CBX4 deletion promotes tumorigenesis under KrasG12D background by inducing genomic instability

Chromobox protein homolog 4 (CBX4) is a component of the Polycomb group (PcG) multiprotein Polycomb repressive complexes 1 (PRC1), which is participated in several processes including growth, senescence, immunity, and tissue repair. CBX4 has been shown to have diverse, even opposite functions in different types of tissue and malignancy in previous studies. In this study, we found that CBX4 deletion promoted lung adenocarcinoma (LUAD) proliferation and progression in KrasG12D mutated background. In vitro, over 50% Cbx4L/L, KrasG12D mouse embryonic fibroblasts (MEFs) underwent apoptosis in the initial period after Adeno-Cre virus treatment, while a small portion of survival cells got increased proliferation and transformation abilities, which we called selected Cbx4−/−, KrasG12D cells. Karyotype analysis and RNA-seq data revealed chromosome instability and genome changes in selected Cbx4−/−, KrasG12D cells compared with KrasG12D cells. Further study showed that P15, P16 and other apoptosis-related genes were upregulated in the primary Cbx4−/−, KrasG12D cells due to chromosome instability, which led to the large population of cell apoptosis. In addition, multiple pathways including Hippo pathway and basal cell cancer-related signatures were altered in selected Cbx4−/−, KrasG12D cells, ultimately leading to cancer. We also found that low expression of CBX4 in LUAD was associated with poorer prognosis under Kras mutation background from the human clinical data. To sum up, CBX4 deletion causes genomic instability to induce tumorigenesis under KrasG12D background. Our study demonstrates that CBX4 plays an emerging role in tumorigenesis, which is of great importance in guiding the clinical treatment of lung adenocarcinoma.


INTRODUCTION
The origin of tumor initiation remains one of the most important questions in modern cancer biology.5][6] Chromobox (CBX) family proteins are the core components of PRC1, which has been proved to be a transcription suppressor and play an important role in organ development and tumorigenesis. 7,8[12] CBX4 has been proved to participate in several processes including growth, senescence, immunity, and tissue repair. 7hrough the combination of Polycomb-and SUMO E3 ligasedependent functions via N-terminal chromodomain and two SUMO-interacting motifs (SIM), CBX4 can regulate some cellular processes. 13,14CBX4 maintains nucleolar homeostasis and regulates the expression of certain genes to resist cell senescence. 15,16During cell differentiation, CBX4 interacts with P63 transcription factors to play a regulatory role. 17Besides, CBX4 also contributes to HIV-1 latency through forming phase separation. 180][21] Previous studies have proved that CBX4 serves as a vital regulator for thymic epithelial generation and maintenance as well as the development of thymocyte. 22CBX4 has been demonstrated to promote metastasize and progression in lung cancer through regulating BMI-1. 23Overexpression of CBX4 recruits GCN5 to sustain H3K27Ac of Runx2 promoter, upregulates its transcription and promotes lung metastasis in osteosarcoma subsequently. 19CBX4 is also found to promote breast cancer via miR-137 via Notch1 signaling, and to be stimulative to tumorigenesis in lung adenocarcinoma (LUAD) possibly through Wnt/β-catenin pathway. 20,21AS is a membrane protein with GTPase activity and can transmit signals from cell membrane to nucleus in activated state, which regulates cell growth and differentiation. 24Oncogenic mutations including Kras G12D are frequently observed in ~20% of all types of human cancers including carcinomas of the lung, colon, and pancreas. 25][31] We used the LSL-Kras G12D activation induced lung tumor mice model in this study and found that CBX4 deletion promoted lung tumor progression in Kras G12D background.Further, our in vitro and RNA-Seq data showed that CBX4 loss caused instability of chromosome and most cells tended to apoptosis in the initial stage.Then minority of cells survived from genomic instability due to several changes of signaling pathway including Hippo pathway and gained stronger proliferation and invasion ability, thereby promoting tumorigenesis.It reminds that the function of genes may be related to the contexts, pressure, and identity of tumors, which should be considered when developing the target therapies.In summary, we tended to investigate the role of CBX4, a key component of PRC1 complex, in lung adenocarcinoma tumorigenesis under Kras G12D background with Cre-induced LSL-Kras G12D mouse model.G12D mice model CBX4 is a component of the Polycomb group multiprotein and has been proved to participate in cell growth, senescence, and other processes else.6][17][18][19][20][21] As mere CBX4 deficient does not induce LUAD (Supplementary Fig. 1), we chose Kras G12D mouse model for this study, widely used in lung cancer studies, and Adeno-Cre could be delivered via nasal inhalation to initiate Kras G12D -dependent lung cancer. 25We crossed Cbx4 L/L with LSL-Kras G12D mice to get Cbx4 L/L , LSL-Kras G12D mice for subsequent experiments.Immunofluorescence with Lenti-Cre-GFP demonstrated more tumor-like Kras G12D cell aggregations due to loss of CBX4 (Supplementary Fig. 2).

CBX4 deletion promotes cell proliferation and invasion in longterm cell culture
Then we sought to explore the mechanism through in vitro experiments with Mouse Embryonic Fibroblast (MEF).
However, 4 weeks soft agar assay indicated dramatic increase in colony formation and cell transformation abilities in Cbx4 −/− , Kras G12D group (Fig. 2a, b).We further extended the 2D culture to 4 weeks, and in consistence with the soft agar assay results, we found that a small portion of Cbx4 −/− , Kras G12D MEFs survived in long-term (28 days) culture, which we called the selected cells (Fig. 2c, d).To ensure the authenticity of the result, we performed the Cre-GFP cell tracing experiments in Cbx4 L/L , LSL-Kras G12D MEFs and found that majority of Cre-GFP positive (Cbx4 −/− , Kras G12D ) cells underwent apoptosis, while a small amount of Cre-GFP positive cells survived and divided (Supplementary Fig. 7).Further experiments showed that selected cells acquired stronger abilities in cell proliferation and transformation compared with Kras G12D cells (Fig. 2e, f).RT-qPCR and genotype of lung tissues confirmed that these selected cells and the lung tumors were indeed CBX4deleted (Fig. 2g, h).These data unravel that a minuscule part of Cbx4 −/− , Kras G12D cells acquire stronger proliferation and transformation abilities during the long-term culture, which accounts for the increased tumorigenesis in Cbx4 −/− , Kras G12D mice.
Typical selected Cbx4 −/− , Kras G12D clones gain cell proliferation and clone formation abilities in vitro Next, to further validate the cell proliferation and clone formation abilities of selected Cbx4 −/− , Kras G12D cells in vitro, we selected 5 typical single clones from the selected pool for further experiments.Genotype result confirmed that those clones were Cbx4 deficient (Fig. 3a).MTT assay presented that all 5 Cbx4 −/− , Kras G12D clones exhibited faster growth rates compared with Kras G12D MEFs control (Fig. 3b).FACS analysis revealed a similar result by the larger percentage of proliferating cells in every selected Cbx4 −/− , Kras G12D clone (Supplementary Fig. 8).Compared with the Kras G12D MEFs, Cbx4 −/− , Kras G12D clones in soft agar yielded greater colony numbers, indicating they got promotion in vitro transformation (Fig. 3c, d).
To notarize whether these alterations in the Cbx4 −/− , Kras G12D were caused by loss of environmental CBX4 or irreparable genome change, we over-expressed mCBX4 in two Cbx4 −/− , Kras G12D clones.Real-time PCR and WB confirmed the ectopic expression of mCBX4 in these two clones (Supplementary Fig. 9).However, it was indicated that cell growth couldn't be restored by CBX4 reintroduction.So ectopic CBX4 expression was irrelevant to cell proliferation in selected Cbx4 −/− , Kras G12D clones (Fig. 3e).Colony formation assays also demonstrated that CBX4 had no reverse on the phenotype of these clones (Fig. 3f, g).This means that for selected Cbx4 −/− , Kras G12D cells, the changes in proliferation and invasion are irreversible cellular changes, rather than conditionally correlated with CBX4 expression, probably due to occurrences of oncogenic mutations and signals during the selection.
We performed Bulk RNA Sequencing to further decipher the differences between the primary Cbx4 −/− , Kras G12D cells, the selected Cbx4 −/− , Kras G12D cells and the Kras G12D cells.Gene Ontology (GO) Analysis revealed thousands of genes were significantly altered in primary and selected Cbx4 −/− , Kras G12D cells compared with Kras G12D cells (Fig. 4d-f and Supplementary Table 1).Further, RNA-seq data revealed significant changes in cell cycle, chromosome maintenance and hemostasis in primary Cbx4 −/− , Kras G12D cells (Fig. 4g), implying genomic instability, which was suggested by Karyotype analysis (Fig. 4c).Totally, the result shows that CBX4 loss causes chromosome instability in Kras G12D background.
P15 and P16 are upregulating in primary Cbx4 −/− , Kras G12D cells causing cell apoptosis According to the results above, we hypothesized that the loss of CBX4 regulated the expression of some apoptosis-related genes (also tumorigenesis-related genes) during the initial cell culture process.P16 has been proved as an activating factor of tumor suppressor pathways and inactivated in many different human tumor types. 33To understand the above phenomenon in detail, we first performed QPCR and found that expressions of P15 and P16 were upregulated in primary Cbx4 −/− , Kras G12D cells.WB and IF also supported this observation (Fig. 5a-d), which suggested that absence of CBX4 promoted cell apoptosis in early culture by upregulating the expression of tumor suppressor genes.Moreover, we found that apoptosis-related genes were highly expressed in atypical adenomatous hyperplasia (AAH, the initial stage of LUAD), then decreased during tumor progression (Fig. 5e, f and Supplementary Fig. 10), whereas the expression of Ki67 was in the opposite trend (Fig. 5g, h).
These results provide evidence for a predominant apoptotic trend at the initial stage of CBX4 deletion and then a promotion of tumorigenesis as the few surviving cells acquires greater proliferative and invasive abilities.
Multiple pathways involved in tumorigenesis including Hippo are changed in selected Cbx4 −/− , Kras G12D cells Tumor initiation can be launched by epigenetic regulation, which participates in various processes of cells.There are often genomic changes that occur during cell apoptosis, and studies have shown that cytogenetic genome instability may inhibit programmed apoptosis through a variety of ways, so we took that into account. 34In addition, multiple pathways including Hippo were found to be altered in selected Cbx4 −/− , Kras G12D cells (Fig. 4d-f).The Hippo pathway has also been widely studied in lung cancer, and its activation can inhibit the occurrence and development of lung cancer. 35,36Abnormalities in signaling pathways lead to alteration of epigenetic modulators and unsubstantiated gene expression, which may result in tumorigenesis.Western blot showed that phosphorylated MST, MOB and YAP were decreased, and total YAP was almost unchanged, which meant Hippo signal pathway was turn-off in selected Cbx4 −/− , Kras G12D MEFs (Fig. 6a).YAP fusion has been shown to exclude the PRC2 complex in ependymoma, 37 therefore in our study suggest CBX4 is likely to have a moderating effect on Hippo signaling pathway as well.In our study, the YAP nuclear localization increased in the Cbx4 −/− , Kras G12D mice lung tumors compared with the Kras G12D mice (Fig. 6b, c).At the same time, we also noticed basal cell cancer-related signatures in selected Cbx4 −/− , Kras G12D MEFs (Fig. 6d), and we speculated that cell identity may have changed.It is a conceivable direction for later mechanism research.
We also noticed 18 Hox genes, Mmp13, Tbx, Pdk4, Bmp and other proven tumor-related genes in the list.Alterations in the expression of these genes can also promote tumorigenesis in different aspects [38][39][40][41][42][43] (Fig. 6e).We performed QPCR to assess the expression of the other members of the PcG group and found that PHC1 and CBX2 were significantly upregulated in the selected Cbx4 −/− , Kras G12D MEFs (Supplementary Fig. 11).MTT and soft agar assays further confirmed overexpression of mPHC1 and mCBX2 promoted cell proliferation and transformation in selected Cbx4 −/− , Kras G12D cells, though knockdown of these two had the opposite effect (Supplementary Figs. 12 and 13).
In general, we believe that when CBX4 is deficient, most cells with Kras G12D mutation undergo apoptosis for chromosome instability in the initial stage, but a small population of cells survive from genomic instability acquiring stronger abilities in proliferation and transformation, due to changes of multiple pathways including Hippo (Fig. 6f).Our study provides a possible mechanism of lung cancers with low CBX4 level and suggests new clues for CBX4-targeted therapies in cancer treatment.

DISCUSSION
Previous studies have shown that CBX4 functions as a protooncogene during tumorigenesis. 21In this study, we performed CBX4 IHC staining on the previous tumor samples, 21 and found that a subset of LUAD patients had high CBX4 expression but there were also a significant number of LUAD cases with low CBX4 expression, leading to a worse prognosis under the background of Kras mutation, restricted to stage III (Supplementary Fig. 14).This indicated that the study was clinically instructive under certain condition.We found that most cells got apoptosis due to instability of chromosome in the initial stage caused by CBX4 loss, but minority of cells survived and improved the ability of proliferation and transformation, promoting lung tumors under Kras G12D activation background.This dichotomy effect can also be seen in other CBX family members, such as CBX7, which is known to suppress cervical cancer, hepatocellular carcinoma and lung cancer, [44][45][46] but the study also shows it could inhibit P16 and activate AKT-NF-κB-miR-21 pathway to promote cell survival and stem cell-like characteristics in gastric cancer. 47e found that CBX4 loss triggered cell apoptosis at the early stage of the Kras G12D MEFs by chromosome instability, possibly through dysregulation of P15 and P16.Cell apoptosis is a form of programmed cell death that orderly and efficiently remove the damaged cells, which can be regarded as a self-contained mechanism against tumors. 48The expression of P16 mediates cell Fig. 3 Typical selected Cbx4 −/− , Kras G12D clones gain cell proliferation and clone formation abilities in vitro which cannot be rescued by ectopic CBX4. a Genotype (with or without Cre recombinase) of selected Cbx4 −/− , Kras G12D clones.Wild-type (W), heterozygote (T) and mutant (M) type were used as control.b MTT assay of 5 selected Cbx4 −/− , Kras G12D clones and the control (Kras G12D MEFs).c, d Colony formation and transformation abilities of five selected Cbx4 −/− , Kras G12D clones were shown by soft agar assay and the quantitative analysis.e MTT analysis to show cell proliferation with or without ectopic expression of CBX4 with the control of Kras G12D cells.f, g Colony formation ability of selected Cbx4 −/− , Kras G12D cells (clone1 and clone3 representatively) with or without ectopic expression of CBX4 is determined by soft agar assay compared with the control of Kras G12D MEFs.Scale bar: 200 μm (left) and 50 μm (right).Data are shown as means ± SEM.Ns p > 0.05, **p < 0.01, ***p < 0.001 apoptosis, and therefore represses tumors in the early stage of cell culture.There's evidence that PRC1 has crucial impact on the initiation of cellular senescence by combining with the promoter of P16 and mediating the transcription. 49Studies have manifested that ectopic expression of some components of PRC1 will suppress the expression of P16, 33 which hints at a possible mechanism of P16's up-regulation.
PcG proteins, as a known epigenetic transcriptional regulator, can recruit different components to form different PRC complexes through histone modification. 5,50,51Studies on the roles of these complexes in different tissues show different effects. 52As a model proposed in Drosophila from previous studies, PRC complexes induce chromatin compaction leading to gene silencing. 7,53owever, the transcription factors involved in this process are not conserved in mammalian cells, so there are still no in-depth research results in related fields.
An important part of this study is the discovery of "tumorization" changes through genome instability in the survival cells of CBX4-related apoptosis described above.Our study finds CBX4 deletion promotes the genomic instability by karyotype analysis.Then 249 genetic mutations are identified in Cbx4 −/− , Kras G12D cells altogether via bioinformatics analysis (Supplementary Table 1).We notice several Hippo pathway related genes and find Hippo pathway is repressed in Cbx4 −/− , Kras G12D cells through WB.At the same time, we notice Wnt signaling pathway appear into the list.Previous articles also indicate that Wnt signaling pathway is involved in the regulation of CBX4 on LUAD formation. 21CBX4 has been shown associated with Wnt signaling pathway in lung cancer, and many other studies have reported that Wnt pathway activation can promote lung cancer by promoting angiogenesis and the expression of related proteins. 54,55The Hippo pathway has also been widely studied in lung cancer, and its activation can inhibit the occurrence and development of lung cancer. 35,36he interaction between CBX4 and these signaling pathways can be a probable direction for later research of mechanism.Though, the exact regulatory mechanism remains to be elucidated.
Our results so far point to the chromosomal instability caused by CBX4 deletion and the selection of MEFs in the Kras G12D background, causing majority of cells to undergo apoptosis, and the survival selected cells will acquire enhanced proliferation and invasion ability, and trend toward tumor formation.If further studies can identify the pathway by which CBX4 affects genomic instability or how the altered genes or signaling pathways affect tumor formation, targeting which will be the clues for LUAD treatment.
In summary, we have shown that loss of CBX4 (as a component of PRC1) influences the chromosome stability, causing cell apoptosis mostly in the initial stage by regulating some apoptosis-related genes' expression under Kras G12D mutation background.CBX4 loss also alter a variety of tumorassociated genes and signaling pathways including Hippo pathway in the survival selected cells, finally inducing tumorigenesis.Our study can provide new clues for future studies on relative targeted therapies.

METHODS AND MATERIALS
Mouse cohorts and treatment LSL-Kras G12D mouse were generous gifts from labs of Kwok-Kin Wong.CBX4 L/L mice were generated by Guoliang Xu lab.All mice were cultivated at Fudan University in the environment of pathogen-free and were treated by protocols granted by the Department of Laboratory Animal Science Fudan University.All mice' genotype was done at the age of 8 ~10 days using the following primers (Supplementary Table 2).All LSL-Kras G12D and Cbx4 L/L , LSL-Kras G12D mice were treated with 2 × 10 6 PFU Adeno-Cre or Lenti-Cre-GFP via nasal inhalation at the age of 8 weeks. 21oss inspection and pathologic examination were conducted accordingly.Analyses of lung tumors of all cultured mice after the dissection were performed.The lung tissue of mice was inflated and fixed in 4% formalin, paraffin embedded, sectioned, and stained with hematoxylin and eosin (H&E) afterwards.Tumor numbers and sizes were analyzed using stained consecutive sections via microscopy and quantitated with ImageJ software.
Fluorescence activating cell sorter (FACS) assay Virus-infected cells was collected at 80% confluency and fixed with 75% ethanol.Propidium iodine (PI) staining was conducted, and flow cytometry was applied for cell cycle analysis.
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay Virus-infected cells were plated in 96-well plate and cell viability were measured daily for 5 days.Twenty μl of MTT working solution (5 mg/ml) were added into each well and incubated for 4 h at 37 °C.Remove the supernatants and dissolve the resultant MTT formazan in 100 μl of dimethyl sulfoxide (DMSO) before reading.The absorbance at 570 and 630 nm were measured subsequently.

3T3 proliferation assay
To evaluate different MEFs' cell proliferative ability, 8 × 10 4 Kras G12D and selected Cbx4 −/− , Kras G12D MEFs were seeded onto 6-well plates.The cells were split into new plates at suitable density every 3 days.Cell numbers were counted and with GraphPad Prism 4 Demo software.

Soft agar assay
In total, 5000 ~10,000 virus-infected cells were re-suspended in culture medium with an additional 0.2% agar and layered them onto 1% agar beds in 6-well plates.The culture medium was changed every 3 days for 2 ~4 weeks, and the samples were subjected to 0.005% crystal violet staining.The numbers of colonies were quantitated by microscopic pictures.
Real-time polymerase chain reaction (PCR) assay RNA was extracted by Trizol (Invitrogen).First strand cDNA was generated using Revert Aid First Strand cDNA Synthesis Kit (Fermentas) following manufacturer's instruction.Genomic DNA samples were extracted with Gentra Puregene Tissue Kit (Qiagen).cDNA was subjected to quantitative real-time PCR with genespecific primers via the 7500 Fast Real-Time PCR System (Applied Biosystems) and the SYBR Green Master PCR mix (Invitrogen) (Supplementary Table 3).Beta actin was used as internal control.
Western blot analysis Total protein lysate was prepared by homogenization in protein loading buffer.Equal amounts of protein were separated by electrophoresis on an SDS-PAGE gel (SDS is from Abcone, L77480) and transferred onto PVDF membranes.Western blot analysis was performed using the following antibodies (Supplementary Table 4).

RNA-seq data analysis
There were three MEFs (Kras G12D , primary Cbx4 −/− , Kras G12D MEFs and selected Cbx4 −/− , Kras G12D MEFs) collected for RNA-seq analysis.RNA-Seq (SE) read raw data obtained from HiSeq platform.FastQC was applied to evaluate the quality of sequencing.Raw reads were mapped by Tophat; the gene counts were conducted by htseq-count; and differentially expressed genes were selected by DESeq with FDR < 0.1.
Immunofluorescence staining analysis Cells were fixed in 4% formaldehyde in PBS buffer at room temperature for 10 min, washed with PBS, and permeabilized by PBST (PBS with 0.25% Triton X-100 (Abcone, X10010)).Cells and frozen sections were blocked for 30 min and incubated at 4 °C with primary antibody (Supplementary Table 5) overnight in PBSA (PBS and 3% BSA).After three times PBST washes at 15 min interval the samples were incubated with Alexa Fluor 552 conjugated goat anti-rabbit secondary antibody at a dilution of 1:1000 for 1 h at a room temperature.Nuclei were stained by DAPI.Samples were mounted with Aqua-Poly/Mount (Polysciences).
Human LUAD samples analysis A total of 72 pathologically confirmed human LUAD specimens and 7 normal lung specimens were collected in Fudan University Shanghai Cancer Center between January 2008 and December 2009 with written consents of patients and the approval from the Institute Research Ethics Committee.All tumor specimens were taken at the time of surgical resection.Patients were also divided into high and low CBX4 expression subgroups with its median expression value as the cut-off. 21atistical analysis Data were presented as mean ± standard error unless otherwise indicated.Student's t test or one-way analysis of variance (twosided) was applied in multiple groups to determine the differences, and Tukey-Kramer multiple comparison test was applied for post hoc comparisons.Kaplan-Meier analysis with log-rank test was used to assess patients' and mice' survival between subgroups.GraphPad Prism 5 software was applied for all statistical analyses, and if p value < 0.05, it was statistically significant.
Fig. 6 CBX4 deletion influences multiple signaling pathways including Hippo pathway and ultimately promotes Kras G12D tumorigenesis.a Western Blot of CBX4, MST1, pMST1/2, MOB1, pMOB1, YAP, pYAP and β-tubulin in Kras G12D and selected Cbx4 −/− , Kras G12D MEFs.b Representative immunohistochemical staining of YAP in lung sections from Kras G12D and Cbx4 −/− , Kras G12D mice.Scale bar: 500 μm (up), 200 μm (down).c Quantitative analysis of nuclear YAP positive cell percent in IHC staining of lung sections from Kras G12D and Cbx4 −/− , Kras G12D mice.d GO enrichment analysis focused Hippo signaling pathway based on independent analysis.e Expression condition of several representative genes in selected Cbx4 −/− , Kras G12D cells compared with Kras G12D and primary Cbx4 −/− , Kras G12D groups.Upper one/row is Kras G12D , the middle one/row is primary Cbx4 −/− , Kras G12D group, the lower one/row is selected Cbx4 −/− , Kras G12D group.Red indicates more expression and blue means less expression.f Mechanism of CBX4 deletion in promoting tumorigenesis with Kras G12D mutation.At the early stage of cell culture, P15, P16 and other apoptosis-related genes are upregulated in the Cbx4 −/− , Kras G12D cells due to chromosome instability, which lead to a large population of cell apoptosis.At the late stage of cell culture, a small population of cells survive from genomic instability acquiring stronger abilities in proliferation and transformation called "selected Cbx4 −/− , Kras G12D ", due to changes of multiple pathways including inactivation of Hippo pathway, which ultimately result in tumorigenesis.Data are shown as means ± SEM. **p < 0.01, ***p < 0.001 Signal Transduction and Targeted Therapy (2023) 8:343