High BTG2 expression was associated with favorable platinum-based chemotherapy

To investigate whether these in vitro observations have clinical relevance, the response to platinum-based chemotherapy and prognosis of patients were analyzed according to primary tumor expression level of BTG2. At first, BTG2 expression status was screened in various normal and paired tumor tissues with immunohistochemistry (IHC) to validate BTG2 antibody and select proper type of cancer for further study. As previous reports using same kind of BTG2 antibody for IHC, strong and specific expression of BTG2 in purkinje cells of cerebellum (Figure S7A) and same staining protocol was applied for further IHC. In head and neck region, basal BTG2 expression was observed in stratified squamous epithelium (Figure S7B), while no or weak BTG2 expression was observed in squamous cell carcinoma of head and neck (Figure S7C, D). In thyroid gland, normal follicular epithelium (Figure 7SE) and papillary thyroid cancer cells (Figure S7F, G) showed no BTG2 expression. In lung tissue, normal alveolar epithelium showed no BTG2 expression (Figure S7I.), while airway epithelium showed moderate BTG2 expression (Figure S7H). In squamous or adenocarcinoma of lung, more decreased BTG2 expression was observed compared to that of normal upper airway epithelium despite heterogeneous expression (Figure S7J). BTG2 expression of breast gland (Figure 7SK) and liver (Figure 7SN) is relatively higher as previous reports, while that of breast carcinoma (Figure 5SL, M) and hepatocellular carcinoma (Figure S7O, P) was significantly reduced as previous reports. In gastric epithelium, strong BTG2 expression was observed (Figure S7Q), while in colonic epithelium, significant BTG2 expression was not observed (Figure S7T.). BTG2 expression of gastric cancer (Figure S7R, S) was more variable compared to that of colon cancer (Figure S7U, K.). In kidney tissue, tubular epithelium showed strong BTG2 expression as previous report (Figure S7W), while renal clear cell carcinoma showed no BTG2 expression (Figure S7X, Y).

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Figure S7. BTG2 expression in various normal and cancer tissues by immunohistochemistry.

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For further study, lung cancer was selected since Bcl-XL is studied as important mediator for survival of lung cancer [68] and platinum based-chemotherapy is standard chemotherapy regimen in advanced NSCLC. Based on our IHC observation, BTG2 expression of normal airway epithelium was decreased in their counterpart, squamous cell carcinoma (SCC). In addition, while prognosis of adenocarcinoma was significantly improved after discovery of different genetic background and therapeutic advances with target therapy such as EGFR, ALK tyrosine kinase inhibitors, that of advanced SCC of lung is median OS of less than 1 year. After all, improving efficacy of platinum based chemotherapy was still important in this histologic type of lung cancer. Therefore, among all advanced NSCLC patients treated with chemotherapy in our institution, a total of 70 patients only with squamous cell histology who received platinum-based doublet chemotherapy as first-line palliative treatment were analyzed. BTG2 expression was investigated with IHC and tumors with H-scores equal or higher than median value (6) were classified as high expression (Figure8).

Interestingly, BTG2 expression was significantly lower in recurrent disease, whereas the base line characteristics had no correlation with BTG2 expression (Table 1). BTG2 expression level of tumors with progressive disease (PD) after platinum-doublet chemotherapy was significantly lower in that of controlled tumors after chemotherapy (Figure 7I). The median progression-free survival (PFS) and overall survival (OS) of all patients were 6.1 and 8.0 months, respectively. The median PFS and OS of patients with high expression of BTG2 were significantly longer than that of patients with low expression (6.7 vs. 3.2 months, p=0.002, Figure 8A and 10.7 vs. 6.0 months, p=0.039, Figure 8B). In addition to univariate analysis (Table 2), high BTG2 expression was independently associated with favorable PFS (hazard ratio=0.28, p<0.0001) and OS (hazard ratio=0.42, p=0.006) in multivariate analysis, (Table 3). Taken together, these data suggested that expression status of BTG2 in NSCLC could be potential prognostic biomarker for platinum-based chemotherapy.

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Figure 7. BTG2 expression measured by immunohistochemistry in advanced squamous lung cancer treated with platinum-based doublet chemotherapy and H-score according to therapy response. (A) BTG2 expression in normal bronchial airway epithelium. (B) No expression of BTG2 in normal alveolar epithelium. No (C), weak (D), moderate (E), and strong (F) expression of BTG in squamous lung cancer samples. (G) Note heterogeneous expression pattern of BTG2 in different area of same lung cancer tissue. (H) BTG2 expression is observed in keratinized area. (I) Significant lower BTG2 expression was observed in tumor sample from patients experiment disease progression after platinum-based doublet chemotherapy.

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Figure 8. Progression-free and overall survival according to the BTG2 expression. Kaplan–Meier progression-free (A) and overall survival curve (B) according to the BTG2 expression. Longer PFS and OS was observed in high BTG2 expression group.

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Figure 9. Proposal function of BTG2 in cancer cell death via regulating Bcl-XL mRNA stability through mRNA binding protein hnRNP C.

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Table 1. Patients characteristics according to the BTG2 expression Characteristics Total N (%) Low BTG2

N, number; PS, performance status; ECOG, Eastern Cooperative Oncology Group.

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PFS, progression-free survival; OS, overall survival; PS, performance status; ECOG, Eastern Cooperative Oncology Group.

４６ PFS, progression-free survival; OS, overall survival; HR, hazard ratio; CI, confidence interval; PS, performance status; ECOG, Eastern Cooperative Oncology Group.

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Figure S8. BTG2 and BTG1 mRNA expression pattern in cancer and paired normal tissue from TCGA database.

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4. DISCUSSION

It has been demonstrated that BTG2, as a founding member of anti-proliferative (APRO) gene family including six different genes in vertebrates (TOB1,TOB2, BTG1 BTG2^/TIS21/PC3, BTG3 and BTG4), can arrest cells at G1/S, G2/M transition, increase apoptosis upon DNA damage, inhibit expansion of thymocytes and involve in the development and differentiation of nerve cells and hematopoietic cells. Transcriptional regulation and post-translational regulations of BTG2 have been well understood from several studies. While, the functional mechanism of BTG2 is not fully understood and further investigation is required. Since BTG2 protein, itself, has no catalytic activity, it is important that finding interacting proteins and understanding the function of interacting partners in the context of cellular phenotype mediated by BTG2. Several binding proteins have been discovered by various methods. PRMT1 as the one of the BTG2 interacting proteins was most actively validated and investigated in several studies. In recent study, the importance of BTG2 and PRMT1 complex in normal B cell differentiation was discovered in BTG2 knock mouse model.

However, in our study, Bcl-XL mRNA regulation by BTG2 did not seem to be dependent on PRMT1.

Another important interacting protein to BTG2 is CNOT7 (caf1a) which has human mRNA deadenylase activity. 3D crystal structure between BTG2 and CNOT7 was discovered along with important amino acid residue of BTG2 for binding CNOT7 [69]. Nonetheless, further studies are required to elucidate whether BTG2 can regulate CNOT7 activity due to the opposite results. TOB, sharing same N terminal A, B box lesion which is important for binding CNOT7, can localize target mRNA directly through the PABP interacting PAM2 motifs located on its C-terminal and can mediate target mRNA deadenylation and degradation. While since BTG2 has no PAM2 motifs and no known mRNA binding activity, another binding partner which has RNA binding ability is needed to explain how BTG2 can mediate CNOT7 activity. According to recent study, BTG2 can interacts with cytoplasmic poly(A) binding protein (PABPC)1 and stimulates CAF1 deadenylase activity [51].

Therefore, BTG2 was suggested general activator of mRNA deadenylation and degradation. However, as our result (Figure S2), BTG2 could decrease the stability of not in general but in more specific target mRNAs. Recently, the molecular mechanism that TOB can enhance cMyc mRNA degradation specifically not by its PAM2 motifs but by interaction with another RNA binding protein, CPEB, was reported. Therefore, we suggested hnRNP C, known RNA binding protein, as one of the potential mediators interacting BTG2 and specific mRNA in this study. HnRNP C, a member of the hnRNP family, binds to nascent RNA transcripts and affects pre-mRNA splicing, export, and translation [16–

20]. Although hnRNP C was reported to affect mRNA stability, the molecular mechanism is not to be fully understood. According to previous studies, baseline hnRNP C may have a role of maintaining mRNA stability. However, as our results, when cells are stressed upon DNA damage and BTG2 is upregulated, hnRNP C could lose its protective role of maintaining mRNA stability by binding to

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increased BTG2. Therefore, considering only in the context of cancer cells and target mRNA of anti-apoptotic gene, Bcl-XL, BTG2 and hnRNP C could have a role as tumor-suppressor and oncogene, respectively. Interestingly, overexpressed hnRNP C in tumor tissue has been suggested as oncogene and negative regulation of p53 activity by hnRNPC was reported through the interacting with lncRNA SNHG1 [70]. These data can suggest the hypothesis that basal hnRNP C can contribute to maintain cellular homeostasis by repressing p53 activity and BTG2, one of its downstream genes and when p53 activity sustained stressful condition, hnRNP C can be an indicator of specific mRNA for regulation by BTG2. Therefore, further study will be interesting to investigate whether BTG2, as a downstream of p53, can regulate mRNA stability of another genes discovered by chip-seq using hnRNP C antibody and elucidate their physiologic and pathologic meanings.

In addition to anti-proliferative function, the involvement of BTG2 in process of cellular survival and death has been suggested. In hippocampal neurons, BTG2 and Bcl6 were non-biasedly identified to contribute to synaptic NMDAR-dependent neuroprotection by gene expression microarray analysis after NMDA receptor activation [27]. Stereotaxic delivery of activity-regulated Inhibitor of death genes including BTG2 to the hippocampus by recombinant adeno-associated viruses showed protection in vivo against seizure-induced brain damage [28]. BTG2 -/- embryonic stem cell showed decreased cell cycle arrest and increased apoptosis after adriamycin treatment [4]. Our group also reported that BTG2 promoted the repair of DNA damage and reduced apoptosis by blocking the damage signal from p-ATM to p-Chk2-p53 [71]. However, like as another p53 downstream gene, BTG2 can also contribute cell death under irreversible damage status. In fact, previous studies reported that BTG2 can enhance cell death during various kind of stimulation. For example, BTG2 augmented EGF-induced U937 cell death [31], adriamycin-induced HeLa cell death [32], H2O2-induced cardiomyoblast cell death [33]. Moreover, during the formation of free digits in the developing limbs, Btg/Tob gene family was up-regulated and Btg2 overexpression induced oxidative stress, arrest of cell cycle progression, senescence and caspase-mediated apoptosis in the regressing interdigits [34]. Likewise, overexpressed BTG2 showed shifting p53-mediated EJ cell senescence to cell death [35]. In addition, C-reactive protein (CRP)-induced apoptosis of monocytes was not observed in monocyte from BTG2-knockout male C57BL/6 mice even though p53 activation was occurred in BTG2-KO monocytes after CRP treatment [22].

Nonetheless, the mechanism of enhanced cell death by BTG2 has not been fully elucidated. In this study, one of the potential explanations that BTG2 can regulate Bcl-XL mRNA level by mediating interaction between hnRNP C and mRNA deadenylase, CNOT7 was suggested. After treatment of cytotoxic agents, rapidly upregulated BTG2 could influence on determination of cell fate by cooperating various cell death inducing signaling. In addition, among BTG2 interacting proteins discovered by protein chip array, AlkB homolog 7 (ALKBH7), showing strongest binding intensity,

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has been reported as having pivotal role in DNA-damaging agent-induced programmed necrosis by triggering the collapse of mitochondrial membrane potential [72]. Considering that some of BTG2 protein can localize in mitochondria [33, 73] and its localization was increased during necrosis [33], further study about relationship between BTG2 and ALKBH7 in the process of programmed necrosis will be interesting for elucidating another molecular mechanism of enhanced cell death by BTG2.

Despite molecular function of BTG2 has not been fully elucidated, its tumor suppressive role such as inhibition of proliferation, invasion and metastasis and promoting cancer cell death after chemotherapy has been suggested in several studies. Among their APRO gene family, BTG2 was the only representative downstream gene of p53 and target of representative onco-miRNA, miR-21. In addition, in several cancer types, TCGA database confirmed that BTG2 expression was relatively decreased compared to normal counterpart and showed the expression pattern of BTG1 was not identical to that of BTG2 (Figure S6) [74]. In several datasets of breast cancer before TCGA project era, low BTG2 expression was correlated with increased lymphatic and blood vessel invasion and metastasis and high local and metastatic recurrence rate and decreased overall survival [37]. In TCGA cohort including 998 patients data, adverse prognosis of low BTG2 expression in breast cancer was revalidated with decreased overall survival and metastasis-free survival [41]. Although BTG2 expression was decreased in cancer, cancer tissues and cells still express basal BTG2, which is regulated by p53 dependent or independent manner. Despite few recurrent missense mutations of BTG2 were reported in TCGA data of lymphoma, in most of malignancy, recurrent genomic and epigenomic alteration suggesting tumor suppressive roles in carcinogenesis were not reported.

Moreover, considering the contradictory report that increased BTG2 expression was correlated with invasiveness of bladder cancer and worse prognosis [44], BTG2 can be oncogene or tumor suppressor gene in different tissues and cellular context during carcinogenesis, like as TGF-beta. Therefore, despite the interaction with CNOT7 has been demonstrated as major molecular mechanism of anti-proliferative function of BTG2 in cell culture system [51], more delicate studies will be required to understand above contradictory situations in variable disease models. From the global RNA sequencing on 106 formalin-fixed, paraffin-embedded prostatectomy samples from 100 patients, BTG2 was discovered as a member of 24-gene signature panel for predicting biochemical recurrence [75]. High expression of BTG2 was associated with low recurrence rate in that study. In our study, the rate of high BTG2 expression in recurrent disease was also significantly lower than that of high BTG2 expression in primary metastatic disease. Since high recurrence rate after radical surgery indicates that tumor has acquired metastatic potential relatively earlier, BTG2 could interfere with tumor metastasis regardless of its anti-proliferative function. In metastatic process, acquiring migration and invasion ability was important and BTG2 has been suggested to inhibit cell migration [40, 76, 77]. However, since survival and proliferation after micro-colonization has been known as rate limiting step of

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metastasis, BTG2 could also have a role for inhibiting tumor cell survival in stressful metastatic environment by enhancing cell death.

Although prognostic implication of BTG2 and another APRO family proteins expression was reported in different types of tumors [74], their role as prognostic factor for chemotherapy was not fully elucidated yet. In previous study, suppression of BTG2 in breast cancer resulted in disease progression through activation of HER pathway, and shBTG2/H-RasV12 tumor cells was more sensitive to lapatinib (HER2/HER3 inhibitor) treatment compared to H-RasV12 tumor cells in mouse xenograft model [37]. However, predictive significance of BTG2 expression level was not validated in tumor samples of patient treated with lapatinib in that study. To the best of our knowledge, this study is the first report to analyze relationship between BTG2 expression of tumor tissue and response of chemotherapy. Only 7.3% of patients with high BTG2 expression were experienced disease progression after platinum-based doublet chemotherapy, while 51.7% of patients with low BTG2 expression showed disease progression (data not shown). In multivariate analysis, high BTG2 expression was independent favorable prognostic factor for PFS and OS. This study is the first report to show that BTG2 expression could be a potential prognostic factor for platinum-based doublet chemotherapy in non-small cell lung cancer patients, as especially with squamous cell histology.

However, further validation studies are needed because the result was from a retrospective analysis of a single institution including relatively small number of patients.

In summary, BTG2 regulated mRNA level of anti-apoptotic gene, Bcl-XL via its well-known binding partner, mRNA deadenylase, CNOT7. In this process, mRNA binding protein, hnRNP C was suggested as mediator between BTG2-CNOT7 complex and 3' UTR of Bcl-XL mRNA, as newly discovered interacting partner of BTG2 in this study. Bcl-XL is decreased after platinum treatment and BTG2 expression can augment platinum-induced cell death. In addition, high BTG2 expression was associated with increased response to the platinum-based chemotherapy and favorable overall survival in advanced squamous cell lung cancer patients.

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