LncRNA-encoded peptides in cancer

Abstract

Long non-coding RNAs (lncRNAs), once considered transcriptional noise, have emerged as critical regulators of gene expression and key players in cancer biology. Recent breakthroughs have revealed that certain lncRNAs can encode small open reading frame (sORF)-derived peptides, which are now understood to contribute to the pathogenesis of various cancers. This review synthesizes current knowledge on the detection, functional roles, and clinical implications of lncRNA-encoded peptides in cancer. We discuss technological advancements in the detection and validation of sORFs, including ribosome profiling and mass spectrometry, which have facilitated the discovery of these peptides. The functional roles of lncRNA-encoded peptides in cancer processes such as gene transcription, translation regulation, signal transduction, and metabolic reprogramming are explored in various types of cancer. The clinical potential of these peptides is highlighted, with a focus on their utility as diagnostic biomarkers, prognostic indicators, and therapeutic targets. The challenges and future directions in translating these findings into clinical practice are also discussed, including the need for large-scale validation, development of sensitive detection methods, and optimization of peptide stability and delivery.

Introduction

Long non-coding RNAs (lncRNAs) were initially defined as a class of RNAs longer than 200 nucleotides that do not encode proteins [1,2,3]. Initially regarded as "noise" from genome transcription, lncRNAs have increasingly been shown to play important roles in the regulation of gene expression at the epigenetic, transcriptional, and post-transcriptional levels [4,5,6,7,8]. They have also been found to be intimately linked with the occurrence and progression of a spectrum of human diseases, with a particularly significant association observed in the context of cancer [9,10,11,12,13,14,15,16].

With the advancement of proteomics and translation technologies, it has been discovered that some lncRNAs have the ability to encode small peptides or micropeptides [17,18,19,20,21]. These peptides are encoded by small or short open reading frames (sORFs) and can range in length from tens to over a hundred amino acids (aa) [22,23,24,25]. Furthermore, there is growing evidence that peptides derived from lncRNAs have specific biological functions and can act as oncogenic drivers or tumor suppressors [26,27,28,29]. They play important roles in various cancer processes, such as transcriptional regulation, post-transcriptional regulation, translation and post-translational regulation, signal transduction, and cancer metabolism [8, 30,31,32,33,34].

Despite numerous reviews on lncRNA-encoded peptides published in the past, most are outdated as they were released several years ago [26, 35, 36]. LncRNA-encoded peptides have emerged as a hot topic in recent years, with many new discoveries identifying novel lncRNAs that encode for peptides and their significant functions in cancer, along with new regulatory mechanisms. In this review, we review the methods for detecting lncRNA-encoded peptides, comparing their differences. Additionally, we systematically summarize the lncRNAs known to encode peptides, their roles and mechanisms in cancer, with a particular focus on regulatory mechanisms that have not been systematically reviewed before. Finally, we explore the potential applications of these peptides. Overall, this review aims to provide a comprehensive and systematic resource for future researchers in the field of lncRNA-encoded peptides.

Biogenesis and detection of lncRNA-encoded peptides

Biogenesis of lncRNA-encoded peptides

The biogenesis of peptides encoded by lncRNAs is a multifaceted process that encompasses the transcription of lncRNAs by RNA polymerase II, followed by their maturation, which includes the addition of a 5' cap (m7G) and a polyadenylated tail. After undergoing alternative splicing, these transcripts are exported to the cytosol, where they harbor the potential to be translated into peptides [37, 38]. Notably, the work of Yu et al. has shown that DNA damage can prompt ribosomes to associate with the internal ribosome entry site (IRES) region within the lncRNA CTBP1-DT. This interaction bypasses the inhibitory effects of upstream open reading frames (uORFs) and triggers the cap-independent translation of a novel microprotein termed DNA damage-upregulated protein (DDUP) [39]. Some researchers have raised skepticism, suggesting that mere RNA structure and ribosome binding are not definitive indicators of a transcript's translatability, making the elucidation of their translational mechanisms a challenging endeavor [40]. Moreover, the translation of lncRNA-encoded peptides, despite their brevity, is also contingent upon the presence of open reading frames (ORFs). As we know, ORFs are nucleotide sequences that span from a start codon to the nearest stop codon within a nucleotide sequence. A translatable ORF is typically recognized as the coding DNA sequence (CDS) on an mRNA that gives rise to its principal protein product. In mRNA, codons-triads of nucleotides-correspond to specific amino acids, with the AUG codon typically serving as the start signal and UAA, UAG, and UGA being the traditional stop codons in eukaryotic organisms. sORFs, typically less than 100 codons in length [41], are sometimes extended to include sORFs of 200–250 codons as described in various studies [42,43,44]. These sORFs are distinguished by their size from all other ORFs, but not all sORFs are translated or are indeed translatable. Identifying any ORF within genomic DNA is straightforward, but differentiating between coding and non-coding sORFs is more complex. Most de novo gene prediction algorithms differentiate coding from non-coding sequences by recognizing genomic patterns indicative of features (such as start codons, stop sites, splice junctions, promoters, and polyadenylation signals) or by analyzing intrinsic DNA sequence properties (including codon usage bias, nucleotide composition, and in-frame hexamer frequency) [45, 46]. However, these algorithms are not optimized for sORFs, as they focus on longer ORFs with a higher prevalence of these features [47, 48]. As a result, many gene annotation tools overlook ORFs shorter than 100 codons, often dismissing them as insignificant [49, 50]. However, with the advancement of technology, the challenge has begun to be addressed effectively. Several approaches have been taken to systematically predict sORFs with coding potential (Table 1). For example, Lin et al. presented PhyloCSF, a new computational method examining evolutionary conservation of a sORF across species [51]. Camargo et al. employed RNAsamba, a sophisticated bioinformatics tool that predicts the coding potential of RNA molecules from sequence information alone [52]. Utilizing a neural network-based algorithm, RNAsamba identifies patterns that distinguish coding transcripts from non-coding ones, offering a promising avenue for sORF prediction [52]. These resources have significantly expanded our understanding of the coding potential within sORFs.

Table 1 Tools for predicting sORFs

While the current research has shed preliminary light on this topic, it is clear that the regulatory mechanisms by which ORFs within lncRNAs are translated into peptides are not yet fully understood. There is a pressing need for future studies to delve deeper into these mechanisms, providing a more comprehensive understanding of the translation process of lncRNA-encoded peptides. To date, a considerable number of lncRNA-encoded peptides have been identified. In response to this growing body of information, several databases have been developed and are now accessible to researchers seeking data related to lncRNA-encoded peptides (Table 2). These databases serve as valuable repositories, enabling investigators to rapidly access information on lncRNA-encoded peptides of interest and providing a rich resource for the scientific community.

Table 2 Databases for lncRNA-encoded peptide

Detection of lncRNA-encoded peptides

With technological progress, there are several methods available to predict and validate the coding potential of translated small open reading frames, including bioinformatics, ribosome profile sequencing (Ribo-seq), reporter tag, epitope tagging, antibody-based validation and mass spectrum (MS) (Fig. 1). These methods are often combined with protein detection procedures such as western blotting, immunocytochemistry, or immunoprecipitation steps to verify the translation of sORFs [71, 72].

Fig. 1

Detection methods for the coding potential of lncRNAs. A Prediction of short open reading frames (ORFs) within lncRNAs. B Sucrose density gradient separation to detect ribosome enrichment on lncRNAs. C Detection of GFP translation using a GFP fusion with a mutated start codon within a lncRNA ORF. D Integration of a tag at the lncRNA ORF site using gene editing technology to assess the expression of the tagged protein. LHA, left homologous arm; RHA, right homologous arm. E Detection of intracellular lncRNA-encoded peptides using antibodies raised against synthetic peptides. F Mass spectrometry identification of peptide expression. Image created with BioRender.com

Ribosome profile sequencing

Ribosomes are one of the fundamental components of the translation process in eukaryotic cells [73, 74]. Undoubtedly, Ribo-seq is one of the most promising scientific evidences that could point towards answering which lncRNAs are capable of encoding peptides [75,76,77]. It is an emerging technique that offers a glimpse into protein synthesis through the deep sequencing of RNA fragments protected by ribosomes [78, 79]. The core Ribo-seq methodology employs RNase I to digest unprotected, single-stranded RNA, leaving behind ribosome-protected fragments (RPFs). These fragments are then isolated, sequenced, and mapped to the genome, allowing for the assembly of transcripts and the discovery of novel sORFs with coding potential [80, 81]. However, this technique faces challenges, including reliance on next-generation sequencing, which can introduce false positives due to sequencing quality and depth. Additionally, Ribo-seq may not capture all sORFs due to their variable expression across conditions, stages, and tissues [82]. It also requires a significant starting material, such as 10 million cells, to meet sequencing RNA requirements [83]. Notably, Xiong et al. have recently developed an ultrasensitive Ribo-seq method, termed Ribo-lite, which can be applied to ultra-low input oocytes, even single oocytes [84]. Zou and colleagues have successfully applied this method to investigate the translational regulation during human oocyte maturation and early embryonic development [85]. However, the applicability of this method for single-cell translational regulation analysis in other cell types has yet to be reported. Furthermore, In the field of ribosome profiling, the specificity of RNase I for single-stranded RNA is a well-established fact. Notably, this specificity also introduces a potential pitfall. Given that RNase I cannot target double-stranded RNA regions, such as those found in the stem-loop structures of microRNA precursors [86], there exists a risk of inadvertently generating pseudo ribosomal footprints (pseudo-RPFs) from these complex structures. While it is true that double-stranded RNAs are not commonly encountered within the cellular milieu, their presence, albeit rare, cannot be entirely discounted. This rarity does not eliminate the possibility that they might contribute to false-positive signals in ribosome profiling assays, thereby complicating the interpretation of the resulting data. Consequently, researchers must exercise caution when analyzing ribosome profiling data to ensure that the observed ribosomal footprints are indeed indicative of active translation events rather than artifacts stemming from the presence of double-stranded RNA structures. The short length of RPFs, approximately 30 nucleotides, complicates the differentiation of transcript isoforms resulting from alternative splicing [87]. It's important to note that ribosomal occupancy does not automatically indicate translation of the ORF [88], as it has been shown that start codons can regulate translation attenuation of a downstream ORF, mRNA availability through nonsense-mediated decay [40].

Reporter tags

The coding potential of sORFs can be evaluated by fusing them with reporter tags and then detecting the signal through immunoblotting or microscopy [34]. Specifically, a FLAG/HA-tag system is genetically engineered to be cloned immediately preceding the stop codon of the sORF under investigation. This fusion sequence, which includes the FLAG/HA-tag, is subsequently inserted into a plasmid vector, which then serves as the template for in vitro cell transfection. Upon transfection into the target cell line, the expression of the FLAG/HA-tagged micropeptide is quantified using western blotting and immunofluorescence assays with anti-FLAG/HA tag antibodies [89, 90]. As an alternative approach, sORFs derived from lncRNAs can be fused to the N-terminus of GFP vectors. The expression levels of the GFP-tagged micropeptides are then evaluated using western blotting, fluorescence microscope or immunofluorescence assays with anti-GFP antibodies, providing a visual and quantitative assessment of the micropeptide's presence and distribution within the cells [91,92,93]. It should be noted that inserting a reporter tag internally or at the N-terminus of micropeptide carries the risk of disrupting the protein's function, as well as its intramolecular interactions and folding [25, 94,95,96]. This possibility underscores the need for careful experimental design and the interpretation of results with an awareness of potential artifacts introduced by the tagging process.

Epitope tagging

Epitope tagging is a method that incorporates a recognizable epitope tag into a protein sequence, allowing for specific and sensitive detection of sORF using available antibodies [97]. In the context of sORFs encoded within lncRNAs, the CRISPR-Cas9 system presents a powerful tool for the site-specific introduction of epitope tags. The CRISPR-Cas9 system can be programmatically designed to target the stop codon of the lncRNA locus in question within the genome of the cells. By designing a guide RNA that directs the Cas9 nuclease to the desired location, researchers can introduce an epitope tag at the stop codon, effectively tagging the sORF for detection purposes. Once the epitope tag is integrated into the lncRNA locus, the expression of the resulting micropeptides can be assessed using Western blotting, fluorescence microscope or immunofluorescence assays with corresponding anti-tag antibodies [98, 99]. This approach effectively validates the coding potential of lncRNAs. However, several challenges must be considered when using epitope tagging. Firstly, the insertion of an epitope tag has the potential to disrupt the native structure and function of the protein, which could lead to misinterpretation of the protein's behavior in cellular assays. Secondly, the efficiency of tag integration can vary, and off-target effects may occur with the CRISPR-Cas9 system, potentially tagging unintended sites. Additionally, the detection of the tagged protein relies on the availability and specificity of antibodies, which may sometimes result in high background signals or false negatives.

Antibody-based validation

Antibody-based validation is a critical process in the identification and characterization of sORF-encoded polypeptides (SEPs). This approach involves the synthesis of antibodies that are specific to the predicted sequences of SEPs, allowing for the detection and confirmation of these peptides within complex cellular environments through western blotting [25]. For example, Faure et al. employed a monoclonal antibody directed against the Gau protein, a peptide approximately 100 amino acids long, to confirm the existence and functionality of the Gau protein [100]. Nonetheless, developing antibodies against SEPs presents significant challenges, primarily due to the small size of sORFs. Additionally, detecting SEPs can be problematic when they are expressed at low levels, as elevated antibody signals may not be easily discernible [25]. Therefore, ongoing efforts to refine antibody-based validation techniques will be essential for the discovery and characterization of new SEPs and the elucidation of their biological functions.

Mass spectrometry

Mass spectrometry is a sophisticated analytical technique that has proven indispensable in the field of proteomics, offering unparalleled capabilities for the identification and quantification of proteins and peptides, which provide direct evidence of sORFs’ translation into SEPs [88, 101]. This method is often paired with the immunoprecipitation of ORF-GFP fusion peptides, leveraging anti-GFP antibodies to precipitate GFP-tagged SEPs from cell lysates. This approach not only detects unannotated proteins but also confirms the translation of sORFs into peptides, which not only detects unannotated proteins but also verifies the translation of sORFs into peptides [25, 102]. However, while mass spectrometry is adept at peptide detection, it has limitations in identifying SEPs due to their short length and a propensity for producing tryptophan-containing peptides. Furthermore, low-abundance SEPs can be overlooked during sample preparation [76, 88]. Therefore, special attention must be given to the separation and concentration steps of peptides, which are crucial for detecting small and/or low-abundance products in cell lysates [76].

The conditions, difficulty levels, and reliability of the aforementioned methods for detecting lncRNA-encoded peptides are encapsulated in Table 3. It is important to note that affirming the coding potential of an lncRNA necessitates a multifaceted approach, employing multiple methods to ascertain its function and to circumvent the possibility of false positives. This underscores the importance of a rigorous and integrated methodological strategy in validating the biological significance of lncRNA-encoded peptides.

Table 3 Condition, difficulty degree and reliability of methods for detecting lncrNA-encoded peptides

Functions of lncRNA-encoded peptides in cancer

lncRNAs serve multifaceted roles, constructing intricate regulatory systems and engaging in a spectrum of biological activities. While numerous sORFs within lncRNAs and their corresponding short peptides have been detected using the methods previously described, the functional assignments for these peptides remain scarce. Emerging research suggests that the micropeptides derived from lncRNAs could be pivotal in tumorigenesis and tumor progression. In this section, we provide a compilation of lncRNA-encoded peptides that are associated with various cancer-related biological processes (Table 4).

Table 4 Peptides encoded by lncRNA and their functions

Colorectal cancer

Colorectal cancer (CRC), the second most prevalent cancer in women and third in men globally, is a major contributor to cancer-related mortality, accounting for 9.2% of such deaths [133, 134]. The exploration of lncRNA-encoded peptides has unveiled their pivotal role in the molecular intricacies of CRC, influencing its development, progression, and response to treatment (Table 4 and Fig. 2). For example, the HOXB-AS3 peptide, typically down-regulated in colon cancer, can inhibit cancer growth by interfering with PKM splicing and glucose metabolism (Fig. 2A) [33], while the SRSP peptide promotes cancer cell proliferation and metastasis by affecting the splicing of transcription factor Sp4 (Fig. 2B) [105]. The RBRP peptide, upregulated in metastatic CRC, stabilizes c-Myc mRNA by binding to IGF2BP1, enhancing tumor progression (Fig. 2C) [104]. These results underscore the post-transcriptional regulatory potential of lncRNA products in CRC. In the context of tumor metabolism, the overexpression of ASAP boosts ATP synthase activity and mitochondrial oxygen consumption, promoting CRC proliferation (Fig. 2D) [103]. Additionally, pep-AP can modulate CRC's chemotherapy sensitivity by adjusting metabolic pathways, leading to ROS accumulation and apoptosis, which may sensitize cells to treatments like Oxaliplatin (Fig. 2E) [108]. lncRNA-encoded peptides also regulate signaling pathways in CRC. BVES-AS1-201-50aa and MBOP peptides, for instance, activate the Src/mTOR and MEK1/pERK pathways, respectively, to bolster CRC cell viability, migration, and invasion (Fig. 2F–G) [106, 109]. The revelation that E3 ubiquitin ligases MAEA and RMND5A mediate MBOP degradation underscores the complex regulatory networks governing micropeptide metabolism within cells. The FORCP peptide adds another layer of complexity, inhibiting cell proliferation and inducing apoptosis in response to endoplasmic reticulum stress (Fig. 2H) [107]. These findings suggest that lncRNA-encoded peptides could serve not only as diagnostic markers but also as novel targets for therapeutic intervention in CRC, with the potential to improve treatment strategies through a deeper understanding of their mechanisms and regulatory roles.

Fig. 2

The role of lncRNA-encoded peptides in colorectal cancer (CRC). A The peptide HOXB-AS3 encoded by LncRNA HOXB-AS3 interacts with hnRNP A1 to affect PKM mRNA splicing, inhibiting CRC growth and metastasis. B The peptide SRSP encoded by LncRNA LOC90024 interacts with SRSF3 to influence splicing of SP4 mRNA, promoting CRC growth and metastasis. C The peptide RBRP encoded by LINC00266-1 interacts with IGF2BP1 to maintain c-Myc mRNA stability, promoting CRC growth and metastasis. D The peptide ASAP encoded by LINC00467 enhances ATP synthase activity and mitochondrial oxygen consumption by interacting with ATP5A and ATP5C, promoting CRC growth. E The peptide pep-AP encoded by Lnc-AP interacts with TALDO1 to attenuate the pentose phosphate pathway (PPP), inducing apoptosis and drug sensitivity in colorectal cancer cells. F The peptide BVES-AS1-201-50aa encoded by LncRNA BVES-AS1 activates the Src/mTOR signaling pathway, promoting CRC proliferation, migration, and invasion. G The peptide MBOP encoded by LINC01234 interacts with MEK1 to regulate the MEK1/pERK/MMP2/MMP9 axis, promoting CRC proliferation and metastasis. H The peptide FORCP encoded by LINC00675 induces apoptosis and inhibits cell proliferation in colorectal cancer cells under endoplasmic reticulum stress. Image created with BioRender.com

Breast cancer

Breast cancer (BC), projected to have 310,720 new diagnoses and 42,250 deaths in the United States in 2024, is the most prevalent malignancy among women [135, 136]. Within this, triple-negative breast cancer (TNBC), characterized by the absence of progesterone, estrogen, and human epidermal growth factor receptors, presents a particularly aggressive subtype with a lower survival rate and a complex molecular profile [137, 138]. LncRNA-encoded peptides are emerging as significant contributors to BC progression, with the peptide MRP, overexpressed in highly malignant BC cells, promoting invasion and metastasis by stabilizing EGFR mRNA and activating the PI3K pathway by binding to HNRNPC (Fig. 3A) [30]. The lncRNA product LINC00511-133aa enhances invasive properties and stem-like characteristics of BC cells by modulating the wnt/β-catenin pathway (Fig. 3B) [110], while HCP5-132aa is implicated in resistance to adriamycin and can trigger excessive autophagy through the ERK/mTOR pathway, and promote TNBC progression by regulating GPX4-induced ferroptosis (Fig. 3C) [112, 113]. Additionally, ASRPS, a peptide encoded by LINC00908, suppresses tumor angiogenesis by inhibiting the STAT3/VEGF pathway (Fig. 3D) [99], and CIP2A-BP, encoded by LINC00665, suppresses TNBC invasion and metastasis by inhibiting the PI3K/AKT/NF-κB pathway (Fig. 3E) [114]. Another peptide MAGI2-AS3-ORF5 interacts with the extracellular matrix to restrict BC cell viability and migration, though its mechanisms require further investigation (Fig. 3F) [111]. The discovery of these lncRNA-encoded peptides and their roles in BC, especially TNBC, opens new avenues for understanding disease progression and resistance to therapy. Their multifaceted influence on cellular processes suggests potential for targeted interventions. For instance, the modulation of MRP to destabilize EGFR mRNA could be a strategy to combat BC metastasis. Similarly, understanding the mechanisms by which peptides like ASRPS and CIP2A-BP inhibit key signaling pathways could lead to the development of new therapeutics that enhance the efficacy of existing treatments or overcome resistance. The interplay between lncRNA products and the extracellular matrix also presents an opportunity to explore the tumor microenvironment's role in BC progression.

Fig. 3

The role of lncRNA-encoded peptides in breast cancer. A The peptide MRP encoded by LncRNA LY6E-DT regulates EGFR mRNA stability and translation by interacting with HNRNPC, promoting breast cancer metastasis. B The peptide LINC00511-133aa encoded by LINC00511 facilitates β-catenin nuclear translocation to activate the transcription of Bax, c-Myc, and CyclinD1, promoting invasiveness and stem-like properties of breast cancer. C The peptide HCP5-132aa encoded by LncRNA HCP5 inhibits autophagy and ferroptosis to promote breast cancer proliferation and migration. D The peptide ASRPS encoded by LINC00908 inhibits STAT3 phosphorylation, leading to the suppression of VEGF transcription and thus inhibiting tumor metastasis and angiogenesis. E The peptide CIP2A-BP encoded by LINC00665 competes with PP2A for binding to CIP2A, reducing AKT phosphorylation to inhibit the PI3K/AKT/NFκB pathway, leading to the downregulation of MMP2, MMP9, and Snail, thus inhibiting breast cancer invasion and metastasis. F The peptide MAGI2-AS3-ORF5 encoded by LncRNA MAGI2-AS3 interacts with extracellular matrix proteins to inhibit breast cancer cell proliferation and migration. Image created with BioRender.com

Liver hepatocellular carcinoma

Liver hepatocellular carcinoma (LIHC) is the most prevalent form of primary liver cancer, constituting 90% of all hepatic cancers [139, 140]. The molecular landscape of LIHC is complex and involves lncRNAs encoded peptides, which play crucial roles in the pathogenesis and progression of the disease. For instance, HBVPTPAP induces apoptosis in LIHC cells via activation of the JAK/STAT signaling pathway, potentially through interaction with PILRA (Fig. 4A) [32]. The peptide SMIM30 is upregulated in LIHC tissues and promotes cell proliferation, migration, and invasion by interacting with SRC and YES1, activating the MAPK pathway, and being transcriptionally regulated by c-Myc (Fig. 4B) [89]. SMIM30 also enhance cell proliferation by promoting the G1/S transition via the Rb pathway and modulate the cyclin/CDK-Rb-E2F1 pathway and cytosolic calcium levels [116], which extends the impact of SMIM30 in LIHC. PINT87aa overexpressed in senescent LIHC cells, inhibits growth and induces cellular senescence by blocking FOXM1-mediated transcription of PHB2 (Fig. 4C) [115], while C20orf204-189AA enhances cell proliferation by stabilizing nucleolin and promoting ribosomal RNA transcription (Fig. 4D) [31]. The presence of additional functional lncRNA-encoded peptides such as CIP2A-BP and Linc013026-68AA in LIHC further underscores the diversity of their roles, with CIP2A-BP enhancing HCC cell proliferation and metastasis in LIHC (Fig. 4E) [117], contrasting its suppressive role in TNBC by inhibiting the PI3K/AKT/NF-κB pathway [114], as previously mentioned. The divergent roles of CIP2A-BP in LIHC and TNBC may be attributed to several factors. These include variations in the cellular microenvironment, differences in the signaling pathways active within each cancer type, and the potential for CIP2A-BP to interact with distinct binding partners across various tissues. These considerations highlight the importance of accounting for tissue-specific and context-specific actions when assessing the contributions of lncRNA-encoded peptides to cancer pathogenesis. Additionally, Linc013026-68AA, has been shown to augment LIHC proliferation (Fig. 4F) [118], of which the precise mechanism also warrants further investigation.

Fig. 4

The role of lncRNA-encoded peptides in liver cancer. A The peptide HBVPTPAP encoded by LncRNA HBVPTPAP promotes membrane localization of PILRA by interacting with it, activating the JAK/STAT signaling pathway to induce apoptosis and inhibit liver cancer development. B The peptide SMIM30 encoded by LINC00998 activates the MAPK signaling pathway and regulates the G1/S phase transition to promote liver cancer proliferation and metastasis. C The peptide PINT87aa encoded by LINC-PINT interacts with FOXM1 to inhibit PHB2 transcription, inducing cellular senescence and suppressing liver cancer growth. D The peptide C20orf204-189AA encoded by LINC00176 promotes liver cancer cell proliferation by stabilizing Nucleolin and enhancing rRNA transcription. E The peptide CIP2A-BP encoded by LINC00665 promotes liver cancer growth and metastasis. F The peptide Linc013026-68aa encoded by LINC013026 enhances the in vitro proliferation of HCC cells. Image created with BioRender.com

Lung cancer

Lung cancer (LC) remains the primary cause of cancer-related mortality worldwide, with a grim prognosis and an estimated 234,580 new cases in the United States alone for 2024 [133, 136, 141, 142]. The disease is generally categorized into two main types: non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), each with distinct clinical features and treatment approaches [143,144,145,146]. Recent advances in molecular research have shed light on the role of lncRNA-encoded peptides in NSCLC, such as ATMLP, which is upregulated in NSCLC tissues and disrupts mitophagy by interacting with NIPSNAP1, thereby promoting malignant transformation and tumorigenesis (Fig. 5A) [120]. Interestingly, ATMLP's expression is regulated by N6-methyladenosine (m⁶A) methylation of its encoding lncRNA AFAP1-AS1 [120], introducing a new perspective on the post-transcriptional regulation of lncRNA-encoded peptides, and suggesting that epigenetic modifications, such as m⁶A methylation, may serve as key regulators in the expression and function of these peptides. Additionally, a peptide encoded by lncRNA DLX6-AS1 has been shown to activate the Wnt/β-catenin pathway, enhancing NSCLC cell proliferation and metastasis (Fig. 5B) [121]. The lncRNA product UBAP1-AST6 also enhances LC cell proliferation and clone formation, although its mechanisms of action require further investigation (Fig. 5C) [122].

Fig. 5

The role of lncRNA-encoded peptides in lung cancer. A The peptide ATMLP encoded by lncRNA AFAP1-AS1 disrupts autolysosome formation by interacting with NIPSNAP1, hindering its transport, leading to lung cancer development and progression. B The peptide encoded by lncRNA DLX6-AS1 enhances the proliferation, migration, and invasion of NSCLC cells by activating the Wnt/β-catenin signaling pathway. C The peptide UBAP1-AST6 encoded by an LncRNA promotes the proliferation of lung cancer cells in vitro. Image created with BioRender.com

Esophageal cancer

Esophageal cancer, predominantly manifesting as esophageal squamous cell carcinoma (ESCC), is the sixth most common cause of cancer mortality worldwide, with a significant incidence in China where it represents over 50% of global cases [147,148,149]. Late-stage symptoms like dysphagia and cervical lymph node enlargement contribute to a low 5-year survival rate and a poor prognosis for ESCC patients [150]. Recent studies have shed light on the role of lncRNA-encoded peptides in ESCC, offering a promising avenue in the battle against this aggressive cancer. Pep-KDM4A-AS1, a peptide encoded by the lincKDM4A-AS1, has been shown to diminish ESCC cell viability and migration by modulating the oxidation–reduction process and fatty acid metabolism. Another peptide (Fig. 6A) [123]. Pep-LINC01116, exhibits similar effects on cell viability and migration (Fig. 6B) [123]. Additionally, YY1BM, a peptide encoded by LINC00278, influences ESCC progression by disrupting the AR signaling pathway, leading to altered expression of eEF2K and impacting cell adaptability under nutrient-deprived conditions (Fig. 6C) [91].

Fig. 6

The role of lncRNA-encoded peptides in esophageal cancer. A The peptide Pep-KDM4A-AS1 encoded by LincKDM4A-AS1 inhibits the proliferation and migration of esophageal cancer cells by regulating intracellular redox processes and fatty acid metabolism. B The peptide Pep‐LINC01116 encoded by LINC01116 reduces the viability of ESCC cells and inhibits their migration. C The peptide YY1BM encoded by LINC00278 promotes apoptosis in esophageal cancer cells by disrupting the binding of YY1 and AR, leading to reduced eEF2K expression. Image created with BioRender.com

Pancreatic cancer

The global burden of pancreatic cancer has seen a sharp escalation over recent decades, with a grim projection that it will remain the leading cause of cancer-related mortality [151, 152]. Recent molecular research has identified the lncRNA-encoded peptide RASON, encoded by LINC00673, as a critical factor in pancreatic cancer pathology. Overexpressed in pancreatic cancer tissues, RASON promotes the proliferation of pancreatic ductal adenocarcinoma by interacting with the oncogenic KRAS^G12D/V mutant protein (Fig. 7A) [124]. This interaction inhibits KRAS^G12D/V'S GTPase activity and GTP hydrolysis by GTPase activating protein (GAP), leading to the stabilization of KRAS^G12D/V in a GTP-bound, hyperactive state—a key driver of pancreatic cancer [124]. The modulation of KRAS activity by RASON, considering KRAS's frequent mutation in cancer, underscores the peptide's potential as a therapeutic target.

Fig. 7

The role of lncRNA-encoded peptides in pancreatic and renal cancers. A The peptide RASON encoded by LINC00673 promotes the growth of pancreatic cancer by stabilizing KRASG12D/V in an active GTP-bound state through interaction with KRASG12D/V. B The peptide SMIM26 encoded by LINC00493 inhibits the proliferation and migration of renal cell carcinoma by enhancing mitochondrial localization of AGK, thereby inhibiting AGK-mediated AKT phosphorylation. C The peptide MIAC encoded by LncRNA AC025154.2 inhibits the proliferation and migration of renal cell carcinoma by interacting with AQP2 to suppress the expression of EREG/EGFR. Image created with BioRender.com

Renal cell carcinoma

Renal cell carcinoma (RCC), with its most aggressive subtype being clear cell renal cell carcinoma (ccRCC), represents a significant health burden, contributing to an estimated 400,000 new cases and 175,000 deaths globally in 2018 [153, 154]. Recent research has shed light on the role of lncRNA-encoded peptides in the pathology of RCC. The peptide SMIM26 is downregulated in RCC tissues and has been shown to inhibit tumor proliferation and metastasis by interacting with AGK and SLC25A11, thereby affecting mitochondrial glutathione import and respiratory efficiency (Fig. 7B) [125]. Additionally, MIAC is down-expressed in ccRCC and, when overexpressed, inhibits tumor proliferation and migration while promoting apoptosis through the modulation of the PI3K/AKT and MAPK pathways by binding to the AQP2 protein and inhibiting EREG/EGFR expression (Fig. 7C) [126].

Ovarian cancer

Ovarian cancer (OV), encompassing malignancies of the ovary, fallopian tube, and peritoneum, is a significant health concern with an annual global incidence of 313,959 cases and 207,252 deaths [155]. Despite declining incidence rates and improving survival rates in regions like the United States and Europe, partly due to the use of oral contraceptives [136, 156], the prognosis for OV remains poor, with most patients diagnosed at advanced stages and lacking effective early detection strategies [157]. However, the role of the lncRNA-encoded peptide DDUP, derived from CTBP1-DT, has emerged as a key player in OV's molecular pathology, particularly in DNA damage repair. DDUP's upregulation is associated with enhanced DNA repair mechanisms and cisplatin resistance in ovarian cancer cells. The use of the ATR inhibitor Berzosertib has been shown to disrupt DDUP foci formation, thereby sensitizing these cells to DNA-damaging chemotherapeutics. The phosphorylation of DDUP in response to DNA damage induces a conformational change that strengthens its interaction with RAD18, supporting DNA repair through homologous recombination (HR) and post-replication repair mechanisms (Fig. 8A) [39]. The upregulation of DDUP following cisplatin treatment further confirms its role in promoting cellular resistance to chemotherapy, emphasizing its significance in OV's therapeutic resistance. Another research team has revealed that DDUP is upregulated in patient-derived OV cells following cisplatin treatment, enhancing the cells' capacity for DNA repair and resulting in cisplatin resistance through RAD51C-mediated HR and PCNA-mediated post-replication repair [127], which further confirm the significant role of lncRNA-encoded peptide in DNA damage repair.

Fig. 8

The role of lncRNA-encoded peptides in ovarian and glioblastoma cancers. A The peptide DDUP encoded by LncRNA CTBP1-DT enhances DNA damage repair and cisplatin resistance in ovarian cancer cells by interacting with H2A.X and RAD18. B The peptide NBASP encoded by LncRNA increases the degradation of FABP5, leading to the inactivation of the MAPK pathway and inhibiting the proliferation and migration of glioblastoma cells. C The peptide sPEP1 encoded by LncRNA HNF4A-AS1 promotes the transcriptional upregulation of hepatocyte-related genes by enhancing the interaction with SMAD4, leading to the occurrence and metastasis of glioblastoma. Image created with BioRender.com

Neuroblastoma

Neuroblastoma (NB) is the most common extracranial solid tumor in children, originating from the developing peripheral sympathetic nervous system and representing approximately 8% of all childhood cancers [158,159,160]. Despite progress in targeted therapies, the long-term survival rate for high-risk children remains under 40%, highlighting the need for innovative treatment strategies [160]. LncRNA-encoded peptides have emerged as potential players in NB pathology. NBASP is downregulated in NB tissues and inhibits cell proliferation, and metastasis by interacting with FABP5 and reducing its expression through the ubiquitin proteasome pathway, resulting in the inactivation of the MAPK signaling pathway (Fig. 8B) [128]. On the other hand, sPEP1, a peptide encoded by HNF4A-AS1 and upregulated in NB stem cells, promotes tumor progression interacting with eEF1A1, enhancing its binding to SMAD4, and leading to the transcriptional upregulation of stem cell genes associated with tumor progression (Fig. 8C) [129].

Osteosarcoma

Osteosarcoma (OS), while a rare cancer, is the most common bone malignancy affecting children and adolescents [161]. It is believed to originate from osteoblastic mesenchymal cells [162]. The prognosis for patients with OS varies significantly depending on the stage of the disease; the 5 year survival rate for patients with localized OS is approximately 70%, but this figure drops to less than 30% for those with metastatic disease, indicating a poor survival outcome [163]. Recent research has highlighted the potential role of lncRNA-encoded peptides in the pathology of OS. One such peptide, LINC00665_18aa, suppresses the viability, proliferation, and migration of human OS cells in vitro and diminishes tumor growth in vivo. The mechanistic insight behind these effects reveals that LINC00665_18aa impairs the transcriptional activity, nuclear localization, and phosphorylation of the CREB1 and disrupts the interaction between CREB1 and RPS6KA3 [130].

Oral squamous cell carcinoma

Oral cancer, predominantly oral squamous cell carcinoma (OSCC), ranks as the sixth most common malignancy globally, yet the 5-year overall survival rate remains under 50%, underscoring an urgent need for innovative therapeutic targets [133, 164]. Research into lncRNA-encoded peptides in OSCC has identified HOXB-AS3 as a significant factor; it is upregulated in OSCC tissues and facilitates cell proliferation and viability by interacting with IGF2BP2 to stabilize the mRNA of c-MYC, a key driver in cell cycle progression and cancer development [131, 165, 166]. This indicates a potential oncogenic role for HOXB-AS3 in OSCC. Interestingly, contrasting roles for HOXB-AS3 have been observed in CRC, where it is downregulated and inhibits cancer progression by interfering with PKM splicing, a key regulatory step in glucose metabolism and the Warburg effect characteristic of cancer cells [167,168,169]. The dualistic behavior of HOXB-AS3 in different cancers, similar to that of CIP2A-BP in liver and breast cancers, highlights the complexity of lncRNA-encoded peptides and their tissue-specific roles in cancer.

Acute myeloid leukemia

Acute myeloid leukemia (AML) is one of the most common clinically fatal malignancies, characterized by differentiation block and clonal expansion of immature cells at various stages. The genetic complexity and highly heterogeneous nature of AML contribute to diverse subtypes with poor prognosis, leading to the limited effects of specific therapies [170,171,172]. The regulatory influence of lncRNA-encoded peptides on protein translation has been discerned in AML. The micropeptide APPLE is notably enriched in ribosomes, where it modulates the initiation phase of translation. This modulation enhances the synthesis of oncoproteins, thereby sustaining elevated rates of translation essential for the malignant phenotype. Mechanistically, APPLE fosters the interaction between PABPC1 and eIF4G, thereby facilitating mRNA circularization and the assembly of the eIF4F initiation complex. This assembly underpins a specific translational program that is conducive to cancer progression [173].While the current body of research is indeed limited, the role of lncRNA-encoded peptides in other hematologic malignancies, such as chronic myeloid leukemia, remains an uncharted territory ripe for exploration.

Although research on lncRNA-encoded peptides has unveiled their potential roles in several types of cancer (Fig. 9), the precise mechanisms by which certain peptides exert their functions remain to be fully elucidated. The complexity of the role of m⁶A modification in lncRNA-encoded peptides is also increasingly evident. For instance, as previously mentioned, m⁶A methylation in the lncRNA AFAP1-AS1 controls the translation of the micropeptide ATMLP in lung cancer [120], while the peptides RBRP can bind to IGF2BP1 and HOXB-AS3 binds to IGF2BP2, important readers of m⁶A modification [174, 175], to increase m⁶A recognition in c-Myc mRNA in CRC and OSCC respectively [104, 131]. These studies suggest that lncRNA-encoded peptides can not only regulate by m⁶A modification but also cooperate with m⁶A modification to influence downstream molecules. However, whether other peptides are regulated by RNA modifications and the intricate interplay between them requires further investigation. Moreover, it is noteworthy that some peptides, including CIP2A-BP and HOXB-AS3, may play opposing roles in different tumors, highlighting the importance of describing a peptide's action within the specific context of a particular cancer. The functional duality of these peptides underscores the need for a nuanced understanding of their roles in various cancerous environments. Furthermore, the exploration of these peptides in other cancer types is currently lacking, such as in the more common malignancies like gastric and prostate cancer. Expanding our research to include these prevalent cancers is crucial for gaining a comprehensive understanding of the breadth of lncRNA-encoded peptides' impact on cancer biology and their potential as therapeutic targets. The investigation into the roles of these peptides in a wider range of cancers could reveal novel insights into cancer pathogenesis and identify new opportunities for targeted cancer therapies.

Fig. 9

lncRNA-encoded peptides identified in various human tumor types. NB, Neuroblastoma; BC, Breast cancer; PDAC, Pancreatic ductal adenocarcinoma; LIHC, Liver cancer; OS, Osteosarcoma; OSCC, Oral squamous cell carcinoma; ESCC, Esophageal squamous cell carcinoma; LC, Lung cancer; RCC, Renal cell carcinoma; CRC, Colorectal cancer; OV, Ovarian cancer. Image created with BioRender.com

Functional mechanisms of lncRNA-encoded peptides in cancer

LncRNA-encoded peptides, despite their short lengths, exert significant regulatory effects in cancer through various mechanisms.

Transcriptional regulation

LncRNAs engage in transcriptional regulation by interacting with transcription factors, influencing the expression of specific genes. For example, PINT87aa interacts with FOXM1 to disrupt the transcription of tumor suppressor [115], and YY1BM interacts with YY1 to affect the androgen receptor signaling pathway, influencing gene transcription [91]. Additionally, lncRNAs can indirectly participate in transcriptional regulation [129].

Post-transcriptional regulation

LncRNA-encoded peptides can directly bind to splicing factors and participate in RNA splicing. For example, SRSP interacts with SRSF3 to affect the production of different protein isoforms [105]. Some peptides can also interact with RNA-binding proteins and RNA modification enzymes, impacting RNA splicing and stability [30, 33, 104, 131]. For example, HOXB-AS3 interacts with IGF2BP2 to stabilize c-MYC mRNA stability [131].

Translation and post-translation regulation

LncRNA-encoded peptides, like APPLE in AML, are involved in the translation initiation phase, enhancing the synthesis of oncoproteins [173]. Additionally, NBASP and ATMLP illustrate how peptides can mediate protein degradation and regulate protein transport and activity, respectively [120, 128].

Bind to metabolic proteins

Moreover, lncRNA-encoded peptides regulate metabolism by binding to metabolic proteins [108, 125]. For example, ASAP promote metabolic processes by interacting with proteins like ATP synthase, affecting cellular metabolism and energy production [103].

Bind to signaling pathway-related proteins

The modulation of signaling pathways by lncRNA-encoded peptides is another critical area of influence. lncRNA-encoded peptides can both activate and inhibit signaling pathways. For instance, MBOP activates the MEK1/pERK/MMP2/MMP9 axis [109], while CIP2A-BP inhibits the PI3K/AKT/NF-κB pathway, impacting cancer progression and metastasis [114].

Genomic stability

LncRNA-encoded peptides also involved in DNA damage repair. For example, DDUP, upon phosphorylation induced by DNA damage, interacts with RAD18 to facilitate repair mechanisms, including RAD51C-mediated homologous recombination and PCNA-mediated post-replication repair [39].

In summary, lncRNA-encoded peptides contribute to cancer development and progression through diverse regulatory roles, including transcriptional and post-transcriptional regulation, modulation of translation and protein activity, metabolic regulation, signaling pathway modulation, and maintenance of genomic stability. These functions are executed through their interactions with a range of protein partners, emphasizing their importance in cellular regulation and cancer biology.

Clinical applications of lncRNA-encoded peptides

An escalating number of studies have substantiated the pervasive involvement of lncRNA-encoded peptides in pivotal physiological processes, with an intimate connection to tumorigenesis and tumor progression. This nascent field within lncRNA research holds the key to unlocking the profound implications of these peptides in cancer biology. As such, their clinical deployment as biomarkers or targets for intervention is anticipated to shed new light on their cardinal role in oncology (Table 5, Fig. 10).

Table 5 The applications of lncRNA-encoded peptides

Fig. 10

Potential applications of lncRNA-encoded peptides. LncRNA-encoded peptides can be utilized in various aspects of oncology, including cancer diagnosis, prognosis, therapeutic target, drug development, immune regulation, and regenerative medicine. SEP, sORF-encoded peptide. Image created with BioRender.com

Diagnosis biomarker

The quest for novel tumor biomarkers within oncology research is driven by the need for markers that are highly sensitive, specific, reproducible, and ideally non-invasive [176,177,178,179]. In this context, circulating micropeptides encoded by lncRNAs emerge as a promising class of biomarkers with the potential to revolutionize cancer diagnostics. The discovery of functional peptides encoded by lncRNAs has opened new avenues in the search for diagnostic biomarkers. These peptides, with their differential expression patterns in malignant versus normal cells, are strong candidates for diagnostic biomarkers. ATMLP, a peptide overexpressed in tumor tissues compared to paracancerous tissues in NSCLC, exemplifies this potential. Its elevated levels in the serum of NSCLC patients, with an AUC of 0.852, suggest its effectiveness as a serum biomarker. Remarkably, ATMLP can prognosticate lung cancer development prior to PET-CT imaging, emphasizing its significant diagnostic value [120]. Similarly, MRP, which intensifies in expression in highly malignant breast cancer cells, has been shown to distinguish patients with and without lymph node metastasis, with an AUC of 0.7112 [30] indicating its potential as a diagnostic tool in breast cancer. However, the diagnostic potential of other lncRNA-encoded peptides and their utility in various biofluids, including urine, warrant further exploration. The promise of these biomarkers lies in their potential for early cancer detection, which is vital for improving patient outcomes. Future research aimed at identifying additional peptides could transform early cancer detection and provide new strategies for timely and effective intervention. As the field advances, the challenge will be to validate these biomarkers in large-scale, multicenter clinical trials to ensure their reliability and utility across diverse patient populations. The successful integration of lncRNA-encoded peptide biomarkers into routine clinical practice will require not only scientific validation but also the development of robust and accessible diagnostic platforms capable of accurately measuring these peptides in patient samples.

Prognosis biomarker

The prognostic utility of lncRNA-encoded peptides in cancer is an emerging field that offers significant promise in predicting disease progression and patient outcomes. These peptides, when identified and characterized, can serve as valuable markers that correlate with late-stage clinical pathological features and poor prognoses, thereby guiding treatment strategies and patient management. Certain peptides have been linked to tumor aggressiveness and survival rates. For instance, in TNBC, specific peptides such as ASRPS and HCP5-132aa have demonstrated a positive correlation with poor OS [99, 113], suggesting their potential as indicators of aggressive tumor behavior and treatment response [99, 113]. Conversely, the presence of the peptide CIP2A-BP has been found to inversely associate with metastasis and OS, indicating its potential as a protective factor or a marker of less aggressive cancer [114]. In CRC, peptides like ASAP, RBRP, and SRSP have been linked to poor OS, with RBRP and SRSP emerging as independent prognostic factors for survival, correlating with advanced clinical stages and higher histological grade [103,104,105]. The prognostic significance of lncRNA-encoded peptides extends beyond breast and colorectal cancers, with implications in pancreatic ductal adenocarcinoma [124], renal cell carcinoma [125], ovarian cancer [39], etc. These peptides enable patient stratification, leading to more personalized treatment plans and improved survival rates. As research continues to elucidate the complexities of lncRNA-encoded peptides, their role in cancer prognosis becomes increasingly clear, offering a unique perspective into tumor biology and informing clinical decision-making.

Therapeutic target

Over the past few decades, cancer treatment has evolved significantly with the introduction of various therapies, including small molecule drugs that target specific signaling pathways, antiangiogenic medications, monoclonal antibodies, and gene therapy [180,181,182,183]. The specificity, efficacy, and reduced side effects associated with peptide or protein-targeted drugs make them particularly promising for clinical application. LncRNA-encoded peptides, with their diverse mechanisms of action, are attractive candidates for therapeutic intervention. The peritumoral administration of sh-RASON, which targeting the peptide RASON, exemplifies the therapeutic potential of SEPs. Studies have shown that sh-RASON can inhibit the growth of xenografted tumors and enhance the sensitivity of KRAS-mutant pancreatic cancer cells to epidermal growth factor receptor inhibitors, such as cetuximab, in a murine model [124], highlighting the potential of lncRNA-encoded peptides as therapeutic agents tailored to target specific molecular aberrations in cancer.

Other potential applications

The specificity, high activity, low cytotoxicity, and diminished immunogenicity of lncRNA-encoded peptides make them prime candidates for drug development. Intratumoral injection of ASRPS has been demonstrated to significantly improve survival in TNBC mouse xenograft models [99]. Synthetic MIAC peptides, administered intravenously, have shown promise in inhibiting tumor growth in RCC models [126]. Additionally, cancer vaccines, which can elicit long-term immunological memory, have garnered significant attention [184,185,186]. Several cancer vaccines are currently utilized in clinical therapy, including Melacine for melanoma and Cima Vax EGF for lung cancer [187, 188]. Laumont et al. highlighted that tumor-specific antigens (TSA) are ideal targets for immunotherapy and found that most TSA derived from non-coding regions [189], suggesting that TSA derived from non-coding regions could be a promising avenue for cancer immunotherapy. The landscape of cancer vaccines is also being reshaped by these peptides, offering the advantage of long-term immunological memory and sustained antitumor effects. Notably, lncRNA-derived peptides have been shown to elicit a potent antigen-specific CD8 + T lymphocyte response, as evidenced by Barczak et al., suggesting their utility in cancer vaccine development [190].

Other studies have explored the role of lncRNA-encoded peptides in immune modulation [191, 192]. Jackson et al. demonstrated that the translation of a novel ORF within the lncRNA Aw112010 is essential for coordinating mucosal immunity during bacterial infection and colitis [193], expanding our understanding of the protein-coding genome and the importance of proteinaceous products from lncRNA in in vivo immune responses. Kikuchi et al. identified a peptide encoded by the lncRNA PVT1 that is predominantly enriched in multiple CRC tissues. The PVT1 peptide was recognized by patient CD8 + tumor-infiltrating lymphocytes and peripheral blood mononuclear cells, indicating the presence of patient immune surveillance [194]. These findings suggest that peptides translated from lncRNAs and presented by HLA class I can be sensed by cancer patient T cells, highlighting their potential in noncoding genomic aberration detection.

As research delves deeper, the regulatory role of lncRNA-encoded peptides in tissue regeneration and stem cell differentiation is coming to light [129, 195, 196]. Matsumoto et al. found that the lncRNA encoding SPAR is downregulated in skeletal muscle upon acute injury. Using a SPAR-polypeptide-specific knockout mouse model created by CRISPR/Cas9, they established that SPAR downregulation enables efficient activation of mTORC1, promoting muscle regeneration [195, 197]. This suggests that lncRNA-encoded peptides could be applied in regenerative medicine, with significant implications for therapeutic approaches following surgical procedures such as hepatectomy.

Prospect and conclusion

The field of lncRNA-encoded peptides in cancer research is burgeoning with potential, offering new insights into the intricate mechanisms underlying tumorigenesis and progression. In the past, the misannotation of genes containing non-canonical ORFs as non-coding RNAs has obscured the significant roles these protein-coding genes play in cancer. However, recent advancements in peptide identification methods, such as Ribo-seq and mass spectrometry, have catalyzed the discovery of SEPs, shedding light on their previously underappreciated functions. To fully harness the potential of SEPs, it is imperative to experimentally validate their translation into functional proteins before delving into their functional studies. In this process, a critical consideration is point-mutation, which may lead to the creation of new ORFs [198, 199]. These peptides often interact with proteins, impacting RNA splicing, and stability, and engaging in cellular metabolism and signaling pathways, thereby participating in biological processes crucial to cancer development. Furthermore, discerning the functions of lncRNA-encoded peptides from those of their parental RNA sequences is imperative. This distinction can be achieved through the overexpression of the full-length lncRNA and its start codon mutant forms, followed by functional assays to determine whether the lncRNA itself or its encoded peptide is responsible for observed biological activities. Post-translational modifications of lncRNA-encoded peptides, analogous to those of mRNA-encoded proteins, are another area that warrants investigation [200, 201]. The interaction between these lncRNA-encoded peptides and the tumor microenvironment, as well as their role in tumor drug resistance, is a relatively unexplored domain that could significantly enhance our understanding of cancer mechanisms and their therapeutic applications [202,203,204,205]. Despite the elucidation of the potential applications of lncRNA-encoded peptides in cancer diagnosis, prognosis, therapeutic targeting, immune modulation, drug development, and regenerative medicine, several unresolved questions and challenges must be addressed before their clinical translation. One of the primary challenges in the application of lncRNA-encoded peptides is the development of an effective delivery system. This system must effectively circumvent the possibility of provoking undesirable immune responses, which can arise from the recognition of these peptides as foreign antigens by antigen-presenting cells and T-cells via the major histocompatibility complex. Such unwanted immune reactions may undermine the therapeutic efficacy of the peptides or even result in detrimental side effects. Extracellular vesicles, with their low immunogenicity and high in vivo stability, are promising candidates for targeted drug delivery [206,207,208]. Furthermore, recombinant technologies and other advancements have facilitated the production of antibodies that can evade immune surveillance and response [209], which is a field that requires further exploration. The development of optimization of peptide stability and half-life to ensure sustained therapeutic effects is also important. While some lncRNA-encoded proteins have emerged as key regulatory factors in the transcriptional networks of human tumors, the functionality, regulation, and mechanisms of the majority remain elusive. Moving forward, there is a pressing need for large-scale validation to substantiate their biological relevance, development of sensitive detection methods, and optimization of peptide stability and delivery.

Effective resolution of the aforementioned issues will not only refine our understanding of the roles of lncRNA-encoded proteins but also provide a roadmap for future research methods and clues. It is undeniable that the mechanism of lncRNA-encoded micropeptides will spearhead a new wave of research enthusiasm and propel the advancement of the life sciences field. The novel perspectives offered by these findings will undoubtedly contribute to the development of future anti-cancer drugs and tumor biomarkers, offering a new frontier in the battle against cancer.