Background
SIRT6 is a member of the sirtuin family and important in the regulation of cellular physiology and tumorigenesis [
1‐
5]. SIRT6 is best known for its roles in the regulation of cellular proliferation, energy metabolism, aging, and DNA damage repair [
1‐
3,
5‐
8]. Especially, the functions of SIRT6 in the repair of DNA damage raise the possibility that SIRT6 may be involved in tumorigenesis [
9]. However, there are controversial reports on the role of SIRT6 in tumorigenesis and tumor progression. Especially, the effect of SIRT6 in tumor growth has been reported controversially according to the type of cancer [
4,
10‐
13]. Despite the roles of SIRT6 as a sensor to repair DNA damage, higher expression of SIRT6 is related to increased proliferation and invasiveness of cancer cells [
14‐
16]. Furthermore, elevated expression of SIRT6 was associated with shorter survival of cancer patients in the breast, stomach, lung, ovary, and lymphoma [
14,
16‐
19]. In contrast, loss of SIRT6 is a factor associated with shorter survival of breast and liver cancer patients in other reports [
13,
20]. Even with the same MDA-MB-231 breast cancer cells, there are conflicting reports of SIRT6 either increasing or decreasing the proliferation of these cells [
13,
14]. These controversial reports on the role of SIRT6 in cancer growth suggest that the effect of SIRT6 expression might differ according to the condition of the cancer itself or the cancer-associated environment.
One of the critical factors determining the survival of cancer patients is the responsiveness of cancer to anti-cancer therapy. Regarding resistance to anti-cancer therapy of cancer cells, various factors such as activation of the DNA damage repair pathway, immune evasion, metabolic change, and the stemness of cancer cells have important roles [
21,
22]. Especially, the DNA damage repair pathway is tumor suppressive in normal cells, but it induces resistance of cancer cells to genotoxic anti-cancer therapies [
23,
24]. Based on these paradoxical roles of the molecules of the DNA damage repair pathway, inhibitors of this pathway, such as Poly (ADP-ribose) polymerase (PARP) inhibitors, have been developed as anti-cancer agents [
11,
21]. Therefore, when based on the role of SIRT6 in the DNA damage repair pathway [
1,
8,
25], SIRT6 might be an inducer of therapeutic resistance [
26].
Osteosarcoma is the most common primary bone malignancy [
27]. Despite recent advances in the understanding of human cancers, osteosarcomas are often refractory to treatment [
28]. Recently, there are increasing reports focusing on the roles of the sirtuins, especially on SIRT6, in human cancers [
4,
29,
30]. Furthermore, the DNA damage repair pathway is a promising target of cancer treatment [
11]. In osteosarcoma cells, the PARP inhibitor, olaparib, potentiates the therapeutic efficacy of doxorubicin [
31]. However, there have been limited studies on the roles of SIRT6 in osteosarcoma [
32‐
35]. Furthermore, the role of SIRT6 in the progression of osteosarcoma has been reported controversially [
32,
33,
35]. However, when considering the role of SIRT6 in the repair of DNA damage, SIRT6 might be involved in the effectiveness of anti-cancer treatment [
2,
8,
25,
26,
29]. Therefore, this study evaluated the role and effect of SIRT6 expression on osteosarcoma by investigating the expression of SIRT6 in human osteosarcomas and assessing the role of SIRT6 in resistance to the treatment of doxorubicin in conjunction with the role of SIRT6 on the repair of DNA damage.
Discussion
In this study, we demonstrate that high expression of SIRT6 is significantly associated with shorter survival of osteosarcoma patients as an independent indicator of shorter OS and RFS of osteosarcoma patients. In line with these results, the expression of SIRT6 was higher in osteosarcoma tissue compared with normal bone tissue [
35]. Furthermore, high expression of SIRT6 was significantly associated with more metastasis and shorter OS and disease-free survival of osteosarcoma patients [
35]. Consistently, the expression of SIRT6 was elevated in cancers compared with normal counterpart cells in the colon, esophagus, melanocytes, thyroid, and lymph node [
12,
15,
19,
40,
41]. In addition, high expression of SIRT6 was associated with lymph node metastasis of colorectal cancers and thyroid papillary carcinomas [
12,
15] and was associated with poor prognosis of breast cancer, ovary cancer, gastric cancer, lung cancer, and diffuse large B-cell lymphoma patients [
14,
16‐
19]. However, controversially, higher expression of SIRT6 was associated with favorable prognosis of breast cancer patients [
13], and loss of SIRT6 was associated with progression of hepatocellular carcinomas [
20] and colon carcinomas [
5]. These controversial reports on the role of SIRT6 in human cancers suggest that the impact of SIRT6 expression in cancer patients might differ depending on cancer type and/or characteristics of cancer patients included in the study [
4,
14]. Therefore, further study is needed to clarify the various roles of SIRT6 in tumorigenesis and cancer progression.
The prognostic significance of SIRT6 expression might be related to the role of SIRT6 on the proliferation of cancer cells, and SIRT6 is known as an important regulator of cellular proliferation [
13‐
16]. However, the impact of SIRT6 on the proliferation of cancer cells has been reported controversially [
13‐
16]. Regarding a role for SIRT6 as an oncogenic molecule, SIRT6 stimulated the proliferation of cancer cells by stimulating the Wnt/β-catenin pathway [
16]. In addition, SIRT6 stimulated the invasiveness of cancer cells by activating the epithelial-to-mesenchymal transition pathway in breast cancer, ovarian cancer, lung cancer, and osteosarcoma cells [
14,
16,
35,
42]. Especially, increased invasiveness of SIRT6-overexpressing cancer cells was associated with activation of the ERK1/2-MMP9 pathway [
35] and secretion of IL8 and TNF cytokines [
43]. In non-small cell lung cancer, SIRT6 induced epithelial-to-mesenchymal transition by regulating the snail/KLF4 pathway [
42]. However, controversially, elevated expression of SIRT6 inhibited the proliferation and invasiveness of osteosarcoma cells [
32], and breast cancer cells [
13]. Overexpression of SIRT6 inhibited the proliferation of HepG2 liver cancer cells by suppressing the ERK1/2 pathway [
44]. In a model of idiopathic pulmonary fibrosis, SIRT6 inhibited epithelial-to-mesenchymal transition [
45]. However, in this study, despite the prognostic significance of SIRT6 on the survival of osteosarcoma patients, the regulation of expression of SIRT6 did not significantly influence the proliferation of osteosarcoma cells. Therefore, further study focusing on the role of SIRT6 according to the specific type of cancer is needed.
In our results, despite the prognostic significance of SIRT6 expression, there was only one potential prognostic clinicopathological variable significantly associated with SIRT6 expression. A factor significantly associated with SIRT6 expression was latent distance metastasis of osteosarcoma. In addition, SIRT6-positivity was significantly associated with shorter OS and RFS in subgroups of osteosarcoma patients who received adjuvant chemotherapy. In addition, overexpression of SIRT6 induced resistance to doxorubicin-mediated apoptosis of osteosarcoma cells and knock-down of SIRT6 sensitized doxorubicin-mediated apoptosis of osteosarcoma cells. These results suggest that SIRT6-related differences in the prognosis of osteosarcoma patients might be related to SIRT6-mediated resistance to anti-cancer therapy. Supportively, knock-down of SIRT6 sensitized non-small cell lung cancer cells to paclitaxel treatment [
46], and inhibition of SIRT6 sensitized lymphoma cells to doxorubicin [
19]. Furthermore, regarding responsiveness of cancer cells on anti-cancer therapy, one mechanism of interest is the role of the molecules related with the DNA damage repair pathway in therapeutic resistance [
2,
24]. As it has been reported in breast and ovarian cancers, mutation in BRCA1/2 is correlates with early development of cancer, which are responsive to genotoxic anti-cancer therapies [
21,
23]. In addition, higher expression of molecules associated with the DNA damage repair pathways such as BRCA1/2, PARP, and γH2AX were associated with shorter survival of human cancer of the breast, ovary, and soft tissue [
47‐
49]. Furthermore, based on this rationale, various PARP inhibitors are FDA-approved drugs with cancer patients with defects in BRCA1/2 such as ovary, breast, and prostatic cancer (OncoKB database,
https://www.oncokb.org, accessed July 31, 2020). In osteosarcoma cells, defects in BRCA1/2 make tumors susceptible to the PARP inhibitor talazoparib [
50]. Furthermore, there is an increasing number of reports indicated that SIRT6 mediates the DNA damage repair pathways as a DNA double-strand break sensor by activating PARP1 [
2,
8,
25]. In this study, overexpression of SIRT6 induced the DNA damage repair pathway. Upon treatment with doxorubicin, the interaction between SIRT6 and p-ATM increased, and overexpression of SIRT6 increased expression of p-ATM and p-Chk2 and consequently decreased expression of γH2AX; conversely, knock-down of SIRT6 decreases the expression of p-ATM and p-Chk2. In addition, SIRT6 overexpression-mediated resistance to doxorubicin was attenuated with co-treatment of the ATM inhibitor KU-55,933 or the PARP inhibitor olaparib. Furthermore, treatment with KU-55,933 or olaparib synergizes with the anti-cancer effect of doxorubicin in both control osteosarcoma cells and osteosarcoma cells with induced overexpression of SIRT6. In line with our results, inhibition of PARP
via knock-down of PARP1 or PARP inhibitor sensitized osteosarcoma cells and Ewing sarcoma cells to radiation or chemotherapeutic drugs [
31,
51,
52]. In contrast, knock-down of SIRT6 increased viability of breast cancer cells treated with trastuzumab [
13]. However, despite controversial reports on the role of SIRT6 as a therapeutic target of human cancer, our results indicate an important role of SIRT6 in the treatment of osteosarcoma in blocking of the SIRT6-mediated DNA repair pathway synergizes with the anti-cancer effect of the genotoxic anti-cancer agent doxorubicin. Furthermore, although a novel small molecular inhibitor of SIRT6, OSS_128167, suppressed
in vivo growth of diffuse large B-cell lymphoma cells [
19], the study of well-established therapeutic agents targeting SIRT6 have been limited [
38], our result suggests that using of an agent targeting the DNA damage repair pathway such as a PARP inhibitor might be helpful for patients in the poor prognostic group of osteosarcoma patients which express high levels of SIRT6. In addition, when considering the reports that elevated expression of SIRT6 is associated with poor prognosis of human cancers, agents which block the DNA damage repair pathway might be useful for the treatment of other human cancer expressing high levels of SIRT6.
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