1 1 2 1 1 2 1 1 1 2 2 2
0 0 0 0 0 0 0 0 0 0 0 + 0 + 0 + 0 0 0 0 0 0 + 0 + + +
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 0 1 0 0 1 0
1 1 1 1 1 1 1 1 1 1 1 1 1 Not evaluable 1 1 0 Not evaluable Not evaluable 1 1 1 1 1 1 1 1
80% 90% 50% 60% 70% 90% 70% 90% 70% 95% 10% 80% 90% Not evaluable 60% 8% 0% Not evaluable Not evaluable 30% 80% 80% 100% 10% 40% 80% 80%
1 0 0 1 1 1 1 0 0 1 0 0 0 Not evaluable Not evaluable 0 0 1 Not evaluable 0 1 0 0 0 0 1 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
1:200, 30 min, citrate buffer), and rabbit anti-phospho-Y466 cortactin antibody (polyclonal; Abcam, Cambridge, UK; working dilution 1:200, 30 min, citrate buffer). Sections were then slightly counterstained with hematoxylin. Appropriate positive and negative controls were run concurrently. Cortactin and p-Y466-cortactin expressions were jointly scored by two pathologists (S.B. and E.V.) who were given no clinical information. Cortactin staining in the cytoplasm was scored semi-quantitatively on a two-tiered scale: 0 = negative, 1 = positive. In positive cases, the proportion of positive cells was calculated as a percentage. The same above-mentioned two-tiered scale was used to assess phosphorylated cortactin expression (residue tyr466). 2.3. Statistical analysis The statistical tests applied were Fisher's exact test and the MannWhitney U test, as appropriate. The odds ratios (ORs) - TBSCC recurrence (1/0) versus variable - with 95% confidence intervals (95% CIs) were also calculated. The log-rank test was used to compare diseasefree survival (DFS) (in months), stratified by the different parameters considered. As a cutoff for the immunohistochemical expression of cortactin in the TBSCC specimens, we chose the median value for the cohort of patients, considered as the analytically best-fitting value, while phosphorylated cortactin (residue tyr466) expression was dichotomized (0 = negative, 1 = positive staining). A p-value b 0.05 was considered significant. The STATA™ 8.1 (Stata Corp, College Station, TX, USA) statistical package was used for all analyses.
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whereas there was no staining in bone matrix or cartilage matrix. Cortactin expression in the neoplastic cells was always cytoplasmic (Fig. 1C), while the expression of phosphorylated cortactin (residue tyr466) was diffuse, in the cytoplasm (Fig. 1D) and membrane (Fig. 1E). The cortactin reaction was more intense in the cytoplasm of carcinoma cells than in the adjacent normal tissue (Fig. 1F). Twenty-three of the 27 TBSCCs were immunohistochemically cortactin-positive. One specimen showed no cortactin immunoreactivity, while 3 were not evaluable due to fixation artifacts. The mean cortactin expression was 63.1% ± 30.0% (median 75.0%). Of the 27 TBSCCs, 15 specimens showed no phosphorylated cortactin (residue tyr466) immunoreactivity, while 3 could not be assessed due to fixation artifacts. Of the remainder, 4 specimens showed only cytoplasmic expression (range 10%–35%), 4 showed only membranous expression (range 3–30%), and one showed both. The Mann-Whitney U test ruled out any significant differences in cortactin expression between the positive TBSCC cases stratified by pT (p = 0.81), N status (p = 0.81), grade (p = 0.69), dura mater involvement (p = 0.43), or disease relapse after treatment (p = 0.97). Our statistical analysis found no significant differences in DFS (in months) when patients were stratified by the cortactin expression in their TBSCC, using a cutoff of 75%, which was the median cortactin expression level for our cohort (log-rank test, p = 0.39). Fisher's exact test also identified no significant association between phosphorylated cortactin (residue tyr466) immunoreactivity (positive vs negative) and pT (p = 0.67), N status (p = 0.35), grade (p = 0.40), dura mater involvement (p = 0.61), or carcinoma relapse (p = 0.67). The log-rank test found no significant association between phosphorylated cortactin (residue tyr466) immunoreactivity and DFS (p = 0.41).
3. Results 4. Discussion 3.1. Patients' clinical outcomes Eight patients developed local recurrences of their TBSCC after a mean 4.9 ± 4.3 months. Seven of the 27 patients died of the disease after their treatment; and 9 died of other causes. When patients were divided into two sub-cohorts according to whether or not their disease recurred locally after treatment, Fisher's exact test found a difference in the distributions for pT stage (pT1-T2 vs pT3-T4: p = 0.03, OR 8.4, 95% CI 1.0–102.0), but not for cT (cT1-T2 vs cT3-T4: p = 0.14), lymph node status (N0 vs N+: p = 0.14), or pathological grade (G1 vs G2: p = 0.41). Dura mater involvement (Fisher's exact test, p = 0.004; OR 3.83) and sacrifice of the facial nerve during surgery (Fisher's exact test, p = 0.006; OR 16, 95% CI 1.6–207.0) correlated significantly with a higher TBSCC recurrence rate. The log-rank test showed a significant difference in DFS (in months) when patients were stratified by cT (p = 0.03), pT (p = 0.0025), or lymph node status (p = 0.028), but not if they were stratified by pathological grade (p = 0.17). A significant difference in DFS emerged when patients were stratified by dura mater involvement (log-rank test, p = 0.0003). Facial nerve sacrifice correlated significantly with DFS too (log-rank test, p = 0.0001). When the approach to treatment was considered, parotidectomy, neck dissection, and postoperative radiotherapy did not correlate significantly with either recurrence rate (Fisher's exact test, p = 0.51, p = 1 and p = 1, respectively), or DFS (log-rank test, p = 0.55, p = 0.28, and p = 0.78, respectively). 3.2. Associations between cortactin, phosphorylated cortactin (residue tyr466) immunoreactivities, clinicopathological features, and prognosis for TBSCC (see Table 1) The normal tissue examined consisted of skin, sometimes with glands. The skin of the external auditory canal showed diffuse, moderate, cytoplasmic staining for cortactin and phospho-Y466 cortactin, as did the adnexal cutaneous glands. Cortactin and its phosphorylated form stained some chondrocytes and osteocytes in the cartilage and bone tissue underlying and remote from the tumor (Fig. 1A and B),
One of the most relevant issues relating to TBSCC is the lack of generally-accepted prognostic factors. This is probably due to: (i) the small number of cases reported, even in the largest available series; (ii) the histological heterogeneity of several case series (because some authors included squamous cell carcinoma arising from the skin of the pinna, or non-epithelial tumors, which have a very different prognosis); (iii) failure to report information on the pathological status of surgical margins; and (iv) the heterogeneity of the treatments adopted at different centers [15,16]. The main strength of the present study lies in the homogeneity of the series of patients considered because: (i) the histological diagnosis was SCC in all cases; (ii) all tumors originated in the temporal bone (SCCs in the periauricular area and adjoining sites were not considered); (iii) all patients underwent primary en-bloc temporal bone surgery; (iv) all patients were treated consecutively by the same surgical team; (v) on final histopathological examination, the surgical margins were negative in all cases; and (vi) only surgical specimens (not biopsies) of TBSCC were considered for immunohistochemical examination. The main weaknesses of our investigation concern: (i) the retrospective setting; and (ii) the limited number of primary SCC cases considered. Although the prognostic value of the available clinicopathological systems for staging TBSCC is often debated [17], and temporal bone cancer has no recognized American Joint Committee on Cancer or International Union Against Cancer staging system of its own, the revised Pittsburgh staging system [12,13] proved quite consistent when applied to the patients considered in the present series: cT stage correlated with DFS (p = 0.03); and pT stage correlated with recurrence rate (p = 0.03), and DFS (p = 0.0025). Lymph node status (N0 vs N+) was also associated with a shorter DFS in our series (p = 0.028). It is worth adding that, although dura mater involvement was diagnosed on histopathological examination in a limited number of cases in our series, univariate analysis found this feature significantly associated with a higher recurrence rate (p = 0.004), and a shorter DFS (p = 0.0003). In recent years, oncological research has focused on tumor biology and discovered biomarkers, the expression of which may be associated
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
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G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
Fig. 1. Cortactin (A) and phospho-Y466 cortactin (B) only stain the chondrocytes and osteocytes in cartilage and bone tissue, while the cartilage and bone matrices remain unstained; C) strong, diffuse cytoplasmic staining for cortactin in TBSCC; D) moderate cytoplasmic staining for phospho-Y466 cortactin in TBSCC; E) moderate membranous staining for phospho-Y466 cortactin in TBSCC; F) strong cytoplasmic staining for cortactin in TBSCC, and no staining in non-neoplastic cartilage.
with the behavior of several malignancies, or predict their response to therapy. Unlike the case of most other human malignancies, a recent detailed review by our group showed that only a handful of such studies have dealt with this topic in TBSCC [4]. Cortactin is a cytoskeletal actin-binding protein and src kinase substrate [18]. It accumulates in peripheral, actin-rich cell structures, giving the impression that it facilitates actin network formation. Recent evidence points to cortactin regulating various aspects of cell dynamics. It is overexpressed in a number of epithelial carcinomas, including breast cancer and head and neck cancer. It has been claimed that this overexpression in human tumors results in an increased cell migration, invasion (with a crucial role in invadopodia [19]), and metastatic potential [20]. Cortactin is post-translationally modified by many different kinases: the best-characterized sites are in the C-terminus and include Y421, Y466, and Y482, which are phosphorylated by src family kinases. Protein phosphorylation serves as an important switch for many cell signaling events. It could act as a switch to regulate the conformation of a protein, thus modifying its cellular and biochemical properties and/or serving as a signature for the recruitment of other proteins in close proximity. The influence of cortactin phosphorylation on cell functions remains largely unknown, but its importance clearly emerges from
the strong positive correlation between high level of tyrosine phosphorylation with enhanced cell migration and tumor metastasis [8]. In the present cohort of TBSCCs, 23 of the 24 immunohistochemically evaluable cases were cortactin-positive, revealing a median 75.0% cortactin expression. Cortactin reaction was more intense in the cytoplasm of carcinoma cells than in the adjacent normal tissue. Our statistical analysis ruled out any significant differences in cortactin expression between TBSCC cases stratified by pT, N status, grade, or dura mater involvement. Cortactin expression was also found not significantly related to TBSCC prognosis (in terms of recurrence rate or DFS). When phosphorylated cortactin (residue tyr466) immunoreactivity was considered in the same series of TBSCC, 4 specimens were positive only in the cytoplasm, 4 only in the membrane, and one in both. Statistical analysis revealed no significant associations between phosphorylated cortactin (residue tyr466) immunoreactivity and TBSCC recurrence rate or DFS. In the present study, cortactin phosphorylation was only investigated in residue tyr466, so we cannot exclude a role in TBSCC for phosphorylated cortactin in tyr421 or tyr482 because they have yet to be analyzed. Further studies should focus on these issues to further clarify whether phosphorylated cortactin has a relevant role in this malignancy's behavior.
Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012
G. Marioni et al. / American Journal of Otolaryngology–Head and Neck Medicine and Surgery xxx (2017) xxx–xxx
Considering the available literature, significant progress has been made in understanding how cortactin regulates cell migration, invadopodia formation and function, and cancer cell invasion and metastasis [21]. In 2006, investigating cortactin's role in HNSCC, Rothschild et al. [22] reported that down-regulating cortactin expression in CTTNamplified HNSCC cells by means of small interfering RNA impaired HNSCC motility and invasion. They found that treating HNSCC cells with the EGFR inhibitor gefitinib limited HNSCC motility and down-regulated cortactin tyrosine phosphorylation [22]. In 2010, Yamada et al. [23] successfully applied RNA silencing to inhibit cortactin expression, thereby curtailing the invasive potential of oral squamous cell carcinoma. In 2015, Ni et al. [24] conducted immunohistochemical analyses and found cortactin expression higher in colon cancer (70.0%) than in control tissues (30.0%). They concluded that cortactin promoted human colon cancer cell growth and tumorigenicity, and that it might serve as an effective target for gene therapy. Finally, in 2016, Long et al. [25] reported that transfection with miR-542-3p mimic reduced cortactin expression in colorectal cancer cells: it regulated CTTN in a targeted manner to modulate the growth and invasion of colorectal cancer cells. In our cohort of cortactin-positive TBSCCs, cortactin immunoreactivity (median 75.0%) was definitely higher in the cytoplasm of carcinoma cells than in the normal adjacent tissue. Cortactin upregulation in TBSCC supports the conviction that inhibiting cortactin functions could have selective effects on this malignancy. Prospective, multi-institutional studies — preferably on an international scale (given the small numbers of cases treated at single institutions) and adopting shared diagnostic/therapeutic approaches — should be designed to further investigate the role of cortactin and phosphorylated cortactin in the biology of TBSCC, and their potential clinical application in integrated treatment modalities. Funding This study was partly supported by grant No. 60A07-0199/14 (G. Marioni) from the University of Padova, Italy. Conflict of interest declared None. Acknowledgments The authors are indebted to Vincenza Guzzardo, DIMED, University of Padova, Italy for her technical contribution to the histopathological analyses. They also thank Frances Coburn for correcting the English version of this paper.
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Please cite this article as: Marioni G, et al, Cortactin and phosphorylated cortactin tyr466 expression in temporal bone carcinoma, American Journal of Otolaryngology–Head and Neck Medicine and Surgery (2017), http://dx.doi.org/10.1016/j.amjoto.2017.01.012