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Correspondence: Shigaku Ikeda, M.D., Ph.D., Department of Dermatology and Allergology, Juntendo University Graduate School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan. Email: firstname.lastname@example.org
The expression of autophagy-related markers has occasionally been reported to correlate with the clinical stage of disease in patients with solid cancer, indicating autophagy activation. However, there have been no such reports for cutaneous squamous cell carcinoma. In this study, we investigated the expression levels of two autophagy-related markers, microtubule-associated protein IA/IB light chain 3 (LC3) and p62/sequestosome-1 (p62), in cutaneous squamous cell carcinoma specimens and assessed their correlation to clinicopathological factors in patients with this type of cancer. As a marker of the autophagosome, LC3 expression increases with autophagosome formation/accumulation, whereas p62 expression decreases due to selective degradation via autophagy. We performed immunostaining for LC3 and p62 in 50 cutaneous squamous cell carcinoma specimens obtained from patients treated by surgical resection, counted the number of cells that showed positive staining, and calculated the percentage of positive cells per low-power microscopic field. We next investigated the correlations between the expression levels of these markers and various clinicopathological factors. The results indicated that LC3 expression increased significantly with advanced clinical stage (P <0.001) and increased tumor diameter (P =0.046). By contrast, the expression of p62 decreased significantly with advanced clinical stage (P <0.001) and increased tumor diameter (P =0.001). These results suggest that autophagy becomes activated during disease progression in patients with cutaneous squamous cell carcinoma.
Cancer cell metabolism and growth are supported by the continuous flow of nutrients from surrounding blood vessels that emerge under conditions promoting angiogenesis. However, this new vasculature is insufficient for sustaining cancer cell proliferation and the supply of oxygen and glucose. As an additional or supplementary metabolic pathway, autophagy has been proposed as a potential mechanism for the survival of the cancer cells. Autophagy is a major cellular degradative system involved in the turnover of cellular constituents, including organelles such as the endoplasmic reticulum and mitochondria. Autophagy is enhanced under nutrient-deprived conditions in which degradation products, such as sugars, fatty acids and amino acids, are recycled for both anabolic and catabolic pathways.[1, 2] A number of reports have recently shown that autophagy is significantly activated in cancers such as pancreatic cancer, colorectal cancer and breast cancer, and this activity has been positively correlated with the progression of clinical disease stage and patient outcome.[3-5] These results suggest that autophagy may play a role in promoting cancer growth. In the field of dermatology, however, it remains unknown whether autophagy is activated in skin cancer, with the exception of malignant melanoma.
Cutaneous squamous cell carcinoma (SCC) is the third most common malignant tumor of the skin after basal cell carcinoma and actinic keratosis. Because actinic keratosis is regarded as an early stage of SCC, SCC together with actinic keratosis account for the largest number of cases of malignant tumors of the skin. Moreover, the percentage of cases in which progression is rapid and distant metastasis is present is higher in patients with cutaneous SCC compared to basal cell carcinoma. The clinical features of cutaneous SCC are diverse, treatment can be difficult, and it is important for dermatologists to understand the clinical proclivities of this disease. We therefore investigated whether autophagy was enhanced in cutaneous SCC, particularly in relation to the clinical features of patients. To this end, we evaluated two autophagy-related markers: microtubule-associated protein IA/IB light chain 3 (LC3) and p62/sequestosome-1 (p62). Because the lipidated form of LC3 (LC3-II) is recruited to autophagosomal membranes, the cellular LC3-II level can be used to indicate the quantity of autophagosomes.[3, 5, 6] Furthermore, p62 is selectively degraded via autophagy, and its level is therefore markedly increased when autophagy is suppressed. In this study, using LC3 and p62 antibodies, we conducted immunohistochemical analyses to assess autophagy in cutaneous SCC samples obtained from patients.
The specimens used in this study were obtained from patients who underwent surgical resection for cutaneous SCC at Juntendo University Hospital (Tokyo, Japan) between January 2006 and December 2011. In all cases, the pathological diagnosis was made by diagnostic pathologists and dermatologists. Staging was performed according to the Union for International Cancer Control (UICC), version 7.0; there were 25 stage I cases, 15 stage II cases, eight stage III cases and two stage IV cases. The patients ranged in age 33–95 years, and the mean age was 75.0 years. The patients consisted of 26 men and 24 women. The institutional review board of Juntendo Hospital approved all protocols.
The surgical specimens were fixed in 10% neutral buffered formalin at room temperature, and their sizes and gross findings were recorded. All of the specimens were embedded in paraffin using the standard method. The tumors were cut into serial sections, and the final diagnosis was made in hematoxylin–eosin-stained sections. Elastica van Gieson staining was performed to confirm blood vessel invasion. The specimens were histologically divided into three groups, consisting of well, moderately and poorly differentiated samples, based on Broders's system. Because many dermatopathologists generally use a variation of this system for investigating the prognostic factors of SCC, the presented cases were divided into three subtypes as follows: well-differentiated SCC with differentiated lesions in more than 75% of the structure (Broders's grade 1); moderately differentiated SCC with differentiated lesions in 25–75% of the structure (Broders's grade 2 and 3); and poorly differentiated SCC with differentiated lesions in less than 25% of the structure (Broders's grade 4).
We investigated the correlations between the following clinicopathological parameters and tumor expression of autophagy-related markers: (i) interval between the onset of symptoms and resection of the lesion (<1 vs ≥1 year); (ii) age (<75 vs ≥75 years); (iii) sex; (iv) tumor diameter (≤2 vs >2 cm); (v) degree of tumor differentiation (well, moderately or poorly differentiated); (vi) lymphovascular invasion (present vs absent); (vii) blood vessel invasion (present vs absent); (viii) UICC stage classification (stage I and II vs stage III and IV); and (ix) lymph node metastasis (present vs absent).
Immunohistochemical staining with antibodies against autophagy-related molecules
Autophagy in the tumor specimens was evaluated by staining with anti-LC3, anti-lysosomal-associated membrane protein 1 (LAMP1) and anti-p62 antibodies. LC3 is an effective marker of autophagy due to its expression both inside and outside of the isolation membrane during autophagosome formation. Once formed, the autophagosome fuses rapidly with the lysosome to form the autolysosome, in which sequestered cytoplasmic components are degraded by lysosomal hydrolases. To evaluate the entire process of autophagic flux (i.e. autophagic degradation), staining for the lysosome marker LAMP1 (ab24170; Abcam, Cambridge, MA, USA) was also necessary. In addition, we included p62 as another marker for autophagic degradation. p62 binds polyubiquitinated proteins through its ubiquitin-binding region and is selectively recognized by autophagosomes through the LC3-binding region, and these characteristics enable p62 to transport ubiquitinated proteins to autophagosomes for select degradation via autophagy. An increase in the level of p62 signifies an impairment in autophagy, whereas decreased expression enables normal autophagic degradation to occur. Two experienced pathologists blinded to the clinical findings performed these evaluations.
All of the sections used for immunohistochemical staining were first fixed in formalin and embedded in paraffin. Serial sections of the tissue were then immunohistochemically stained with LC3B (ab48394; Abcam), LAMP1 (ab24170; Abcam) and p62 (G-P62C; PROGEN Biotechnik, Heidelberg, Germany) antibodies. Immunostaining was performed according to the standard streptavidin–biotin protocol. The reaction products were stained with diaminobenzidine, and the background was counterstained with hematoxylin. After immunostaining, we examined the sections under an optical microscope to measure the number of cells showing positive staining among 1000 cells in a single field of view, and we then calculated the percentage of cells showing positive staining. The staining intensity was classified into three grades: weak; moderate; and strong.
We used Fisher's χ2-test to determine the statistical significance of associations between LC3 and p62 expression and clinicopathological factors. P < 0.05 was considered statistically significant.
LC3 staining of the tumors
Immunostaining for LC3 could be observed at a low level in the normal epidermis. However, in SCC specimens obtained from patients, the tumor cells and stroma showed diffuse but clearly positive staining for LC3. LC3 expression was also seen along the cell membrane of tumor cells (Fig. 2a,c,e,g). Tumor cells in many of the patient samples were positive, although some tumor cells were not stained in some of the samples. The rates of positive tumor cells ranged 0–100%, and the mean ± standard deviation (SD) was 39.4 ± 29.1%. Therefore, we classified the samples into two groups: an LC3-positive group, in which LC3-positive cells were equal to or more than 39% (i.e. the mean value or higher); and an LC3-negative group, in which LC3-positive cells were less than 39% (i.e. less than the mean value). With regard to the staining intensity, the staining was weak in 60%, moderate in 32% and strong in 8% of the cases. The LC3 expression rates (mean ± SD) according to clinical stage were as follows: stage I, 22.6 ± 18.5%; stage II, 43.9 ± 27.5%; and stage III/stage IV, 75.1 ± 16.8%. Thus, the LC3 rate increased significantly as the clinical stage advanced (P =0.0002). There were 25 clinical stage I cases, of which seven demonstrated positive LC3 expression (i.e. expression of LC3 was observed in ≥39% of the tumor cells). Diffuse staining for LC3 throughout the entire tumor was observed in all seven cases, although some of these seven cases showed predominant staining at the tumor margin. In all samples, the intensity of the staining was weak. In 14 of the 18 cases that were evaluated as negative, LC3 expression was observed in less than 39% of the tumor cells. In all of these 14 cases, the LC3-positive cells were predominantly observed at the tumor margin. In the remaining four cases, while positive staining was observed within the stroma, the tumor cells did not show positive staining for LC3. There were 15 clinical stage II cases, of which seven showed positive staining for LC3. Diffuse staining of the entire tumor was noted, and the staining intensity was moderate. In seven of the eight remaining negative stage II cases, the staining was predominantly observed in the tumor margin, similar to the negative stage I cases. All 10 of the clinical stage III/stage IV cases showed positive staining; this staining intensity was moderate to strong and tended to be uniform throughout the tumor.
The results for LAMP1 staining were similar to the results for LC3 staining (Fig. 1). That is, the tumor staining for LAMP1 was low in early cancers, whereas LAMP1 was more strongly expressed in advanced cancers.
p62 staining of the tumors
Both the cytoplasm of the tumor cells and the stroma of the SCC showed positive staining for p62 (Fig. 2b,d,f,h). Immunohistological staining for p62 was also slightly positive in the basal layer of the epidermis in the surrounding non-cancerous tissue. The rates of positive tumor cells ranged 0–47.2%, and the mean ± SD was 12.2 ± 8.0%. We therefore considered the results of the p62 staining to be “positive” when the proportion of cells showing positive staining was 12% or higher (i.e. the mean value or higher) and “negative” when it was less than 12% (i.e. less than the mean value). With respect to the intensity of staining, the staining intensity was weak in 74% of cases, moderate in 22% and strong in 4%; thus, the staining was weak in the majority of cases. The p62 expression rates (mean ± SD) according to clinical stage were as follows: stage I, 24.6 ± 17.1%; stage II, 18.4 ± 15.1%; and stage III/stage IV, 8.2 ± 7.8%. Thus, the expression rate decreased significantly as the clinical stage progressed (P =0.0002). Of the 25 clinical stage I cases, 20 (80%) were positive; of the 15 stage II cases, six (40%) were positive; and of the 10 stage III/stage IV cases, all cases were negative. The p62-positive cases consisted of cases in which positively stained cells were seen sporadically in parts of the tumor as well as cases in which predominantly the tumor margin showed positive staining. The staining tended to be predominantly seen in the tumor periphery in cases where the interval from the onset of symptoms to resection of the tumor was less than 2 years. Staining was more pronounced in the central part of the tumor in cases where the aforementioned interval was 2 years or more. p62 expression in the stroma was seen in only a small number of cases.
Assessment of LC3 and p62 expression in relation to the tumor diameter and degree of tumor differentiation
We divided the cases according to tumor diameter into a group with tumor diameters of 2 cm or less (n = 27) and another group with tumor diameters of more than 2 cm (n = 23). The LC3 expression rate (mean ± SD) was 27.6 ± 19.6% in the 2 cm or less group and 57.2 ± 31.1% in the more than 2 cm group; the LC3-positive rate increased significantly as the clinical disease stage advanced (P =0.046). The p62 expression rate (mean ± SD) was 23.3 ± 17.1% in the 2 cm or less group and 15.2 ± 14.2% in the more than 2 cm group; the p62-positive rate decreased significantly as the clinical stage progressed (P =0.002). We then compared the LC3 and p62 staining rates according to the degree of tumor differentiation (well, moderately and poorly differentiated groups), although there were no significant differences between groups.
Correlations with other clinicopathological factors
The results of comparisons to other parameters are shown in Table 1. In cases of cutaneous SCC where an extended interval of several years had elapsed after the onset of symptoms, LC3 staining tended to be observed throughout the tumor but was predominant at the cell membrane. In those cases, the staining intensity was weak for LC3. In addition, it was noteworthy that LC3 expression was significantly higher, whereas p62 expression was significantly lower, in cases with lymph node metastasis.
Table 1. Correlations between clinicopathological factors and LC3 and p62 expression
LC3 expression positive/negative
p62 expression positive/negative
LAMP1, lysosomal-associated membrane protein 1; LC3, microtubule-associated protein IA/IB light chain 3; p62, p62/sequestosome-1; UICC, Union for International Cancer Control.
Lymphatic vessel invasion
Blood vessel invasion
Lymph node metastasis
The clinicopathological significance of autophagy in cancer remains a subject of controversy. One of the reasons why the significance of autophagy in tumor cells remains ambiguous may be related to different contributions of autophagy to different phases of carcinogenesis. As depicted in a recent review by White et al., autophagy is thought to be suppressed in the initial phase of tumorigenesis, whereas autophagy promotes the growth and proliferation of established cancer. Moreover, while some reports have evaluated the expression of autophagy-related proteins in digestive or breast cancers, few studies have been performed in skin cancer, with the exception of malignant melanoma.
The keratinization of individual cells, cancer pearls, mitotic figures and the breakdown of cell organization are known to be pathological characteristics of cutaneous SCC. Staining for CD147, MCT1 and MCT4, among other markers, has been performed in pathological studies to determine the significance of these proteins in cutaneous SCC. Indeed, while these proteins have been suggested to serve as biomarkers of SCC and as targets of treatment, few studies to date have used antibodies against autophagy-related molecules. In the present study, we performed immunohistochemical staining using LC3 and p62 antibodies in cutaneous SCC specimens resected at our hospital, collated the results and conducted statistical analysis to determine the significance of LC3 and p62 expression.
These results revealed that the expression of LC3 was increased, whereas that of p62 tended to decrease, as the clinical stage of the disease progressed and the tumor diameter increased, lending support to the possibility that cancer cell growth and metabolism are autophagy dependent. Fujii et al. carried out staining of pancreatic cancers for LC3 and reported that LC3 expression increased as the disease stage progressed. Similarly, Rossitza Lazova et al. stained specimens of malignant melanoma and breast cancer for LC3 and observed stronger LC3 expression in tumors obtained from patients with advanced disease and poor outcomes. Our results obtained for cutaneous SCC are consistent with these reports.
We also performed immunostaining for the lysosome marker LAMP1 and compared these results to the LC3 staining results. In accordance with LC3 expression, LAMP expression increased significantly as the clinical stage advanced, which suggests that the autophagosome–lysosome flux (autophagic degradation) is substantially upregulated in LC3-positive SCC.
Moreover, our results showed significantly higher LC3 expression and lower p62 expression in cases of lymph node metastasis, suggesting that autophagy is activated in cases where the malignancy grade is high enough to give rise to lymph node metastasis. That is, the cases in which LC3 expression was increased and p62 expression was decreased may be severe, such that we should suspect lymph node metastasis in those cases. This information may lead to the consideration of sentinel lymph node biopsy as an appropriate clinical measure.
Bowen's disease is an in situ SCC of the skin, which is characterized by a deficiency in tumor polarity. We also performed immunostaining for LC3 and p62 in a few specimens of Bowen's disease and Bowen's carcinoma. Although we did not report the details of this result in the current work, the expression of LC3 and p62 in Bowen's disease and Bowen's carcinoma was similar to that observed in cutaneous SCC, indicating no correlation between the expression of autophagy-related proteins and tumor polarity.
Attempts to develop treatments targeting autophagy have begun, and a clinical trial for the use of chloroquine, an inhibitor of lysosomal acidification, in malignant melanoma is currently under way in the USA.[10, 11] Based on the results of the present study, we propose that it also may be possible to develop a similar treatment for SCC of the skin. The treatment of cutaneous SCC on the face by resection often decreases patient quality of life, and we hope that novel treatments emerge for such cases instead of surgery. To this end, this was the first report to assess the correlation between the expression of autophagy-related markers and disease progression in patients with cutaneous SCC.
We are grateful to Dr Takashi Yao of Department of Human pathology and Mr Atsushi Furuhata of the Laboratory of Biomedical Imaging Research, Juntendo University Graduate School of Medicine, Tokyo, Japan, for technical assistance. This work was supported in part by the Research on Measures for Intractable Diseases Project matching fund subsidy (H23-028) from the Ministry of Health, Labor and Welfare, Japan (A. T., S. I.); a Grant-in-Aid for Scientific Research on Priority Areas (18076005 to T. U.); and a Research Grant from the Takeda sScience Foundation (T. U.).