LncRNA HCP5 Encoding Protein Regulates Ferroptosis To Promote The Progression of Triple Negative Breast Cancer

Long non-coding RNAs (lncRNAs) is widely described as a class of RNA longer than 200 nucleotides without encoding capability. But recent years, more and more open reading frames (ORFs) have been found in lncRNAs which indicate they have coding capacity. But the mechanisms of the encoding products in cancer are mostly unknown. We have previously shown lncRNA HCP5 is an oncogene in triple negative breast cancer (TNBC), and the aim of the current study was to investigate if lncRNA HCP5 encoding protein promotes TNBC by regulating ferroptosis. Methods We use bioinformatics to predict coding capacity. Molecular biology experiments and the xenograft assay in nude mice to study the mechanism of lncRNA HCP5 encoding protein. And the protein expression was evaluated in a tissue microarray of 140 invasive breast tumors and 45 pared precancerous breast tissues. Association between the protein expression and clinicopathologic features of breast cancer patients was analyzed.


Introduction
A new report shows that the most commonly-diagnosed cancers worldwide were female breast cancer (about 2.26 million cases) [1]. In China, the incidence of breast cancer also ranks the rst among female malignant tumors. Triple negative breast cancer (TNBC), which accounts for 10%-17%, is characterized by high aggressiveness and poor prognosis due to the lack of therapeutic targets [2]. However, the underlying molecular mechanisms responsible for TNBC tumorigenesis are still not fully understood.
Ferroptosis is a kind of programmed cell death, which leads to the shrinkage of mitochondria, the increase of membrane density and the decrease or disappearance of mitochondrial cristae in cell morphology [3]. This process is characterized by lipid peroxide accumulation, membrane repair and irondependent accumulation of reactive oxygen species (ROS) [4,5]. Several genes and pathways responsible for the regulation of iron and ROS metabolism have been implicated in ferroptosis, such as the Xc − /GSH/GPX4, ACSL4/LPCAT3/15-LOX and FSP1/CoQ10 pathway [6][7][8].
LncRNAs are widely found in organs and systems of the body [9][10][11]. Although once considered as the "noise" in the human genome, with the development of bioinformatics, 2% of lncRNAs have been found to encode proteins or peptides [12]. Studies had shown that tumor-related lncRNA coding proteins or peptides can be combined with traditional anticancer drugs or chemoradiotherapy to improve the therapeutic effect of cancers and reduce mortality [13]. For example, lncRNA coding peptide ASRPS involved in malignant progression of TNBC and HOXB-AS3 coding peptide inhibited the growth of CRC [14,15]. Also a 73aa peptide encoded by CircPPP1R12A can help colon cancer metastasize [16].
In this study, we discovered that the lncRNA HLA complex P5 (HCP5), which was previously reported as an oncogene in TNBC [17], encoded a conserved 132-amino acid small protein which named HCP5-132aa. Knockdown HCP5 ORF caused low HCP5-132aa expression, induced ferroptosis by decreasing GPX4 and increasing lipid ROS in TNBC cells. High levels of the HCP5-132aa protein were associated with a poor survival rate in breast cancer patients. Collectively, we revealed that a protein encoded by lncRNA HCP5 promoted the malignant progression of TNBC through inhibiting ferroptosis.

Material And Methods
Prediction of lncRNA HCP5 encoding products using bioinformatics analysis To identify the lncRNA HCP5 encoded peptides or proteins in breast cancer, we integratively analyzed HCP5 open reading frame (ORF), ribosome pro le (Ribo-seq) and MS/MS data of MDA-MB-231 cell line (Fig. S1). LncRNA HCP5 sequence (GRCh38 fasta format) was obtained from NCBI Gene [18]. Ribo-seq data (GSE69923) and MS/MS data (PXD008222) were downloaded from Gene Expression Omnibus (GEO) database [19] and EMBI-EBI-PRIDE database [20], respectively. HCP5 ORFs were identi ed by ORF nder [21] in NCBI based on HCP5 exon sequence. We screened ORFs with start codon "ATG" in the positive strand as candidates. Then, we obtained Ribo-seq les of SRA format and converted them to fastq format by using fastq-dump tool (https://ncbi.github.io/sratools/fastq-dump.html). Next, adaptor sequences were trimmed using Trim Golare (https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/). Reads shorter than 25 bp after adaptor trimming were discarded. rRNA sequences were ltered by using RNAcentral database [22]. Furthermore, the remaining reads were aligned to reference genome GRCh38 using TopHat2 [23] and bam format le was obtained. The ORFs fewer than 400 nt with reads mapped were regarded as convinced HCP5 small ORFs (sORFs). In addition, MS/MS raw data was converted to mgf format by MSConvert [24]. Then, Peppy was used to get amino acid (aa) sequences for peptides or proteins and aligned to GRCh38. Finally, the convinced HCP5 sORFs with MS/MS peptides mapped were validated as highly convinced HCP5 sORFs. Immunohistochemistry staining (IHC) The tissue sections were dried at 60°C for 1 h then dewaxed in xylene and rehydrated through graded alcohol concentrations using standard procedures. Antigen retrieval was performed in citrate buffer (pH 6.0) and autoclave at 121°C for 90 s. The procedure of lentivirus infection is as follows: the plate containing cells was added with appropriate amount of lentivirus in concentration gradient, followed by adding 1/1000 polybrene to enhance infection. The sequence of the HCP5-132aa shRNA was showed in Table S1. Lentivirus vector LV5 containing full-length HCP5-132aa or empty vector were purchased from GenePharma Co., Ltd. (Shanghai, China).
Later, the cells were harvested by trypsin and washed three times with ice-cold PBS followed by resuspending in PBS plus 1% BSA. The amount of ROS within cells was examined by ow cytometry analysis (FACSCantoTM II, BD Biosciences).

Iron assays
For the iron assay, we used an Iron Assay Kit (MAK025-1KT, Sigma Aldrich) to measure Fe 2+ or total iron in each cell line. First, 2 × 10 6 of cells was rapidly homogenized in 4 ~ 10 volumes of Iron Assay buffer. Samples were centrifuged at 13,000 × g for 10min at 4°C to remove insoluble material. To measure ferrous iron, 5 µl of iron assay buffer was added to each well. Samples were mixed well using a horizontal shaker or by pipetting and the reactions were incubated for 30 min at room temperature in dark conditions. Then, 100 µL of Iron Probe was added to each well containing standard or test samples. Samples were mixed well using a horizontal shaker or by pipetting and the reactions were incubated for 60 min at room temperature in dark conditions. Finally, the absorbance was measured at 593 nm (A593).

Immuno uorescence analysis
Cells were xed in 4% formaldehyde for 30 min at room temperature before cell permeabilization with 0.1% Triton X-100 (4°C, 10 min). Cells were saturated with PBS containing 2% bovine serum albumin for 1 h at room temperature and processed for immuno uorescence with 1mg/ml C11-BODIPY followed by 10mg/ml Hoechst 33258 (Invitrogen, Carlsbad, CA, USA

Statistical analysis
All statistical analyses were performed using SPSS 19.0 software (IBM Corp, Armonk, NY, USA). Results are expressed as the mean ± standard deviation (SD). Group means were compared using Student's t-test for independent data. All P-values are two-tailed, and P < 0.05 was considered to indicate statistical signi cance. The chi-square test was used to compare HCP5-132aa expression between breast cancer tissues and paired breast tissues and the association with clinicopathologic parameters. Survival analyses were estimated using the Kaplan-Meier method.

Results
LncRNA HCP5 encoded a protein and upregulated in TNBC cell lines HCP5 was originally annotated as an lncRNA gene located in chr6 (p21.33) in Homo sapiens and we previously reported lncRNA HCP5 promoted TNBC progression [17]. Here, we predicted that there are ve convinced HCP5 sORFs with Ribo-seq reads mapped identi ed in exon by ORF nder. After mapped with MS/MS peptides, three highly convinced HCP5 sORFs were remained in the following analysis (Fig. S2). The peptides encoded by HCP5 sORFs were named as small peptides (sPEPs). The lengths of identi ed sPEPs were 132aa, 43aa and 22aa. The sequences were shown in the Table S2. We further identi ed a 399bp ORF with the potential to encode a highly conserved 132aa protein which was named HCP5-132aa ( Fig. 1A-C).
To determine whether HCP5-132aa was endogenously expressed, we produced antibody against the HCP5-132aa and detected via western blot analysis in MCF-10A and breast cancer cell lines. The results showed that HCP5-132aa protein expression was higher in TNBC cell lines (MDA-MB-231, MDA-MB-468 and HCC-1937 cells) than other cell lines (Fig. 1D). Next we did protein subcellular localization and we found HCP5-132aa expressed both in nucleus and cytoplasm (Fig. 1E).

HCP5-132aa high expression indicates a poor prognosis for breast cancer patients
To examine the role of the HCP5-132aa in breast cancer patients, protein levels in tissue microarray analysis of 140 pairs of breast cancer tissues and matched precancerous tissues were analyzed by IHC assay. HCP5-132aa levels increased in the TNBC tissues compared with those in the non-TNBC and precancerous tissues (Fig. 1F, Table S3 and S4, P = 0.042 and P < 0.001, respectively). Increased HCP5-132aa levels were positively associated with more advanced clinical stages of breast cancer (P = 0.002; Table S3). Kaplan-Meier survival analyses revealed that patients with higher HCP5-132aa levels were at increased risk of breast cancer related death compared with patients with lower HCP5-132aa expression levels ( Fig. 1G-H, P < 0.001, log-rank test). Therefore, increased HCP5-132aa levels were correlated with a poor prognosis in breast cancer patients.

Knockdown of HCP5-132aa inhibited TNBC cell malignant phenotypes
To determine whether HCP5-132aa had a function in TNBC, HCP5-132aa ORF expression was knockdown or overexpressed in MDA-MB-231 and MDA-MB-468 cells (Fig. S3A-D). Knockdown HCP5-132aa ORF expression inhibited TNBC cell growth, colony formation, migration and promoted apoptosis ( Fig. 2A-D). Also, HCP5-132aa ORF knockdown could make cell cycle arrested in S stage. The proportion of cells at G2/M phase was not changed (Fig. 2E). These results demonstrated that the HCP5-132aa is a positive regulator of TNBC progression.

HCP5-132aa knockdown promoted ferroptosis
In order to verify the regulatory mechanism of HCP5-132aa in TNBC, RNA sequencing was performed to search for the differentially expressed genes (DEGs) after HCP5-132aa ORF knockdown. We applied DEseq2 for differential expression analysis and identi ed 720 DEGs (q < 0.05 and |log2 (Fold Change) | > log2(1.5)) (Fig. 3A). Then, we performed functional enrichment analysis for the DEGs by clusterPro ler R package. The result demonstrated that the DEGs were signi cantly enriched in 39 pathways (P < 0.05), which included the ferroptosis pathway (Fig. 3B).
Furthermore, we chose ferroptosis activators Erastin and RSL3 to induce ferroptosis since sulfasalazine didn't work in MDA-MB-231 cells (Fig. S4). Mitochondrial morphological changes in MDA-MB-231 were observed using TEM. After speci cally knocking down HCP5-132aa ORF, the mitochondrial membrane density was increased and the mitochondrial crest was reduced, and the mitochondria damage worse when adding Erastin. But there was no obviously change of mitochondria morphology when overexpressed HCP5-132aa in the same dose Erastin stimulating MDA-MB-231 cells (Fig. 3C).
Ferroptosis can cause lipid reactive oxygen species (ROS) accumulation, we measured the lipid ROS by C11-BODIPY staining through owcytometry and confocal after HCP5-132aa ORF knockdown and stimulating with ferroptosis stimulator or inhibitor. The results validated that the lipid ROS level increased in HCP5-132aa ORF knockdown cells and getting much higher after Erastin or RSL3-induced. Like Fer-1 and Lip-1, co-treatment with HCP5-132aa overexpression plasmid suppressed lipid ROS level in response to Erastin or RSL3 (Fig. 3D-F). But there was no signi cant difference in iron levels between these groups (Fig. S5A-B). Thus, we inferred that HCP5-132aa ORF downregulation could promote ferroptosis through inducing lipid ROS production.
Western blot showed that the GPX4 protein expression was reduced after knockdown of HCP5-132aa ORF or stimulated with ferroptosis activators Erastin and RLS3 compared with the control group in MDA-MB-231 and MDA-MB-468 cells. But there was no difference of other ferroptosis associated protein ACSL4, AMID and FTH between the control and HCP5-132aa knockdown groups. Ferroptosis inhibitors Fer-1 and Lip-1 could not reverse GPX4 level in MDA-MB-231, but the GPX4 protein expression could be reversed by Fer-1 in Erastin stimulated and Lip-1 in RLS3 induced HCP5-132aa knockdown MDA-MB-468 cells (Fig. 3G-J). These results indicate that knockdown HCP5-132aa ORF speci cally regulates GPX4 and induced ferroptosis in TNBC cells.

HCP5-132aa knockdown synergized with ferroptosis activators inhibiting tumor growth in vivo
To assess the effect of HCP5-132aa on TNBC in vivo, we treated subcutaneous xenograft tumor of silencing HCP5-132aa ORF in nude mice with Erastin or RSL3. As shown in Fig. 4A-C, HCP5-132aa ORF knockdown inhibited tumor growth in vivo. Erastin or RSL3 inhibited the growth of tumor cells in the knockdown groups signi cantly. The differences in tumor weight among these groups were signi cant. Hematoxylin and eosin (HE) stained tissue sections showed that there were no damages in the organs (lungs, livers, kidneys and hearts) at the given dose (Fig. S6). The reduction of tumor cells was more obvious in the shRNA HCP5-132aa group and Erastin treatment groups than shNC group (Fig. 4D). These data suggested that HCP5-132aa downregulation could synergize with ferroptosis activators to inhibit tumor growth in vivo. We draw the schematic diagram of lncRNA HCP5 encoding protein HCP5-132aa induced ferroptosis through regulating GPX4 and ROS (Fig. 4E).
In this study, we provided the rst evidence that lncRNA HCP5 encoding a 132aa protein, which we named HCP5-132aa. This protein can promote the malignant progression of TNBC through regulating the ferroptosis pathway in vitro and in vivo. Mechanistically, these effects are dependent on GPX4 and lipid ROS levels, no other ferroptosis associated pathways. The HCP5-132aa levels were increased in TNBC cell lines and primary cancer tissues compared with those in their corresponding parental cell lines and precancerous tissues, respectively. Moreover, the HCP5-132aa high expression was associated with poorer patient prognoses which indicated that HCP5-132aa might have the potential to be a prognostic factor for TNBC.
Recent advances in bioinformatics and biochemical methodologies have revealed that lncRNAs may harbor concealed peptides or proteins; however, only a few have been functionally veri ed and characterized [16], and fewer articles reported lncRNA-encoded proteins/peptides function in cancer progression (8-9) [29]. Here, we have identi ed and characterized the functions of a conserved protein encoded by the lncRNA HCP5 during tumorigenesis. HCP5-132aa promotes TNBC cell growth, colony formation, migration and induced S phase cell cycle arrested. Besides, it can suppress cell apoptosis.
These results indicate that HCP5-132aa is a tumor promotor.
Moreover, RNA sequencing results between the control and HCP5-132aa ORF knockdown cells suggested that the DEGs could be enriched in the ferroptosis pathway. The ferroptosis was recently discovered as an apoptosis-independent form of programmed necrosis [30]. It is characterized by the iron-dependent lethal accumulation of lipid ROS. The ferroptotic cells exhibit smaller mitochondria, diminished or vanished of mitochondria crista, and condensed mitochondrial membrane densities [6]. We veri ed that HCP5-132aa ORF knockdown alone could induce cell ferroptosis, suggesting that it might act as a driver of ferroptosis. So far, we are the rst to propose that the protein encoded by lncRNA can promote the development of cancer through regulating ferroptosis. We found that knocking down HCP5-132aa ORF increased the mitochondrial membrane density, reduced the mitochondrial crest, and even worse when adding Erastin.
But HCP5-132aa ORF overexpression could suppress the morphological changes of mitochondria induced by Erastin. We also demonstrated that HCP5-132aa knockdown could directly decrease GPX4 expression and increase ROS level. GPX4 is the only peroxidase known to e ciently reduce esteri ed, hydroperoxy fatty acids into unreactive alcohols. We speculated HCP5-132aa could be a GPX4 activator to promote the antioxidant response, decreasing natural lipid ROS species to accumulate. Recently, ferroptosis suppressor protein 1 (FSP1), also known as AMID, was identi ed as a second ferroptosis suppression mechanism through its recycling of coenzyme Q10, a radical-trapping antioxidant [31]. But the results showed other ferroptosis pathway proteins ACSL4, AMID and FTH expression were unchanged. The xenograft results suggested that knockdown HCP5-132aa ORF could synergize with ferroptosis activators to inhibit tumor growth in vivo.

Conclusions
In summary, we found that the lncRNA HCP5 encodes a protein HCP5-132aa. The HCP5-132aa promoted TNBC growth by regulating GPX4 and subsequent inhibited ROS level, thereby suppressing ferroptosis. TNBC patients with HCP5-132aa high expression exhibit more aggressive clinicopathological features and poorer prognoses. Thus, our ndings expand the understanding of the pathways that regulate ferroptosis and provide an approach for exploiting ferroptosis therapeutically.