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Autophagy-related genes Raptor, Rictor, and Beclin1 expression and relationship with multidrug resistance in colorectal carcinoma
Human Pathology (2015) 46, 1752–1759
www.elsevier.com/locate/humpath
Original contribution
Autophagy-related genes Raptor, Rictor, and Beclin1 expression and relationship with multidrug resistance in colorectal carcinoma☆,☆☆ Wu Shuhua MD a,⁎,1 , Sun Chenbo MSc b,1 , Li Yangyang MSc a , Gao Xiangqian MSc a , He Shuang MSc a , Li Tangyue MSc b , Tian Dong MD a,⁎ a
Department of Pathology, Binzhou Medical University Hospital, Binzhou, Shandong Province, China 256603 Department of Pathology, Binzhou Medical University, Binzhou, Shandong Province, China 256603
b
Received 9 June 2015; revised 10 July 2015; accepted 15 July 2015
Keywords: Colorectal cancer; MDR; Raptor; Rictor; Beclin1; Prognosis
Summary This study aims to evaluate the relationship between the expressions of autophagy-related genes Raptor, Rictor, and Beclin1 and the expression of multidrug resistance (MDR) gene in colorectal cancer (CRC) patients. Immunohistochemistry and real-time polymerase chain reaction were used to detect the protein and messenger RNA expressions of mammalian target of rapamycin (mTOR), Raptor, Rictor, Beclin1, light chain 3 (LC3), and MDR-1 in 279 CRC specimens. Patients were followed up annually by telephone or at an outpatient clinic. Results revealed that the protein and messenger RNA expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are significantly higher than in adjacent tissues. LC3 expression in poorly differentiated CRC is higher than that in welldifferentiated CRC, and the expression of mTOR, Raptor, Rictor, and LC3 in lymph node metastasis is higher than that obtained in the absence of lymph node metastasis. The expression of LC3 is positively correlated with those of Beclin1 and Rictor and negatively correlated with Raptor and mTOR in CRC. The expression of Raptor is negatively correlated with Rictor. The expression of MDR-1 is positively correlated with those of Beclin1, LC3, and Rictor and negatively correlated with Raptor and mTOR. Kaplan-Meier analysis revealed that the 5-year survival rate of patients without lymph node metastasis; positive expression of Rictor, Beclin1, and LC3; and negative expression of Raptor and mTOR were higher than those with these characteristics. To conclude, the expressions of Beclin1, Raptor, and Rictor are related to the development and progression of colorectal carcinoma and MDR. (Clinical trial registration number: 2014-009-01.) © 2015 Elsevier Inc. All rights reserved.
☆ Competing interests: There was no financial or personal relationship with other people or organizations in the study. There is no conflict of interest in the publication of this manuscript. ☆☆ Funding/Support: This work was supported by Scientific and Technological Project of Shandong Province, Binzhou, China. ⁎ Corresponding authors. Department of Pathology, Binzhou Medical University Hospital, Binzhou 256603, Shandong Province, China. E-mail addresses: [email protected] (S. Wu), [email protected] (D. Tian). 1 Contributed equally to this work.
http://dx.doi.org/10.1016/j.humpath.2015.07.016 0046-8177/© 2015 Elsevier Inc. All rights reserved.
1. Introduction Colorectal cancer (CRC) is a common digestive cancer in China. At present, chemotherapy occupies an irreplaceable position in the treatment of CRC. However, the resistance of tumor cells to chemotherapeutic drugs seriously affected the clinical effect of chemotherapy drugs. Clinically, chemotherapy resistance occurs in approximately 50% of patients who undergo chemotherapy, thereby making chemotherapy difficult [1].
Raptor, Rictor, and Beclin1 and MDR in colorectal carcinoma Therefore, a study of CRC resistance mechanisms has important clinical implications for CRC treatment. Recent studies reveal that many factors can affect the sensitivity of chemotherapy drugs, including drug pharmacokinetics, drug targets changes, drug-induced DNA damage repair, abnormal changes in cell signal transduction pathways, apoptosis escape, and autophagy. Among these factors, autophagy and its correlation with drug resistance in tumor cells have become a popular research topic in recent years. Autophagy is a “self-digestion” process, in which cytoplasmic components, such as cytosolic proteins and organelles, are enclosed to form double-membrane autophagosomes; these vesicles can degrade the sequestered intra-autophagosomal components by using lysosome hydrolases. A recent study found that different levels of autophagy play a dual role in tumor cell resistance. On one hand, autophagy can prevent apoptosis induced by anticancer drugs, thereby promoting tumor resistance [2]. On the other hand, excessive autophagy can lead to autophagic cell death, which may become the cause of death of apoptosis-tolerant tumor cells [3], thereby decreasing tumor resistance to some extent. However, studies on the correlation between tumor cells' chemotherapy tolerance and autophagy have just started. Many mechanisms underlying autophagy and tumor cell chemotherapy tolerance are still unknown. In this study, we detected the expressions of Beclin1, Raptor, Rictor, and multidrug resistance gene MDR-1 in CRC and analyzed their relationship in combination with clinicopathological factors to investigate the relationship between expression levels and chemotherapy resistance and CRC prognosis.
2. Materials and methods 2.1. Clinical specimens and patient data A total of 279 CRC tissue samples at different stages were collected from the Department of Pathology of Binzhou Medical University Hospital between January 2006 and January 2010. Tissue samples were routinely fixed in 10% buffered neutral formalin. Cancer tissues were cut into wedge shapes, whereas normal tissues were cut at least 5 cm away from the tumor margin. All CRC patients were clinically and pathologically proven to have not received preoperative chemotherapy or radiotherapy before operations. 5-fluorouracil (5-Fu) combined with calcium folinate chemotherapy was performed in all patients after operations. All specimens were collected with the informed consent of the patients, and the Ethical Committee of Binzhou Medical University Hospital approved the protocols used in this study. Clinicopathological classification was performed according to the National Comprehensive Cancer Network classification parameters [4]. Demographic and clinicopathological parameters were prospectively recorded using a chart review. Patients were followed up annually by telephone or at outpatient clinics until January 2015 or until the time of death. Cardiovascular and cerebrovascular diseases, respiratory diseases, and other malignant tumors were excluded in all CRC patients.
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2.2. Immunohistochemistry and scoring Before staining, paraffin-embedded tissue blocks were cut in 4-μm-thick sections. Sections were deparaffinized in an oven at 60°C for 2 hours and then rehydrated with 2 and 3 changes of xylene and ethanol, respectively. Antigen retrieval was performed using the microwave retrieval method. Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide for 10 minutes at room temperature. Nonspecific binding was blocked by incubating sections with 10% normal goat serum in Phosphate Buffered Saline (PBS) for 30 minutes at room temperature. Without washing, sections were incubated with rabbit monoclonal antibody against human mammalian target of rapamycin (mTOR) (1:200; Abcam), Raptor (1:100; Abcam), Rictor (1:100; Abcam), Beclin1 (1:200; Abcam), light chain 3 (LC3) (1:50; Abcam), and MDR-1 (Fuzhou Maixin Biotech, Fuzhou, China) at 4°C overnight. The sections were then incubated with horseradish peroxidase– conjugated secondary goat anti-rabbit antibody (Abcam, San Francisco, CA) for 1 hour at room temperature. Sections were washed with PBS and treated with the Metal-enhanced DAB Substrate Kit (Thermo Scientific, Waltham, USA) to visualize the antigen-antibody complex. Two researchers who were unaware of the clinicopathological statuses of the specimens scored each section separately. The percentage of stained cells on each section was scored as follows: 0 (b5%), 1 (5%-25%), 2 (26%-50%), and 3 (N50%). Staining intensity was scored as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The final score of each specimen was calculated by multiplying stained cell score with the staining intensity score. Thus, the final score ranged from 0 to 9. Low expression was defined as a final score of less than 4, whereas high expression was defined as a score of greater than or equal to 4.
2.3. RNA collection, complementary DNA synthesis, and real-time polymerase chain reaction analysis Total RNA was extracted from fresh frozen tumor specimens and the corresponding noncancerous tissues by using Trizol reagent (Invitrogen, Dalian, China). The amount of RNA was determined with the absorbance at 260 nm, and its purity was estimated using the ratio of the absorbance at A260/280. The total RNA was reverse transcribed using Prime Script RT reagent kit (Takara, DRR037A; , Dalian, China). Real-time polymerase chain reactions (PCRs) were performed on the CFX96TM real-time PCR detection system C1000 (Applied Biosystems, Dalian, China). For SYBR Green I–based real-time PCRs, each 25 μL of the reaction mixture contained 2 μL of primer pairs, 2 μL of complementary DNA, 12.5 μL of SYBR Premix Ex TaqII, and ddH2O to obtain a final volume of 25 μL.
2.4. Statistical analysis All statistical analyses were conducted with the SPSS 19.0 software (SPSS, Chicago, IL). The correlation of protein expressions with clinical parameters was analyzed
1754 Table 1
W. Shuhua et al. Correlation of mTOR, Raptor, Rictor, Beclin1, and LC3 with different clinicopathological parameters
Parameters Age (y) b60 ≥60 Sex Male Female Tumor sizes (cm) b5 ≥5 Tumor site Colon Rectal Histologic grade Grade 1 Grade 2 Grade 3 Histology Tubular Mucinous Depth of invasion Mucosa and submucosa Muscularis propria Subserosa Lymph node metastasis Yes No
n
Beclin1 (+)
P
LC3 (+)
P
122 157
111 141
.742
110 133
.178
61 68
.266
58 61
.146
78 97
.712
146 133
132 120
.958
127 116
.954
72 57
.282
69 50
.141
90 85
.696
127 152
115 137
.906
115 128
.081
66 63
.079
54 65
.967
81 94
.739
115 164
106 146
.381
101 142
.761
58 71
.239
52 67
.468
74 101
.639
80 139 60
70 130 52
.194
55 133 55
0
41 59 29
.423
39 51 29
.133
60 81 34
.258
235 44
213 39
.68
203 40
.411
105 24
.228
100 19
.938
151 24
.222
22 45 212
19 38 195
.242
19 40 184
.925
6 17 106
.058
9 18 92
.903
15 31 129
.514
152 127
136 116
.6
123 120
.001
56 73
.001
54 65
.008
82 93
.001
using χ2 and Fisher exact tests. Overall survival (OS) was plotted using the Kaplan-Meier method. P ≤ .05 was considered statistically significant.
3. Results 3.1. Correlation of mTOR, Raptor, Rictor, Beclin1, and LC3 with different clinicopathological parameters We analyzed the correlation between the mTOR, Raptor, Rictor, Beclin1, and LC3 expression levels of CRC samples and a set of clinicopathological parameters, including age, sex, histologic type, and tumor site (Table 1). The expression of LC3 in moderately and poorly differentiated CRC (95.68% and 91.67%) was higher than in well-differentiated CRC (68.75%). The expressions of mTOR, Raptor, Rictor, and LC3 in the presence of lymph node metastasis (57.48%, 51.18%, 73.23%, and 94.49%) are higher than in the absence of lymph node metastasis (36.84%, 35.53%, 53.95%, and 80.92%, respectively). The overexpression of Beclin1 has no correlation with the degree of differentiation and lymph node metastasis (P N .05).
3.2. Correlation of mTOR, Raptor, Rictor, Beclin1, and LC3 protein with MDR-1 In colorectal carcinoma, the protein expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 90.32%,
mTOR (+)
P
Raptor (+)
P
Rictor (+)
P
87.09%, 46.23%, 42.65%, 62.72%, and 72.75%, respectively, and the messenger RNA (mRNA) expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 2.8060 ± 0.0428, 2.3280 ± 0.0416, 2.4790 ± 0.0398, 2.6480 ± 0.0412, 2.2130 ± 0.0415, and 2.6430 ± 0.0391, respectively, which are significantly higher than in adjacent tissues (P b .05; Fig. 1). As is shown in Table 2, the expression of LC3 is positively correlated with those of Beclin1 and Rictor (rs = 0.513, P b .01; rs = 0.168, P b .01) and negatively correlated with Raptor and mTOR (rs = −0.317, P b .01; rs = −0.158, P b .01; Table 2) in CRC. The expression of Beclin1 was not correlated with those of Raptor and Rictor (P N .05; Table 3). The expression of Raptor is negatively correlated with Rictor (rs = −0.669, P b .01; Table 4). The expression of MDR-1 is positively correlated with that of Beclin1, LC3, and Rictor (rs = 0.181, P b .01; rs = 0.149, P b .05; rs = 0.427, P b .01) and negatively correlated with expressions of Raptor and mTOR (rs = −0.189, P b .01; rs = −0.385, P b .01; Table 5).
3.3. Correlation between mTOR, Raptor, Rictor, Beclin1, LC3, and MDR expression levels and patient OS The median survival time of 279 patients was 59 months, and the 5-year survival rate of patients was 45.2%. Kaplan-Meier analysis (Fig. 2) revealed that Beclin1, Raptor, Rictor, LC3, mTOR, MDR-1 expression, and lymph node metastasis could be relevant predictive factors of OS. The
Raptor, Rictor, and Beclin1 and MDR in colorectal carcinoma
1755
A
B
C
D
E
F
G
Fig. 1 Immunohistochemical staining of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 protein expressions in CRC tissues. High-power view (original magnification ×400) shows strong staining for Beclin1 (A), LC3 (B), mTOR (C), and Rictor (E) in the cytoplasm of cancer cells, Raptor in the nucleus and cytoplasm of cancer cells (D), MDR-1 in the membrane and cytoplasm of cancer cells (F). Significant higher mRNA expression of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 has been found in CRC tissues compared to counterpart normal colorectal mucosa tissues (G).
1756
W. Shuhua et al.
Table 2 Rictor
Correlation of LC3 with Beclin1, mTOR, Raptor, and
−
−
+
P
Raptor −
+
P Rictor
+
−
P
+
Rictor
LC3 − 20 16 0 12 24 .008 6 30 0 21 15 .005 + 7 236 138 105 154 89 83 160
5-year survival rate of patients with positive expression of Beclin1 was 47.6%, and that of patients with negative expression of Beclin1 was 22.2%. The 5-year survival rate of patients with positive expression of LC3 was 48.1%, and that of patients with negative expression of LC3 was 25.0%. The 5-year survival rate of patients with positive expression of Rictor was 56.0%, and that of patients with negative expression of Rictor was 26.9%. The 5-year survival rate of patients with positive expression of Raptor was 35.3%, and that of patients with negative expression of Raptor was 52.5%. The 5-year survival rate of patients with positive expression of mTOR was 38.0%, and that of patients with negative expression of mTOR was 51.3%. The 5-year survival rate of patients with positive expression of MDR-1 was 38.9%, and that of patients with negative expression of MDR-1 was 61.8% (P b .01; Fig. 2).
4. Discussion Recent studies reveal that many factors can affect the sensitivity of chemotherapy drugs, including several that attracted widespread attention, as follows: abnormal changes of cell signal signaling pathway, apoptosis escape, and autophagy. A new study revealed that silencing mutant and wild-type K-ras would increase the resistance of HCT-116 cell line as a model of CRC to fluorouracil [5]. A currently accepted mechanism of tumor cell resistance is MDR1 gene product P-glycoprotein overexpression. P-glycoprotein, a transmembrane transporter protein, can pump the drug out from intracellular to extracellular areas, which could reduce intracellular drug concentration, thereby leading to tumor resistance [6]. Therefore, MDR1 gene is often clinically detected to reflect the tumor cell drug resistance more objectively [7]. The results of our study showed that MDR-1 expression rate was 72.78% in CRC, thereby suggesting the presence of MDR in CRC.
Correlation of mTOR with Beclin1 Beclin1
mTOR
Correlation of Raptor with Rictor Raptor
Beclin1 P mTOR
Table 3
Table 4
− +
P
−
+
12 15
138 114
.307
− +
P
−
+
15 145
89 30
0
Autophagy as a new way of programmed cell death is closely related to chemotherapy resistance [8]. Studies have shown that autophagy is abnormal in the chemotherapy course of liver cancer, leukemia, lung cancer, CRC, and other tumors [9-11]. Autophagy is regulated by many signaling pathways, in which phophoinositide3-kinase (PI3K) signaling pathway serves an important function in the proliferation, cell growth, cycle progression, apoptosis, migration, and survival of cancer cells. Growth factor combined with tyrosine kinase receptor activates type I PI3K, and the activated PI3K can promote the phosphorylation of downstream AKT, which could be activated. AKT activation could activate downstream of mTOR, thereby inhibiting autophagy [12]. mTOR can be divided into mTORC1 and mTORC2. When mTORC1 is inhibited, mTORC2 can induce negative feedback activation of AKT, thereby activating mTORC1 again and reducing the inhibition of autophagy [13]. Therefore, mTOR mainly inhibits autophagy through mTORC1. mTORC2 not only makes the negative feedback loop but also promotes autophagy by inducing amino acid starvation [14]. Raptor and Rictor are the core elements of mTORC1 and mTORC2, respectively, and these elements maintain the integrity of their molecules. Unlike type I PI3K, type III PI3K can positively regulate autophagy. Type III PI3K combines with Beclin1, and the complex promotes other autophagy-related proteins located in the proautophagy pathway, thereby activating autophagy [15]. Microtubule-associated protein 1A/1B–LC3, the homolog of yeast Atg8, is ubiquitously distributed and essential to the formation of autophagosomes in mammalian cells. During the formation, cytosolic LC3 (LC3-I) is transformed into membrane-bound LC3-II by conjugating to phosphatidylethanolamine; this process is catalyzed by the E1-like enzyme Atg7 and the E2-like enzyme Atg3 [9-11]. The intra-autophagosomal LC3-I is degraded by lysosomal hydrolases during the fusion of autophagosomes with lysosomes. Autophagic induction by starvation stimulates the conversion of LC3-I to LC3-II and up-regulates LC3 expression; therefore, this protein is used as a specific marker for autophagy [16,17]. Results showed that LC3 expression is positively correlated with those of Beclin1 and Rictor and negatively correlated with Raptor and mTOR, thereby implying that Beclin1 and Rictor overexpression enhanced autophagy, whereas Raptor and mTOR overexpression inhibited autophagy in CRC. The protein expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 90.32%, 87.09%, 46.23%, 42.65%, 62.72%, and 72.75%, respectively, and the mRNA expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 2.8060 ± 0.0428, 2.3280 ± 0.0416, 2.4790 ± 0.0398, 2.6480
Raptor, Rictor, and Beclin1 and MDR in colorectal carcinoma Table 5
Correlation of MDR-1 with Beclin1, LC3, mTOR, Raptor, and Rictor Beclin1
MDR-1
1757
− +
P
−
+
14 13
62 190
0
LC3
P
−
+
16 20
60 183
.01
mTOR
P
−
+
17 133
59 70
0
Raptor
P
−
+
32 128
44 75
0
Rictor
P
−
+
54 50
22 153
0
B
A
C
D
E
F
Fig. 2 Kaplan-Meier survival curves for Beclin1 (A), LC3 (B), Rictor (C), Raptor (D), mTOR (E), and MDR-1 (F) expression in CRC. In Kaplan-Meier analysis, high Raptor, mTOR, and MDR-1 expressions correlated with a shorter OS in CRC patients than the corresponding controls.
1758 ± 0.0412, 2.2130 ± 0.0415, and 2.6430 ± 0.0391, respectively, which are significantly higher than in adjacent tissues. The analysis in the correlation with clinicopathological factors shows that LC3 expression was positively correlated with differentiation and lymph node metastasis in CRC. We believe that autophagy plays a synergistic role in the development, proliferation, metastasis, and invasion of CRC. Recently several studies have shown that autophagy has a dual effect on the drug resistance of tumor cells. On one hand, autophagy can provide nutrients to the cells to resist the harsh living environment produced by chemotherapy, thereby producing drug resistance. A recent study revealed that, in hepatocellular carcinoma cell lines, cisplatin activates autophagy by activating ATG7, thereby increasing liver cancer cell resistance to cisplatin [9]. However, autophagy can induce apoptosis and promote autophagic cell death, thereby reducing drug resistance. A recent study revealed that the degradation of KRAS could enhance 4-hydroxy tamoxifen–induced autophagic cell death, thereby reversing cell resistance to 4-hydroxytamoxifen [18]. Our previous studies indicated that the increasing expression of Beclin1 in CRC tissues may be correlated with development and multidrug resistance gene MDR of CRC. Thus, MDR is correlated with autophagy. The expression of MDR-1 was positively correlated with that of Beclin1, LC3, and Rictor and negatively correlated with Raptor, thereby implying that the increase of autophagy is closely related to MDR in CRC. We believe that tumor cells can produce stress tolerance to induce autophagy after exposure to chemotherapy drugs. Autophagy could prevent chemotherapy drugs from affecting tumor cells and could reduce the intracellular concentration of chemotherapeutic drugs by decreasing the permeability of cell membranes. Autophagy can also slow down cell metabolism and intracellular material exchange, thereby maintaining homeostasis within cells and weakening the killing effect of chemotherapy on tumor cells, resulting in chemotherapy tolerance. In this study, we followed up 279 cases of CRC patients and found that the prognosis of CRC was not only correlated with the clinical and pathological factors, such as degree of differentiation and lymph node metastasis, but also with mTOR, Raptor, Rictor, Beclin1, and LC3 expression levels in CRC patients. Patients with high expressions of Raptor and mTOR and low expressions of Rictor, Beclin1, and LC3 have higher mortality and worse prognosis. On one hand, the activation of autophagy can induce tumor cell resistance to chemotherapy and reduce the killing effect of chemotherapy on tumor cells. On the other hand, unlimited proliferation of tumor cells often leads to ischemia and hypoxia states because of inadequate blood supply. This long-term ischemia and hypoxia stimulation leads to the continuous increase in tumor cell autophagous activity, eventually leading to tumor cell autophagic cell death, which plays a dominant role in killing tumor cells. The development of targeting medicine and biological markers is proceeding in the direction of personalized therapy, which is the most effective means of cancer clinical
W. Shuhua et al. treatment. Personalized therapy largely improves therapeutic effect. The antitumor therapy involving the blocking of key autophagy-related molecules by targeting medicine has been highly valued by people and has been used in subclinical studies. Rapamycin receptor inhibitors, such as rapamycin and temsirolimus, are new targeting medicines that can specifically bind to the mTOR, thereby activating autophagy and resulting in autophagic cell death. However, a study [19] showed that most patients did not significantly benefit from the treatment, indicating the occurrence of primary or secondary resistance in these patients. Invalid effect of rapamycin to mTORC2 is an important mechanism underlying rapamycin tolerance [20,21]. Studies have shown that mTORC2 can induce a negative feedback activation of AKT, thereby reactivating mTORC1, reducing the inhibition of autophagy, and eventually leading to rapamycin resistance [22,23]. Beclin1 increases the expression of LC3 and enhances autophagy, not only inhibiting the growth of colon cancer cells but also enhancing the antitumor effect of rapamycin [19]. The results of this study show that the expression of Raptor is negatively correlated with Rictor in CRC. We speculate that the negative feedback regulation of mTORC2 is an important reason for rapamycin treatment resistance. However, the regulation mechanism of mTORC2 in autophagy in CRC needs further study. In summary, autophagy-related genes Raptor, Rictor, and Beclin1 are highly expressed in CRC. Gene expression is not only correlated with differentiation, lymph node metastasis, and other clinicopathological factors but also correlated with MDR in CRC. Therefore, the synchronous detection of Raptor, Rictor, and Beclin1 has important significance in the clinical detection of CRC malignancy and the rational screening of effective chemotherapy drugs.
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www.elsevier.com/locate/humpath
Original contribution
Autophagy-related genes Raptor, Rictor, and Beclin1 expression and relationship with multidrug resistance in colorectal carcinoma☆,☆☆ Wu Shuhua MD a,⁎,1 , Sun Chenbo MSc b,1 , Li Yangyang MSc a , Gao Xiangqian MSc a , He Shuang MSc a , Li Tangyue MSc b , Tian Dong MD a,⁎ a
Department of Pathology, Binzhou Medical University Hospital, Binzhou, Shandong Province, China 256603 Department of Pathology, Binzhou Medical University, Binzhou, Shandong Province, China 256603
b
Received 9 June 2015; revised 10 July 2015; accepted 15 July 2015
Keywords: Colorectal cancer; MDR; Raptor; Rictor; Beclin1; Prognosis
Summary This study aims to evaluate the relationship between the expressions of autophagy-related genes Raptor, Rictor, and Beclin1 and the expression of multidrug resistance (MDR) gene in colorectal cancer (CRC) patients. Immunohistochemistry and real-time polymerase chain reaction were used to detect the protein and messenger RNA expressions of mammalian target of rapamycin (mTOR), Raptor, Rictor, Beclin1, light chain 3 (LC3), and MDR-1 in 279 CRC specimens. Patients were followed up annually by telephone or at an outpatient clinic. Results revealed that the protein and messenger RNA expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are significantly higher than in adjacent tissues. LC3 expression in poorly differentiated CRC is higher than that in welldifferentiated CRC, and the expression of mTOR, Raptor, Rictor, and LC3 in lymph node metastasis is higher than that obtained in the absence of lymph node metastasis. The expression of LC3 is positively correlated with those of Beclin1 and Rictor and negatively correlated with Raptor and mTOR in CRC. The expression of Raptor is negatively correlated with Rictor. The expression of MDR-1 is positively correlated with those of Beclin1, LC3, and Rictor and negatively correlated with Raptor and mTOR. Kaplan-Meier analysis revealed that the 5-year survival rate of patients without lymph node metastasis; positive expression of Rictor, Beclin1, and LC3; and negative expression of Raptor and mTOR were higher than those with these characteristics. To conclude, the expressions of Beclin1, Raptor, and Rictor are related to the development and progression of colorectal carcinoma and MDR. (Clinical trial registration number: 2014-009-01.) © 2015 Elsevier Inc. All rights reserved.
☆ Competing interests: There was no financial or personal relationship with other people or organizations in the study. There is no conflict of interest in the publication of this manuscript. ☆☆ Funding/Support: This work was supported by Scientific and Technological Project of Shandong Province, Binzhou, China. ⁎ Corresponding authors. Department of Pathology, Binzhou Medical University Hospital, Binzhou 256603, Shandong Province, China. E-mail addresses: [email protected] (S. Wu), [email protected] (D. Tian). 1 Contributed equally to this work.
http://dx.doi.org/10.1016/j.humpath.2015.07.016 0046-8177/© 2015 Elsevier Inc. All rights reserved.
1. Introduction Colorectal cancer (CRC) is a common digestive cancer in China. At present, chemotherapy occupies an irreplaceable position in the treatment of CRC. However, the resistance of tumor cells to chemotherapeutic drugs seriously affected the clinical effect of chemotherapy drugs. Clinically, chemotherapy resistance occurs in approximately 50% of patients who undergo chemotherapy, thereby making chemotherapy difficult [1].
Raptor, Rictor, and Beclin1 and MDR in colorectal carcinoma Therefore, a study of CRC resistance mechanisms has important clinical implications for CRC treatment. Recent studies reveal that many factors can affect the sensitivity of chemotherapy drugs, including drug pharmacokinetics, drug targets changes, drug-induced DNA damage repair, abnormal changes in cell signal transduction pathways, apoptosis escape, and autophagy. Among these factors, autophagy and its correlation with drug resistance in tumor cells have become a popular research topic in recent years. Autophagy is a “self-digestion” process, in which cytoplasmic components, such as cytosolic proteins and organelles, are enclosed to form double-membrane autophagosomes; these vesicles can degrade the sequestered intra-autophagosomal components by using lysosome hydrolases. A recent study found that different levels of autophagy play a dual role in tumor cell resistance. On one hand, autophagy can prevent apoptosis induced by anticancer drugs, thereby promoting tumor resistance [2]. On the other hand, excessive autophagy can lead to autophagic cell death, which may become the cause of death of apoptosis-tolerant tumor cells [3], thereby decreasing tumor resistance to some extent. However, studies on the correlation between tumor cells' chemotherapy tolerance and autophagy have just started. Many mechanisms underlying autophagy and tumor cell chemotherapy tolerance are still unknown. In this study, we detected the expressions of Beclin1, Raptor, Rictor, and multidrug resistance gene MDR-1 in CRC and analyzed their relationship in combination with clinicopathological factors to investigate the relationship between expression levels and chemotherapy resistance and CRC prognosis.
2. Materials and methods 2.1. Clinical specimens and patient data A total of 279 CRC tissue samples at different stages were collected from the Department of Pathology of Binzhou Medical University Hospital between January 2006 and January 2010. Tissue samples were routinely fixed in 10% buffered neutral formalin. Cancer tissues were cut into wedge shapes, whereas normal tissues were cut at least 5 cm away from the tumor margin. All CRC patients were clinically and pathologically proven to have not received preoperative chemotherapy or radiotherapy before operations. 5-fluorouracil (5-Fu) combined with calcium folinate chemotherapy was performed in all patients after operations. All specimens were collected with the informed consent of the patients, and the Ethical Committee of Binzhou Medical University Hospital approved the protocols used in this study. Clinicopathological classification was performed according to the National Comprehensive Cancer Network classification parameters [4]. Demographic and clinicopathological parameters were prospectively recorded using a chart review. Patients were followed up annually by telephone or at outpatient clinics until January 2015 or until the time of death. Cardiovascular and cerebrovascular diseases, respiratory diseases, and other malignant tumors were excluded in all CRC patients.
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2.2. Immunohistochemistry and scoring Before staining, paraffin-embedded tissue blocks were cut in 4-μm-thick sections. Sections were deparaffinized in an oven at 60°C for 2 hours and then rehydrated with 2 and 3 changes of xylene and ethanol, respectively. Antigen retrieval was performed using the microwave retrieval method. Endogenous peroxidase activity was quenched by incubation with 3% hydrogen peroxide for 10 minutes at room temperature. Nonspecific binding was blocked by incubating sections with 10% normal goat serum in Phosphate Buffered Saline (PBS) for 30 minutes at room temperature. Without washing, sections were incubated with rabbit monoclonal antibody against human mammalian target of rapamycin (mTOR) (1:200; Abcam), Raptor (1:100; Abcam), Rictor (1:100; Abcam), Beclin1 (1:200; Abcam), light chain 3 (LC3) (1:50; Abcam), and MDR-1 (Fuzhou Maixin Biotech, Fuzhou, China) at 4°C overnight. The sections were then incubated with horseradish peroxidase– conjugated secondary goat anti-rabbit antibody (Abcam, San Francisco, CA) for 1 hour at room temperature. Sections were washed with PBS and treated with the Metal-enhanced DAB Substrate Kit (Thermo Scientific, Waltham, USA) to visualize the antigen-antibody complex. Two researchers who were unaware of the clinicopathological statuses of the specimens scored each section separately. The percentage of stained cells on each section was scored as follows: 0 (b5%), 1 (5%-25%), 2 (26%-50%), and 3 (N50%). Staining intensity was scored as follows: 0 (no staining), 1 (weak staining), 2 (moderate staining), and 3 (strong staining). The final score of each specimen was calculated by multiplying stained cell score with the staining intensity score. Thus, the final score ranged from 0 to 9. Low expression was defined as a final score of less than 4, whereas high expression was defined as a score of greater than or equal to 4.
2.3. RNA collection, complementary DNA synthesis, and real-time polymerase chain reaction analysis Total RNA was extracted from fresh frozen tumor specimens and the corresponding noncancerous tissues by using Trizol reagent (Invitrogen, Dalian, China). The amount of RNA was determined with the absorbance at 260 nm, and its purity was estimated using the ratio of the absorbance at A260/280. The total RNA was reverse transcribed using Prime Script RT reagent kit (Takara, DRR037A; , Dalian, China). Real-time polymerase chain reactions (PCRs) were performed on the CFX96TM real-time PCR detection system C1000 (Applied Biosystems, Dalian, China). For SYBR Green I–based real-time PCRs, each 25 μL of the reaction mixture contained 2 μL of primer pairs, 2 μL of complementary DNA, 12.5 μL of SYBR Premix Ex TaqII, and ddH2O to obtain a final volume of 25 μL.
2.4. Statistical analysis All statistical analyses were conducted with the SPSS 19.0 software (SPSS, Chicago, IL). The correlation of protein expressions with clinical parameters was analyzed
1754 Table 1
W. Shuhua et al. Correlation of mTOR, Raptor, Rictor, Beclin1, and LC3 with different clinicopathological parameters
Parameters Age (y) b60 ≥60 Sex Male Female Tumor sizes (cm) b5 ≥5 Tumor site Colon Rectal Histologic grade Grade 1 Grade 2 Grade 3 Histology Tubular Mucinous Depth of invasion Mucosa and submucosa Muscularis propria Subserosa Lymph node metastasis Yes No
n
Beclin1 (+)
P
LC3 (+)
P
122 157
111 141
.742
110 133
.178
61 68
.266
58 61
.146
78 97
.712
146 133
132 120
.958
127 116
.954
72 57
.282
69 50
.141
90 85
.696
127 152
115 137
.906
115 128
.081
66 63
.079
54 65
.967
81 94
.739
115 164
106 146
.381
101 142
.761
58 71
.239
52 67
.468
74 101
.639
80 139 60
70 130 52
.194
55 133 55
0
41 59 29
.423
39 51 29
.133
60 81 34
.258
235 44
213 39
.68
203 40
.411
105 24
.228
100 19
.938
151 24
.222
22 45 212
19 38 195
.242
19 40 184
.925
6 17 106
.058
9 18 92
.903
15 31 129
.514
152 127
136 116
.6
123 120
.001
56 73
.001
54 65
.008
82 93
.001
using χ2 and Fisher exact tests. Overall survival (OS) was plotted using the Kaplan-Meier method. P ≤ .05 was considered statistically significant.
3. Results 3.1. Correlation of mTOR, Raptor, Rictor, Beclin1, and LC3 with different clinicopathological parameters We analyzed the correlation between the mTOR, Raptor, Rictor, Beclin1, and LC3 expression levels of CRC samples and a set of clinicopathological parameters, including age, sex, histologic type, and tumor site (Table 1). The expression of LC3 in moderately and poorly differentiated CRC (95.68% and 91.67%) was higher than in well-differentiated CRC (68.75%). The expressions of mTOR, Raptor, Rictor, and LC3 in the presence of lymph node metastasis (57.48%, 51.18%, 73.23%, and 94.49%) are higher than in the absence of lymph node metastasis (36.84%, 35.53%, 53.95%, and 80.92%, respectively). The overexpression of Beclin1 has no correlation with the degree of differentiation and lymph node metastasis (P N .05).
3.2. Correlation of mTOR, Raptor, Rictor, Beclin1, and LC3 protein with MDR-1 In colorectal carcinoma, the protein expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 90.32%,
mTOR (+)
P
Raptor (+)
P
Rictor (+)
P
87.09%, 46.23%, 42.65%, 62.72%, and 72.75%, respectively, and the messenger RNA (mRNA) expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 2.8060 ± 0.0428, 2.3280 ± 0.0416, 2.4790 ± 0.0398, 2.6480 ± 0.0412, 2.2130 ± 0.0415, and 2.6430 ± 0.0391, respectively, which are significantly higher than in adjacent tissues (P b .05; Fig. 1). As is shown in Table 2, the expression of LC3 is positively correlated with those of Beclin1 and Rictor (rs = 0.513, P b .01; rs = 0.168, P b .01) and negatively correlated with Raptor and mTOR (rs = −0.317, P b .01; rs = −0.158, P b .01; Table 2) in CRC. The expression of Beclin1 was not correlated with those of Raptor and Rictor (P N .05; Table 3). The expression of Raptor is negatively correlated with Rictor (rs = −0.669, P b .01; Table 4). The expression of MDR-1 is positively correlated with that of Beclin1, LC3, and Rictor (rs = 0.181, P b .01; rs = 0.149, P b .05; rs = 0.427, P b .01) and negatively correlated with expressions of Raptor and mTOR (rs = −0.189, P b .01; rs = −0.385, P b .01; Table 5).
3.3. Correlation between mTOR, Raptor, Rictor, Beclin1, LC3, and MDR expression levels and patient OS The median survival time of 279 patients was 59 months, and the 5-year survival rate of patients was 45.2%. Kaplan-Meier analysis (Fig. 2) revealed that Beclin1, Raptor, Rictor, LC3, mTOR, MDR-1 expression, and lymph node metastasis could be relevant predictive factors of OS. The
Raptor, Rictor, and Beclin1 and MDR in colorectal carcinoma
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A
B
C
D
E
F
G
Fig. 1 Immunohistochemical staining of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 protein expressions in CRC tissues. High-power view (original magnification ×400) shows strong staining for Beclin1 (A), LC3 (B), mTOR (C), and Rictor (E) in the cytoplasm of cancer cells, Raptor in the nucleus and cytoplasm of cancer cells (D), MDR-1 in the membrane and cytoplasm of cancer cells (F). Significant higher mRNA expression of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 has been found in CRC tissues compared to counterpart normal colorectal mucosa tissues (G).
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W. Shuhua et al.
Table 2 Rictor
Correlation of LC3 with Beclin1, mTOR, Raptor, and
−
−
+
P
Raptor −
+
P Rictor
+
−
P
+
Rictor
LC3 − 20 16 0 12 24 .008 6 30 0 21 15 .005 + 7 236 138 105 154 89 83 160
5-year survival rate of patients with positive expression of Beclin1 was 47.6%, and that of patients with negative expression of Beclin1 was 22.2%. The 5-year survival rate of patients with positive expression of LC3 was 48.1%, and that of patients with negative expression of LC3 was 25.0%. The 5-year survival rate of patients with positive expression of Rictor was 56.0%, and that of patients with negative expression of Rictor was 26.9%. The 5-year survival rate of patients with positive expression of Raptor was 35.3%, and that of patients with negative expression of Raptor was 52.5%. The 5-year survival rate of patients with positive expression of mTOR was 38.0%, and that of patients with negative expression of mTOR was 51.3%. The 5-year survival rate of patients with positive expression of MDR-1 was 38.9%, and that of patients with negative expression of MDR-1 was 61.8% (P b .01; Fig. 2).
4. Discussion Recent studies reveal that many factors can affect the sensitivity of chemotherapy drugs, including several that attracted widespread attention, as follows: abnormal changes of cell signal signaling pathway, apoptosis escape, and autophagy. A new study revealed that silencing mutant and wild-type K-ras would increase the resistance of HCT-116 cell line as a model of CRC to fluorouracil [5]. A currently accepted mechanism of tumor cell resistance is MDR1 gene product P-glycoprotein overexpression. P-glycoprotein, a transmembrane transporter protein, can pump the drug out from intracellular to extracellular areas, which could reduce intracellular drug concentration, thereby leading to tumor resistance [6]. Therefore, MDR1 gene is often clinically detected to reflect the tumor cell drug resistance more objectively [7]. The results of our study showed that MDR-1 expression rate was 72.78% in CRC, thereby suggesting the presence of MDR in CRC.
Correlation of mTOR with Beclin1 Beclin1
mTOR
Correlation of Raptor with Rictor Raptor
Beclin1 P mTOR
Table 3
Table 4
− +
P
−
+
12 15
138 114
.307
− +
P
−
+
15 145
89 30
0
Autophagy as a new way of programmed cell death is closely related to chemotherapy resistance [8]. Studies have shown that autophagy is abnormal in the chemotherapy course of liver cancer, leukemia, lung cancer, CRC, and other tumors [9-11]. Autophagy is regulated by many signaling pathways, in which phophoinositide3-kinase (PI3K) signaling pathway serves an important function in the proliferation, cell growth, cycle progression, apoptosis, migration, and survival of cancer cells. Growth factor combined with tyrosine kinase receptor activates type I PI3K, and the activated PI3K can promote the phosphorylation of downstream AKT, which could be activated. AKT activation could activate downstream of mTOR, thereby inhibiting autophagy [12]. mTOR can be divided into mTORC1 and mTORC2. When mTORC1 is inhibited, mTORC2 can induce negative feedback activation of AKT, thereby activating mTORC1 again and reducing the inhibition of autophagy [13]. Therefore, mTOR mainly inhibits autophagy through mTORC1. mTORC2 not only makes the negative feedback loop but also promotes autophagy by inducing amino acid starvation [14]. Raptor and Rictor are the core elements of mTORC1 and mTORC2, respectively, and these elements maintain the integrity of their molecules. Unlike type I PI3K, type III PI3K can positively regulate autophagy. Type III PI3K combines with Beclin1, and the complex promotes other autophagy-related proteins located in the proautophagy pathway, thereby activating autophagy [15]. Microtubule-associated protein 1A/1B–LC3, the homolog of yeast Atg8, is ubiquitously distributed and essential to the formation of autophagosomes in mammalian cells. During the formation, cytosolic LC3 (LC3-I) is transformed into membrane-bound LC3-II by conjugating to phosphatidylethanolamine; this process is catalyzed by the E1-like enzyme Atg7 and the E2-like enzyme Atg3 [9-11]. The intra-autophagosomal LC3-I is degraded by lysosomal hydrolases during the fusion of autophagosomes with lysosomes. Autophagic induction by starvation stimulates the conversion of LC3-I to LC3-II and up-regulates LC3 expression; therefore, this protein is used as a specific marker for autophagy [16,17]. Results showed that LC3 expression is positively correlated with those of Beclin1 and Rictor and negatively correlated with Raptor and mTOR, thereby implying that Beclin1 and Rictor overexpression enhanced autophagy, whereas Raptor and mTOR overexpression inhibited autophagy in CRC. The protein expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 90.32%, 87.09%, 46.23%, 42.65%, 62.72%, and 72.75%, respectively, and the mRNA expressions of Beclin1, LC3, mTOR, Raptor, Rictor, and MDR-1 in CRC are 2.8060 ± 0.0428, 2.3280 ± 0.0416, 2.4790 ± 0.0398, 2.6480
Raptor, Rictor, and Beclin1 and MDR in colorectal carcinoma Table 5
Correlation of MDR-1 with Beclin1, LC3, mTOR, Raptor, and Rictor Beclin1
MDR-1
1757
− +
P
−
+
14 13
62 190
0
LC3
P
−
+
16 20
60 183
.01
mTOR
P
−
+
17 133
59 70
0
Raptor
P
−
+
32 128
44 75
0
Rictor
P
−
+
54 50
22 153
0
B
A
C
D
E
F
Fig. 2 Kaplan-Meier survival curves for Beclin1 (A), LC3 (B), Rictor (C), Raptor (D), mTOR (E), and MDR-1 (F) expression in CRC. In Kaplan-Meier analysis, high Raptor, mTOR, and MDR-1 expressions correlated with a shorter OS in CRC patients than the corresponding controls.
1758 ± 0.0412, 2.2130 ± 0.0415, and 2.6430 ± 0.0391, respectively, which are significantly higher than in adjacent tissues. The analysis in the correlation with clinicopathological factors shows that LC3 expression was positively correlated with differentiation and lymph node metastasis in CRC. We believe that autophagy plays a synergistic role in the development, proliferation, metastasis, and invasion of CRC. Recently several studies have shown that autophagy has a dual effect on the drug resistance of tumor cells. On one hand, autophagy can provide nutrients to the cells to resist the harsh living environment produced by chemotherapy, thereby producing drug resistance. A recent study revealed that, in hepatocellular carcinoma cell lines, cisplatin activates autophagy by activating ATG7, thereby increasing liver cancer cell resistance to cisplatin [9]. However, autophagy can induce apoptosis and promote autophagic cell death, thereby reducing drug resistance. A recent study revealed that the degradation of KRAS could enhance 4-hydroxy tamoxifen–induced autophagic cell death, thereby reversing cell resistance to 4-hydroxytamoxifen [18]. Our previous studies indicated that the increasing expression of Beclin1 in CRC tissues may be correlated with development and multidrug resistance gene MDR of CRC. Thus, MDR is correlated with autophagy. The expression of MDR-1 was positively correlated with that of Beclin1, LC3, and Rictor and negatively correlated with Raptor, thereby implying that the increase of autophagy is closely related to MDR in CRC. We believe that tumor cells can produce stress tolerance to induce autophagy after exposure to chemotherapy drugs. Autophagy could prevent chemotherapy drugs from affecting tumor cells and could reduce the intracellular concentration of chemotherapeutic drugs by decreasing the permeability of cell membranes. Autophagy can also slow down cell metabolism and intracellular material exchange, thereby maintaining homeostasis within cells and weakening the killing effect of chemotherapy on tumor cells, resulting in chemotherapy tolerance. In this study, we followed up 279 cases of CRC patients and found that the prognosis of CRC was not only correlated with the clinical and pathological factors, such as degree of differentiation and lymph node metastasis, but also with mTOR, Raptor, Rictor, Beclin1, and LC3 expression levels in CRC patients. Patients with high expressions of Raptor and mTOR and low expressions of Rictor, Beclin1, and LC3 have higher mortality and worse prognosis. On one hand, the activation of autophagy can induce tumor cell resistance to chemotherapy and reduce the killing effect of chemotherapy on tumor cells. On the other hand, unlimited proliferation of tumor cells often leads to ischemia and hypoxia states because of inadequate blood supply. This long-term ischemia and hypoxia stimulation leads to the continuous increase in tumor cell autophagous activity, eventually leading to tumor cell autophagic cell death, which plays a dominant role in killing tumor cells. The development of targeting medicine and biological markers is proceeding in the direction of personalized therapy, which is the most effective means of cancer clinical
W. Shuhua et al. treatment. Personalized therapy largely improves therapeutic effect. The antitumor therapy involving the blocking of key autophagy-related molecules by targeting medicine has been highly valued by people and has been used in subclinical studies. Rapamycin receptor inhibitors, such as rapamycin and temsirolimus, are new targeting medicines that can specifically bind to the mTOR, thereby activating autophagy and resulting in autophagic cell death. However, a study [19] showed that most patients did not significantly benefit from the treatment, indicating the occurrence of primary or secondary resistance in these patients. Invalid effect of rapamycin to mTORC2 is an important mechanism underlying rapamycin tolerance [20,21]. Studies have shown that mTORC2 can induce a negative feedback activation of AKT, thereby reactivating mTORC1, reducing the inhibition of autophagy, and eventually leading to rapamycin resistance [22,23]. Beclin1 increases the expression of LC3 and enhances autophagy, not only inhibiting the growth of colon cancer cells but also enhancing the antitumor effect of rapamycin [19]. The results of this study show that the expression of Raptor is negatively correlated with Rictor in CRC. We speculate that the negative feedback regulation of mTORC2 is an important reason for rapamycin treatment resistance. However, the regulation mechanism of mTORC2 in autophagy in CRC needs further study. In summary, autophagy-related genes Raptor, Rictor, and Beclin1 are highly expressed in CRC. Gene expression is not only correlated with differentiation, lymph node metastasis, and other clinicopathological factors but also correlated with MDR in CRC. Therefore, the synchronous detection of Raptor, Rictor, and Beclin1 has important significance in the clinical detection of CRC malignancy and the rational screening of effective chemotherapy drugs.
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