IntroductionTriple-negative breast cancer (TNBC) represents a distinct subtype of breast cancer which is defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression.1,2,3 Although comprising only 12–17% of total breast cancers, this subtype is the most aggressive, characterized by rapid growth, poor differentiation, drug resistance, high recurrence rates, and significant metastasis capability.4 Due to a lack of specific targets for therapy, the clinical therapeutic approaches for TNBC mainly include surgery, radiation and chemotherapy.5 For a portion of TNBC patients harboring BRCA1/2 germline mutations or with PDL1-positive expression, chemotherapy combined with poly (ADP-ribose) polymerase (PARP) inhibitors or immune checkpoint inhibitors is used in clinical treatment. However, this combination therapy has neither significantly improved the therapeutic efficacy for TNBC nor notably reduced the toxic side effects.6 Therefore, exploring the molecular pathways driving the development of TNBC and identifying specific therapeutic targets remains an urgent and challenging task in TNBC research.Increasing evidence reveals that non-coding RNAs (ncRNAs), which include circular RNAs (circRNAs) and long non-coding RNAs (lncRNAs), are capable of encoding functional polypeptides.7,8 The proteins encoded by ncRNAs contribute significantly to the regulation of both physiological and pathological states.9,10,11 In TNBC, some micropeptides encoded by ncRNAs have been identified as being involved in some critical oncogenic processes, including tumor growth, metastatic spread, and the development of therapeutic resistance.12,13,14 These suggest that the coding function of ncRNAs offers a valuable resource for discovering TNBC-specific therapeutic targets. Additionally, canonical ORFs typically begin with the AUG start codon. However, many coding products in eukaryotic cells use non-AUG codons (such as CUG, UUG, GUG, ACG, AUA, and AUU) for translation initiation.7,15,16,17 These products encoded by non-AUG start codons are worthy of our attention. A prominent illustration of this phenomenon is the oncoprotein c-Myc. Its synthesis commences from a CUG initiation codon located within the transcript of the MYC oncogene,18 and it fulfills essential functions in both the initiation and progression of diverse cancer types.Amplification of the MYC gene is observed in 60% of TNBC cases, which is higher than in other subtypes of breast cancer and indicates a poor prognosis.19 Its encoded protein, c-Myc, significantly promotes TNBC cells to proliferate, migrate, invade, and resist to drug. Therefore, targeting c-Myc is a viable strategy for TNBC treatment.20,21 Several small-molecule inhibitors targeting c-Myc, such as C1572 and MYCi975, have been developed and shown efficacy in pre-clinical or clinical tails of TNBC.22,23 However, long-term use of these inhibitors results in systemic toxicity due to the inhibition of c-Myc physiological functions.24 Thus, further investigation into the specific mechanisms by which c-Myc promotes TNBC is necessary. In cell proliferation, c-Myc is tightly regulated by mitotic signals25 and promotes cell to cross the R checkpoint in the late G1 phase by enhancing the transcription of Cyclin D1/D2 and suppressing the transcription of p15, p16, and p21.26,27 At this stage, with the shrinking of mitotic signals, the T58 and T244 sites of c-Myc are phosphorylated by GSK-3β (glycogen synthase kinase 3β), which are then specifically recognized by FBW7 (F-box and WD repeat domain containing 7) and recruited to the SCF (Skp1-Cullin-F-box-protein) complex for ubiquitination and degradation.28,29,30 In several types of cancer, due to the deletion, mutation and silencing of FBW7 gene, c-Myc is highly expressed.31,32 However, in clinical breast cancer32 and breast cancer cell lines,33 the frequency of FBW7 gene mutation is very low. Although one study showed that the probability of FBW7 gene silencing by promotor methylation is approximately 50% in breast cancer, it only included three cell lines of breast cancer and four samples of primary breast cancer.34 Therefore, the expression level of FBW7 in TNBC and the mechanisms underlying c-Myc overexpression in TNBC remain unclear and warrant further investigation.In this study, to identify specific biomarkers and therapeutic targets for TNBC, we focused on ncRNA-encoded products by removing the restriction that the start codon must be AUG. Consequently, we discovered a noveland unannotated small peptide, named 66CTG, which is encoded by lncRNA CDKN2B-AS1 overexpressed in TNBC using a CUG start codon. This peptide enhanced the transcription of Cyclin D1 by stabilizing c-Myc, thereby promoting cell proliferation and the tumor growth of TNBC. In 89 samples of clinical TNBC, 66CTG was highly expressed, and the expression level of this peptide was positively correlated with that of c-Myc and Cyclin D1. We further demonstrated that 66CTG stabilized c-Myc through competitive interaction with FBW7α during the late G1 phase, while FBW7α mediated the ubiquitination and degradation of 66CTG by recognizing its CPDS56/S60 motif. Our work not only identified a new and potential biomarker and target for TNBC diagnosis and therapy, but also elucidated the mechanisms of c-Myc overexpression in TNBC. This may benefit the treatment of TNBC, especially for those with high expression levels of 66CTG, c-Myc, and Cyclin D1.ResultsA novel peptide 66CTG encoded by CDKN2B-AS1 promotes TNBC cell proliferationIt is reported that LncRNA-encoded proteins have a crucial role in the progression of many kinds of cancer.35 In order to find specific diagnostic and therapeutic targets for TNBC, we focused on the coding functions of ncRNAs. According to a report on ribosome sequencing of breast cancer cells, CDKN2B-AS1 is one of the lncRNAs with coding potential.36 This lncRNA was significantly overexpressed in clinical breast cancer tissues compared with adjacent normal tissue (Supplementary Fig. 1a–c). Notably, in TNBC, this lncRNA showed significant upregulation (Fig. 1a and Supplementary Fig. 1c, d) by using bc-GenExMiner v5.237 and BCIP,38 and was associated with poor prognosis (Supplementary Fig. 1e) by using cBioPortal.39 In order to study the coding function of CDKN2B-AS1, we utilized ORFfinder40 to predict potential ORFs of CDKN2B-AS1 (NR_003529.4) with a specific screening criterion (including: nucleotide length more than 150 nt, exclusion of ATG as the start codon, and non-inclusion of nested ORFs), and identified 19 ORFs in total (Supplementary Table 3). Using SmProt,41 we screened for unannotated coding products of CDKN2B-AS1 (NC_000009.12) that were confirmed by ribosome sequencing. The specific criterion included excluding ATG as the start codon, using data from ribosome RNA sequencing, requiring an amino acid length of more than 50 aa, and the presence of translation initiation sequencing (TISeq) data. As a result, we identified 9 unannotated small peptides translated from CDKN2B-AS1 (Supplementary Table 4). We further conducted overlapping alignment analysis on the results obtained from ORFfinder and SmProt, ultimately identifying ORF1, which translates a 66-amino-acid peptide with CUG as the start codon (Fig. 1b). Then, we cloned ORF1 with a C-terminal 3×Flag tag fusion into the pCDH plasmid and confirmed that ORF1 indeed utilizes CUG to begin encoding a small peptide (Supplementary Fig. 1f). We named this peptide 66CTG.Fig. 166CTG encoded by CDKN2B-AS1 promotes TNBC cell proliferation. a CDKN2B-AS1 expression levels in non-TNBC (n = 578) and TNBC (n = 87) clinical samples from the TCGA database were analyzed using bc-GenExMiner v5.0. b Illustration of the screen process of ORF1 obtained from ORFfinder and SmProt, as well as the localization of ORF1 on CDKN2B-AS1. c Overexpression of 66CTG-3×Flag was detected in MDA-MB-231 cells via Western blotting. d SRB assays assessed the effect of 66CTG-3×Flag overexpression on the proliferation of MDA-MB-231 cells (n = 10). Error bars show the mean ± SD, ***P