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TCF19 promotes cell proliferation and tumor
formation in lung cancer by activating the
Raf/MEK/ERK signaling pathway
Yahui Tian
Air Force Medical Center
Shaowei Xin
Air Force Medical Center
Zitong Wan
Northwestern University
Lu Liu
Jinan University
Zhenzhen Fan
University of Chinese Academy of Sciences, Chinese Academy of Sciences, China National Center for
Bioinformation
Tian Li
Fourth Military Medical University
Fujun Peng
Shandong Second Medical University
Yanlu Xiong
Tangdu Hospital, Fourth Military Medical University
Yong Han ( hanyong_td@163.com )
Air Force Medical Center
Research Article
Keywords: TCF19, cell cycle, Raf/MEK/ERK pathway, lung cancer, tumor gene
Posted Date: January 17th, 2024
DOI: https://doi.org/10.21203/rs.3.rs-3855398/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Additional Declarations: No competing interests reported.
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Abstract
Objective
This study aimed to investigate the role of TCF19 in lung cancer, focusing on its impact on the
development and progression of tumors. Specically, the objective was to elucidate the molecular
mechanisms underlying TCF19-mediated effects, with a particular emphasis on its involvement in the
RAF/MEK/ERK signaling pathway.
Methods
The research involved the analysis of lung cancer tissues to assess the expression levels of TCF19.
In
vitro
experiments were conducted using lung cancer cells (A549 and Hop62) with TCF19 overexpression.
Transgenic mouse models were employed to study the
in vivo
effects of TCF19 on the development of
primary tumors. Transcriptome sequencing was performed to identify alterations in gene expression
proles, and further experiments were carried out to investigate the activation status of the
RAF/MEK/ERK pathway. Functional assays, including cell cycle progression and the levels of cell cycle-
associated proteins, were conducted to understand the underlying mechanisms.
Results
The research ndings demonstrated signicant overexpression of TCF19 in lung cancer tissues.
In vitro
experiments revealed that TCF19 overexpression stimulated the growth of lung cancer cells and
facilitated the development of primary tumors in transgenic mice. Mechanistically, TCF19 overexpression
was associated with an elevation in the Ras and MAPK signaling pathways, as indicated by increased
phosphorylation of Raf1, MEK1/2, and ERK1/2 in A549 and Hop62 cells. However, the inhibition of RAF1
or ERK, either through shRaf1 or ERK inhibitor, led to a reduction in cell cycle-related proteins and inhibited
cell growth in TCF19-overexpressing cells.
Conclusion
In conclusion, this study identied TCF19 as an oncogene in lung carcinoma. The research highlighted its
specic impact on the RAF/MEK/ERK signaling pathway, offering insights into a novel aspect of the
molecular cascade involved in lung cancer development. Targeting TCF19 or its associated signaling
pathways may present a promising avenue for the management of lung cancer characterized by elevated
TCF19 levels.
1. Introduction
Cancer poses a signicant risk to human well-being, with lung cancer remaining among the most fatal
malignancies globally[1–3]. The World Health Organization's International Agency for Research on Cancer
released the most recent global cancer burden statistics in 2020, revealing a total of 9.96million deaths
caused by cancer. Among these, lung cancer accounted for 1.8million deaths, signicantly exceeding
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other types of cancer and claiming the highest number of lives. We have invested heavily in lung cancer
in basic and clinical research and obtained some effective treatment methods for lung cancer, such as
targeted therapy and immunotherapy, and these treatments have certain therapeutic effects[4].
Nevertheless, the survival rate for individuals with lung cancer remains exceedingly poor[5, 6]. Hence,
further comprehensive investigations are required to examine the mechanism behind the onset,
progression, and management of lung carcinoma.
Among the various MAPKs signals, the RAF-MEK-ERK pathway is most deeply studied, and it is mainly
activated by the orderly phosphorylation of Raf, MEK, and ERK kinases[7]. Cell cycling was promoted by
the Ser/Thr kinases ERK1 and ERK2 in the cytoplasm, while the cytosolic kinases MEK1/2 increased the
activity of ERK1/2 and phosphorylated the Thr/Tyr in the activation loop of ERK1/2. Additional
examination of the kinase cascade uncovered that RAF1 serves as the upstream kinase responsible for
phosphorylating MEK1 at Ser222 and MEK2 at Ser218, thereby controlling the functionality of MEK. This
process enables the transmission of MAPK signaling from RAS, RAF, MEK, and ultimately to ERK[8–11].
The RAF-MEK-ERK signaling pathway is strongly associated with the development of different types of
cancers, and several drugs that target important components of this pathway have been effectively
commercialized[10, 12–15]. The importance of this pathway in controlling the malignant development of
tumors can be demonstrated by the proportion of its marketed drugs and the current research situation.
Nevertheless, the precise connections between TCF19 and the Raf/MEK/ERK signaling pathway, which
facilitates the onset and progression of lung cancer, are yet to be determined.
The TCF19 gene, alternatively referred to as SC1, was initially discovered as a growth modulator in
human, mouse, and hamster cells[16]. Early studies have shown that TCF19 is associated with the
occurrence of type diabetes and type diabetes[17], and is also a risk-related site for chronic hepatitis
B[18]. In the past few years, scientists have discovered that TCF19 inhibits WWC1 in colon cancer, leading
to increased cell growth and movement, exhibiting oncogenic properties[16]. Researchers discovered that
TCF19 enhances the advancement of the G1/S phase in the cell cycle in liver cancer by activating
AKT/FOXO1, consequently stimulating cell proliferation[19]. Zi-Hao Zhou et al. The study demonstrated
that TCF19 hinders FOXO1 to enhance the growth of SPC-A1 and SK-MES-1 cells[20]. And our ndings
suggest that TCF19 drives its oncogene function through an entirely new mechanism in lung cancer.
TCF19 promotes lung cancer tumor progression and cell cycle at least in part by activating
Raf1/MEK/ERK signaling pathway. The ndings presented herein provide preliminary evidence
supporting the notion that the overexpression of TCF19 is instrumental in the abnormal activation of the
Raf1/MEK/ERK signaling pathway. At the same time, we discovered that blocking ERK or using shRaf1
can suppress the growth of lung cancer cells induced by TCF19, offering a solid scientic foundation for
treating lung cancer patients with elevated TCF19 levels.
2. Materials and methods
2.1 Plasmid
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The pLVX-TetOne-puro plasmid was purchased from Clontech, and the pLVX-TetOne-human-TCF19-Flag-
tagged-puro plasmid was constructed using standard molecules. The pCDH-EF1a Plasmid was provided
by Dr. Liang Chen (Jinan University, Guangzhou, China), pCDH-EF1a-human-TCF19-3 × Flag and pCDH-
EF1a-GFP × Flag were constructed from standard molecules. Addgene was the source of the acquisitions
of psPAX2 and pMD2. G. Standard subcloning protocols were used to construct the shTCF19#1
(TRCN0000015410), shTCF19#2 (TRCN0000015411)[29], shTCF19#3 (TRCN0000015412), and shRaf1
(TRCN0000001068)[24] in either the pLKO-vector (Addgene) or pLKO-tet-zeocin (Addgene) vectors.
2.2 Cell lines and cell culture
Dr. Liang Chen (Jinan University, Guangzhou, China) supplied NCI-H322, A549, NCI-H460, EKVX, Hop62,
PC-9, NCI-H466, NCI-H1650, and HEK293T. Lung cancer cells were grown in RPMI-1640 medium
supplemented with 10% fetal bovine serum (FBS, Gibco, 42G9274K) and 1%
penicillin/streptomycin/glutamine (Gibco, 15140122). HEK293T was grown in DMEM medium
supplemented with 10% fetal bovine serum (FBS, Gibco, 42G9274K) and 1%
penicillin/streptomycin/glutamine (Gibco, 15140122).
HEK293T cells were transfected with pCDH-EF1a-human-TCF19-3 × Flag/pCDH-EF1a-GFP × Flag/pLKO-
shTCF19/ pLKO-tet-shRaf1-zeocin using psPAX2 and pMD2. G, along with Lipofectamine 3000
(Invitrogen) as the transfection reagent. Fresh media replaced the culture 6–8 hours following
transfection, and then the supernatant was collected after 48 hours and ltered using a 0. 45µm
sterilization lter. The medium containing the modied virus was introduced to the specied cells while
polybrene (8 µg/mL) was present. The infected cells were selected with puromycin or zeocin for 7 days
before additional experiments were performed.
2.3 Human tissues
Approval for the human lung cancer clinical samples collection was granted by the Sunshine Union
Hospital in Shandong province, China. All the patients provided written consent. Pathologists from the
Department of Pathology at Sunshine Union Hospital reviewed all cases to verify the histology and
content of the tumors. The research materials received approval from the Human Ethics Committee at
Weifang Medical University. All work was performed following the approved protocol.
2.4 Western blot analysis
The Western blot analysis in this investigation was conducted according to the methods described
earlier[30]. RIPA lysis buffer (Santa Cruz) along with Protease and phosphatase inhibitor cocktail (Biotech
Roche) were used to lyse cells or clinical samples of lung cancer. In this study, the antibodies used
included anti-TCF19 (diluted 1:1000, YT5078, Immunoway), anti-MEK1/2 (diluted 1:500, sc-81504, Santa
Cruz, USA), anti-p-MEK1/2 (diluted 1:500, sc-81503, Santa Cruz, USA), anti-ERK1/2 (diluted 1:1000,
#4695, Cell Signaling Technology), anti-p-ERK1/2 (diluted 1:1000, #4370, Cell Signaling Technology),
anti-Raf-1 (diluted 1:500, sc-7267, Santa Cruz, USA), anti-p-Raf-1 (diluted 1:500, sc-271919, Santa Cruz,
USA), anti-FLAG (diluted 1:1000, #14793, Cell Signaling Technology), CyclinA1 (diluted 1:1000, CY1027,
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Abways), CyclinD1 (diluted 1:10000, ab228528, Abcam), CyclinE1 (diluted 1:1000, #20808, Cell Signaling
Technology), CDK2 (diluted 1:10000, ab228528, Abcam), anti-GAPDH (diluted 1:1000, #5174, Cell
Signaling Technology), and anti-β-actin (diluted 1:1000, A5316, Sigma). We bought the anti-rabbit and
anti-mouse IgG conjugated with horseradish peroxidase from sigma (A6154, 1 1000; A0168, 1 1000). The
results were obtained using Western blotting substrate (Millipore).
2.5 Real-time PCR
Trizol reagent (Invitrogen) was employed for total RNA extraction, followed by quantitative reverse
transcription PCR with gene-specic primers (5’-3’) as Table 1.
Table 1
Primers for detecting gene transcription.
Gene (Human) Primers (5’-3’)
TCF19-F TCAGCCTGGAAGACCACAGCAG
TCF19-R CCAAAGGTCAGGAGGTCTCCAT
CDK2-F ATGGATGCCTCTGCTCTCACTG
CDK2-R CCCGATGAGAATGGCAGAAAGC
Cyclin A1-F GCACACTCAAGTCAGACCTGCA
Cyclin A1-R ATCACATCTGTGCCAAGACTGGA
Cyclin E1-F TGTGTCCTGGATGTTGACTGCC
Cyclin E1-R CTCTATGTCGCACCACTGATACC
Cyclin D1-F TCTACACCGACAACTCCATCCG
Cyclin D1-R TCTGGCATTTTGGAGAGGAAGTG
β-actin-F CACCATTGGCAATGAGCGGTTC
β-actin-R AGGTCTTTGCGGATGTCCACGT
2.6 Cell proliferation assay
A total of one thousand and three cells were placed in 96-well plates and grown in RPMI medium 1640
supplemented with 10% FBS. The cells were treated with or without Doxycycline hyclate (DOX, 0. 5
µg/mL, Sigma) for a duration of 1 to 5 days. Cell proliferation was assessed by employing the Cell
Counting Kit-8 (CCK8, Dojindo Molecular Technologies) according to the instructions provided by the
manufacturer.
2.7 Colony-forming assay
For the colony forming experiment, 0.5 × 103 cells were placed in 6-well dishes using RPMI medium 1640
supplemented with 10% FBS. Following a period of 14 days, the cells underwent rinsing using 1 × PBS,
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subsequent xation with formaldehyde for a duration of 10 minutes, and nally staining with 0.05%
crystal violet for a period of 30 minutes. The software Image J was used to tally the colonies.
2.8 Soft agar assay
Soft agar was carried out as described in our previous report[21]. Base agar (0.6%, 2 × DMEM + 20% FBS +
2% PS + 1.2% agar) and top agar (0.35%, 2 × DMEM + 20%FBS + 2% PS + 0.7% agar) were prepared
respectively. 1 mL base agar was added into each well of 6-well plates and left for 1 h. 1 × 104 cells were
mixed with 2 mL top agar with or without DOX (0.5 µg/ml), and then the mixture was seeded into 6-well
plates. Every 4 days, 0.2 mL of 1 × liquid medium was applied onto the top gel, with or without DOX (0.5
µg/ml) on its surface. Following incubation in a moist incubator at a temperature of 37°C for a period of
3 to 4 weeks, the number of colonies was determined using an optical microscope placed upside down.
2.9 Cell cycle analysis
At 48 h post-DOX treatment, cells were gathered and subsequently preserved in 70% ethanol for an
overnight period at 4℃. The cell cycle analysis was conducted by employing the cell cycle kit (Beyotime)
according to the instructions provided by the manufacturer. Subsequently, the analysis was carried out
using a BD AccuriTM C6 ow cytometer.
2.10 RNA sequencing
After a 48-hour incubation period, cells were subjected to treatment with or without DOX, followed by
extraction of total RNA using Trizol reagent. The Institute of Genomics conducted RNA sequencing and
created RNA libraries.
2.11
In vivo
xenograft model and KrasG12D/CC10rtTA
transgenic mice model
Every mouse was kept in a sterile setting at Weifang Medical University, and all research procedures
adhered to the Ethical Guidelines for Laboratory Animal Usage and were sanctioned by the Ethics
Committee of Weifang Medical University. All animal research was conducted in strict compliance with
authorized procedures. All surgery was performed under 2∼3% isourane, and every effort was made to
minimize suffering.
Xenograft assay was performed as described in our previous report [21, 22]. A total of twelve 6-week-old
female BALB/c nude mice were subcutaneously implanted with 2 × 106 cells suspended in 100 µL of
Matrigel (Corning) on their right ank. Once the tumors reached a size of approximately 80 mm3, the
mice were divided into two groups using a pair comparison method. This involved carefully matching the
animals based on factors such as age, sex, weight, and others. Essentially identical animals were paired
up, and each pair was then randomly assigned to one of the two groups. The mice fed with DOX food as
experiment group(n = 6), fed with normal food as control group(n = 6). The tumor's size was observed
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every second day for 1–2 weeks until the experiment concluded[21, 22]. At the conclusion of the
experiment, the mice were euthanized, and the tumors were gathered, photographed, and measured.
A transgenic mice experiment was performed as described in our previous report[22]. TetO-
KrasG12D/CC10rtTA transgenic mice were provided by Dr. Liang Chen (Jinan University, Guangzhou,
China). After consuming food containing doxycycline for a period of 2 months, the mice developed lung
adenocarcinoma. Lentivirus overexpression Tetone-TCF19-FLAG(n = 6) or control virus(n = 6) was
administered intranasally to TetO-KrasG12D/CC10rtTA mice on a diet containing doxycycline. Following
a 2-month period, the mice underwent computed tomography (CT) scans using the PINGSENG Healthcare
recorder SNC-100. Subsequently, they were euthanized. Lung tissues were gathered for hematoxylin &
eosin (H&E) staining.
2.12 Statistical analysis
Statistical analyses were carried out using GraphPad Prism 8.0 software. tatistical signicance was
determined by analyzing all experimental data using the two-tailed Student's t-test, with a signicance
level of p < 0. 05. All error bars represent SEM.
3. Results
In lung cancer, TCF19 is a signicant and medically pertinent tumor suppressor gene. The role of TCF19
as a tumor suppressor gene (TSG) has been documented in different types of cancer, although its
specic mechanism in lung cancer remains unknown. Hence, we examined the effect of TCF19 on the
progression of lung carcinoma. By analyzing the TCGA database (http//kmplot.com/analysis), it was
found that the prognosis of NSCLC patients in all stages was signicantly associated with the expression
level of TCF19 (Fig.1A, left). Additionally, this correlation was also observed in stage of NSCLC patients
(Fig.1A, right). According to the UCSC website (https//xenabrowser.net/), Fig.1B demonstrates a notable
increase in TCF19 mRNA expression in lung tumor tissues compared to Para-tumor tissues. Then we
conrmed that TCF19 was expressed at higher mRNA and protein levels in NSCLC tumors than in their
Para-tumor tissues (Fig.1C, D). The ndings indicate a potential association between TCF19 expression
and the onset and progression of lung cancer, highlighting its signicance in clinical research.
3.1 The ectopic expression of TCF19 promotes the proliferation and tumorigenesis of lung cancer cells in
vitro
To explore the function of TCF19, we examined its expression in different lung cancer cell lines (see
Supplementary Fig.1A and 1B). Our ndings revealed that TCF19 expression was comparatively low in
two lung cancer cell lines (H460 and Hop62), while two other lung cancer cell lines (H466 and PC-9)
exhibited relatively high TCF19 expression. Subsequently, we generated stable cell lines that exhibited
elevated levels of TCF19 (H460- TCF19 and Hop62-TCF19), as depicted in Supplementary Fig.1C and 1D,
and reduced TCF19 (H466-shTCF19 and PC9-shTCF19), as illustrated in Supplementary Fig.1E, F, G, and
H). The proliferation and viability of lung cancer cell lines modied with TCF19 were assessed using
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colony-formation and CCK8 assay. The colony-formation experiment demonstrated that the excessive
expression of TCF19 enhances the growth of H460 and Hop62 cells (Fig.2A and C). The CCK8 test
demonstrated that the increased expression of TCF19 improved the survival of H460 and Hop62 cells
(Fig.2B and D). However, according to the colony-formation and CCK8 assay experiments, the stable
lines with TCF19 knockdown grew much more slowly than the control cells (Fig.2E, F, G, and H). The
ndings from all these experiments indicated that the TCF19 protein has the ability to enhance the
growth of lung cancer cells in a laboratory setting.
The soft agar experiment can reect the malignant degree of tumor cells. Then, we performed soft agar
experiments according to the operating procedures of the previous articles[21, 22]. Figure2I demonstrated
that the elevated TCF19 expression greatly enhanced the formation of soft agar colonies in H460 and
Hop62 cells. Moreover, the knockdown of TCF19 inhibited the tumorigenic ability of PC9 and H446 lung
cancer cells (Fig.2J). These results suggested that TCF19 can signicantly promote the tumorigenic
ability of lung cancer cells and has an oncogene phenotype.
3.2 TCF19 promotes the growth of xenograft and primary
lung cancer
in vivo
The effect of TCF19 was also tested
in vivo
. H460-TCF19 cells were injected subcutaneously into nude
mice and then divided into two groups, namely the control group and the TCF19 group, as depicted in
Fig.3A. Notably, the control group (Con) exhibited a comparatively slower rate of tumor growth in
contrast to the TCF19 group (Fig.3B, C). Additionally, the tumor weight in the control group was markedly
lesser than that of the TCF19 group (Fig.3D). Subcutaneously, we injected PC9-shTCF19 cells into nude
mice and then randomly separated them into two groups: the shGFP group and the shTCF19 group.
Based on the test ndings, it was observed that the tumors in the shGFP cohort exhibited accelerated
growth compared to those in the shTCF19 cohort (as depicted in Fig.3E and F). Additionally, the
suppression of TCF19 resulted in a decrease in tumor weight (as shown in Fig.3G).
To further verify the function of TCF19
in vivo
, we tested the tumor formation following TCF19
overexpression in a transgenic lung cancer mouse model. The DOX-induced KrasG12D/CC10rtTA mouse
model was utilized for studying lung cancer. After being fed DOX food for 2–3 months, this mouse model
acquired lung cancer, whereas it remained free from lung cancer when given regular food[21, 22]. By
adhering to this procedure, we administered lentivirus-overexpressing Teto-TCF19-Flag or a control virus
through the nasal route to TetO-KrasG12D/CC10rtTA mice. Consequently, the mice began expressing
KrasG12D and TCF19 or control in their lung epithelial cells upon consumption of a DOX diet (Fig.3H).
The CT scan and HE staining ndings indicated that the lung tumor load was greater in transgenic mice
exhibiting elevated TCF19 expression compared to the control group (Fig.3I, J). Therefore, our data
revealed a potent oncogene function of TCF19
in vivo
.
3.3 TCF19 promotes the progression of the lung cancer cell
cycle
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Our results
in vitro
and vivo conrmed that TCF19 could promote the growth process of lung cancer, and
we usually think that the change in cell proliferation is closely related to cell cycle regulation[23].
Therefore, we used ow cytometry to detect the difference in cell cycle between lung cancer cells
overexpressing TCF19 and the control group. In our ndings, it was observed that the excessive
expression of TCF19 greatly enhanced the progression of the S phase and G2/M phase in the cell cycle,
whereas the G0/G1 phase experienced a notable decrease in H460-TCF19 and Hop62-TCF19 cells (as
shown in Fig.4A, B). The Western blotting and RT-PCR ndings indicated that the upregulation of TCF19
in H460 and Hop62 cells enhanced the levels of proteins associated with the cell cycle (Fig.4C, D). All
these data indicated that the promotion of the cell cycle in TCF19-overexpression cells must be
responsible for its effect on the proliferation of lung cancer cells.
Lung cancer experienced activation of the Raf/MEK/ERK pathway due to TCF19.
In order to further investigate the potential mechanism through which TCF19 enhances the progression of
lung cancer, transcriptome sequencing was conducted (Fig.5A). The Ras and MAPK signaling pathway in
the TCF19-overexpression group were enriched by KEGG enrichment analysis (Fig.5B). It is common
knowledge that Raf1 plays a crucial role as a protein in the Ras signaling pathway, and the conventional
Raf/MEK/ERK signaling pathway is strongly associated with the role of Raf1 in the progression of
cancer[24]. Surprisingly, it was discovered that the phosphorylation status of Raf1, MEK1/2, and ERK1/2
proteins exhibited a notable rise in H460-TCF19 and Hop62-TCF19 cells (Fig.5C). In addition, we
observed a rise in proteins related to the cell cycle and the elevated levels of protein phosphorylation
linked to the Raf/MEK/ERK signaling pathway (Fig.5D). The ndings indicated that TCF19 triggers the
activation of the Raf/MEK/ERK pathway in lung carcinoma.
3.4 The Raf1/MEK/ERK signaling pathway has a signicant impact on the TCF19-driven growth of lung
cancer cells.
The ndings indicated that TCF19 has the ability to enhance the cell cycle progression and elevate the
phosphorylation status of crucial proteins within the Raf/MEK/ERK signaling pathway. Thus, our
hypothesis suggests that TCF19 has the ability to enhance the advancement of the cell cycle via the
Raf/MEK/ERK pathway, consequently fostering the growth of lung cancer cells. To verify this hypothesis,
we used shRaf1 to knock down Raf1 in Hop62-TCF19 cells (Supplementary Fig.2A). From the results, we
found that shRaf1 could signicantly inhibit the cell proliferation caused by TCF19 in Hop62 by Colony-
formation and CCK8 (Fig.6A, B). shRaf1 has the ability to suppress the phosphorylation of proteins such
as Raf1, MEK1/2, and ERK1/2. In addition, cell cycle-related proteins caused by TCF19 were also inhibited
by shRaf1 in Hop62 cells (Fig.6C).
Regulating the expression of cell cycle-related proteins is a crucial function of ERK activation during the
cell cycle process[25]. To validate the role of activated ERK1/2 in promoting cell cycle advancement in
lung cancer cells exhibiting elevated TCF19 levels, we employed an ERK inhibitor (ERKi, GDC-0994) to
hinder the phosphorylation of ERK1/2 induced by TCF19 in lung cancer cells (Supplementary Fig.2B).
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When Hop62-TCF19 cells were treated with ERKi, cell proliferation was signicantly inhibited (Fig.6D and
E), and the protein and mRNA level of cell cycle-related proteins was also signicantly down-regulated
(Fig.6F). The ndings from these experiments indicate that TCF19 enhanced the growth of lung cancer
cells and the development of tumors in both laboratory conditions and living organisms by increasing the
levels of cyclin D1, cyclin E1, cyclin A1, and CDK2 through the Raf/MEK/ERK signaling pathway. Notably,
we analyzed the TCGA database and found that TCF19 was positively correlated with CCNA1, CCNE1,
CDK2, RAF1, MAPK3, and MAPK1 in lung cancer tissue samples (supplementary Fig.3A-F).
In summary, these results further conrmed that Raf/MEK/ERK cascade is required for TCF19 to function
in lung cancer cells.
4. Discussions
From the start of this century, the progress in the clinical management of lung cancer has transitioned
from 'identical medication for various patients' to 'a single medication for each patient', progressing
towards a precise and personalized approach of 'a unique prescription for every patient'. Therefore,
precisely targeted therapy strategies for oncogenes are urgently needed to continuously update and
improve the prognosis of patients. Within this investigation, TCF19 exhibited signicant upregulation in
certain individuals diagnosed with lung cancer, and this elevated expression of TCF19 demonstrated a
direct association with unfavorable outcomes in lung cancer patients. In lung adenocarcinoma cells
H460 and Hop62, TCF19 exhibited signicant expression. The proliferation and malignancy of lung
adenocarcinoma cells were enhanced by the overexpression of TCF19, as demonstrated by Cck8, plate
clone formation, and Soft Agar experiments. Indeed, we showcased the growth-enhancing impact of
TCF19 not just in laboratory settings but also in live organisms by utilizing a xenograft tumor model and
a transgenic mouse model for lung cancer that occurs naturally.
Malfunctioning of the Ras-ERK pathway serves as a primary catalyst for the formation of the majority of
cancer types. The ERK cascade is activated in nearly all cancer types, making mutations that activate this
pathway the most prevalent oncogenic elements across all cancer types[26–28]. The MAPK signaling
pathway is crucial in regulating a range of physiological processes including cellular growth,
development, proliferation, and apoptosis within the network of signaling pathways. ERK belongs to the
MAPK group, and the ERK/MAPK signaling pathway plays a central role in controlling cell growth,
development, and division[26]. This statement also conrmed our ndings. In terms of mechanism, we
discovered that TCF19 stimulates the rise in cyclins by amplifying the phosphorylation of crucial proteins
in the Raf/MEK/ERK signaling pathway, resulting in the proliferation of cancer cells. Notably, TCF19 was
found to be positively correlated with CCNA1, CCNE1, CDK2, RAF1, MAPK3, and MAPK1 in lung cancer
tissue samples in the TCGA database. These ndings suggest TCF19 upregulation affects the prognosis
of lung cancer patients by promoting RAF/MEK/ERK signaling pathway.
Collectively, these ndings suggest that shRaf1/ERKi synergistically blocks TCF19-induced lung cancer
cell proliferation and promotes downregulation of cell cycle checkpoints. Therefore, our results indicate
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that targeting Raf1 or ERK may help suppress the malignant progression of TCF19-overexpressing lung
cancer.
Declarations
Author contributions
YH, YX, and YT designed the project. YT, SX and ZW performed all of the experiments and collected the
data. SX collected the tumor samples of lung cancer paitents. YT, ZF and LL, ZF, TL analyzed the data.
YH, YX, and FP collaborated in the writing and thorough evaluation of the manuscript. The article was
contributed to by all authors and the submitted version was approved by them.
Acknowledgments
We thank Dr. Chen for providing the KrasG12D/+ mice and lung cancer cell lines. We appreciate the
reviewers for their valuable feedback on this manuscript. We thank ying Chen for providing us with the
lung cancer tumor sample from Sunshine Union Hospital in Shandong province.
The accessibility of information and resources
The datasets used and analyzed during the current study are available from the corresponding authors
on reasonable request.
Conict interest
The authors did not report any conicts of interest.
Additional information
Finding
The Natural Science Foundation of Shandong Province (ZR2022QH146, ZR2021QH367), the China
Postdoctoral Science Foundation (2023MT44294), the National Natural Science Foundation of China
(82373102, 82002421 and 82103081), the Youth Talent Lifting Program of Shaanxi Association for
Science and Technology (20230311), and the Key research and development plan of Shaanxi Province
(2023-YBSF-318) provided support for this work.
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Figures
Figure 1
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TCF19 is an important tumor gene in lung cancer. A. Kaplan-Meier survival analysis of TCF19-
overexpression and TCF19-underexpression lung cancer patients. B. Expression level of TCF19 in lung
tumor tissues and Para-tumor tissues (TCGA database). C, D. TCF19 mRNA and protein levels in lung
cancer patient tissue samples (P: Para-tumor tissues, T: Tumor tissues). Data are means ± SEM of three
independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t-test).
Figure 2
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TCF19 overexpression promotes lung cancer cell growth and tumorigenicity
in vitro
. A. Colony-forming
evaluated the effects of TCF19 overexpression on the growth and proliferation of H460. B. CCK8
evaluated the effects of TCF19 overexpression on the growth and proliferation of H460. C. Colony-
forming evaluated the effects of TCF19 overexpression on the growth and proliferation of Hop62 cells. D.
CCK8 evaluated the effects of TCF19 overexpression on the growth and proliferation of Hop62 cells. E.
Colony-forming evaluated the effects of TCF19 knock-down on the growth and proliferation of PC-9 cells.
F. CCK8 evaluated the effects of TCF19 knock-down on the growth and proliferation of PC-9 cells. G.
Colony-forming evaluated the effects of TCF19 knock-down on the growth and proliferation of H446
cells. H. CCK8 evaluated the effects of TCF19 knock-down on the growth and proliferation of H446 cells.
I. Soft-agar assay of H460 and Hop62 cells in the presence of TCF19. J. Soft-agar assay of PC-9 and
H446 cells in the absence of TCF19.
Data are means ± SEM of three independent experiments. * p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001
(Student’s t-test).
Page 17/22
Figure 3
TCF19 promoted tumorigenesis
in vivo
. A. Schematics for the xenograft treatments of nude mice. 2 × 106
cells were subcutaneously inoculated into the right ank of 8-wk-old female nude mice fed with normal
food or DOX food. B,C,D. Tumor growth curves (B) were calculated based on the monitoring data every
other day after subcutaneously injecting Hop62-TCF19 cells into female nude mice. The xenograft
tumors were dissociated, photographed (C), and weighed (D) at the end of the experiments. (n = 6). E, F, G.
Page 18/22
Tumor growth curves (E) were calculated based on the monitoring data every other day after
subcutaneously injecting H446-shTCF19 cells into female nude mice. The xenograft tumors were
dissociated, photographed (F), and weighed (G) at the end of the experiments. (n = 6, shGFP as control
group and shTCF19 as TCF19 knock-down group). H. Schematics of intranasal instillation of retrovirus
for forced expressing TCF19 in Dox inducible Tet-KRASG12D/CC10rtTA lung cancer mouse model. (n = 6;
mice fed with normal food as the control group, mice fed with DOX to induce TCF19 overexpression as
TCF19 overexpression group). I. Ectopic expression of TCF19 promotes lung cancer formation in TetO-
KrasG12D/CC10rtTA mouse model. J. Representative images of Hematoxylin and eosin (H&E) staining of
the lung tissues obtained from lenti-control and lenti-TCF19 treated KrasG12D/CC10rtTA mice.
Data are means ± SEM of three independent experiments. * p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001
(Student’s t-test).
Page 19/22
Figure 4
TCF19 promoted lung cancer cell cycle progression. A. The cell cycle of H460-TCF19 cells was analyzed
by ow cytometry. B. The cell cycle of Hop62-TCF19 cells was analyzed by ow cytometry. C, D. The
expression of cyclin A1, cyclin E1, cyclin D1, CDK2, and TCF19 in H460- TCF19 and Hop62-TCF19 cells
was detected by western blotting.
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Data are means ± SEM of three independent experiments. * p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001
(Student’s t-test).
Figure 5
TCF19 activated Raf/MEK/ERK pathway. A. Heatmap of mRNA expression of Hop62-TCF19 cells. Cells
were extracted for RNA-sequencing analysis. B. KEGG enrichment analysis of signal pathways affected
Page 21/22
by TCF19. C. The effect of TCF19 on the key proteins of Raf/MEK/ERK signaling pathway in H460 and
Hop62 cells by western blotting. D. The relationship between TCF19 and proteins related to Raf/MEK/ERK
signaling pathway or cell cycle in clinical lung cancer tissue samples. Data are means ± SEM of three
independent experiments. * p < 0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t-test).
Figure 6
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TCF19 promoted cell proliferation through the Raf/MEK/ERK signaling pathway. A, B. TCF19 promoted
the growth of lung cancer cells through Raf1 by colony-forming and CCK8 experiments. C, D. TCF19
promoted the growth of lung cancer cells by activating ERK1/2. shRaf1 inhibited the promoting effect of
TCF19 on the lung cancer cell Hop62 by western blotting. E. ERKi inhibited the promoting effect of TCF19
on lung cancer cells Hop62 by western blotting. F. The schematic diagram of TCF19 promoting lung
cancer cell proliferation and tumor growth. Data are means ± SEM of three independent experiments. * p <
0.05, **p < 0.01, *** p < 0.001, **** p < 0.0001 (Student’s t-test).
Supplementary Files
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