Oncogene www.nature.

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ARTICLE
TP53 mutations upregulate RCP expression via Sp1/3 to drive
lung cancer progression
2,3,4 ✉
Caihong Wang1,2,3,4,6, Shaosen Zhang2,3,4,5,6, Boyuan Ma2,3,4, Yan Fu2,3,4 and Yongzhang Luo

© The Author(s), under exclusive licence to Springer Nature Limited 2022

Mutant p53 (mtp53) can exert cancer-promoting activities via “gain-of-function”, which has become a popular research target.
Although lots of researchers focus on the tumor-suppressor role for p53, the regulation of mutant p53 remains unknown. Here, we
report a mechanism by which mtp53 regulate the transcription of Rab coupling protein (RCP) to influence lung cancer behavior.
First, we show that RCP is specifically expressed at high levels in lung cancer tissues and cells, and RCP knockout suppresses tumor
growth and metastasis. Further mass spectrometry and functional analysis identify that Sp1, Sp3 and Stat3 are the transcriptional
activators of RCP. Moreover, p53 is involved in modulating RCP expression in an Sp1/3 dependent manner. Mechanistically, in
contrast to wild-type p53 suppression of RCP transcription by decreasing Sp1/3 proteins, TP53 mutations have changed on Sp1/3
expression via “loss-of-function”. Surprisingly, the DNA contact mutants of p53 further robustly enhance their binding ability with
Sp1/3 to drive RCP expression through the “gain-of-function” activity. Collectively, we reveal a mechanism by which p53 regulating
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the transcription of RCP to influence lung cancer progression, which provides new insights for treating p53 mutant lung cancer.

Oncogene (2022) 41:2357–2371; https://doi.org/10.1038/s41388-022-02260-7

INTRODUCTION Accumulating studies reported oncogenic properties of RCP in

Lung cancer is the first leading cause of cancer-related mortality
around the world [1]. Patients with lung cancer are often
diagnosed with advanced stages of disease, associated with
frequent metastases and a poor 5-year survival rate of about
4–17% [2]. Cancer development is a multi-step pathogenesis, and
the progressive accumulation of genetic and epigenetic altera-
tions invariably lead to dysregulated transcriptional programs.
These dysregulated programs drive tumor malignant progression
by increasing tumor growth and invasiveness. In recent years,
gene-expression patterns, copy number alterations have been
revealed playing pathological roles in human cancers [3]. The
magnitude and frequency of transcriptional reprogramming were
reported to be closely associated with clinical features of human
cancers [4]. It is known as transcriptional addiction, which reveals
vulnerable points in cancer therapy. Tumor progression depends
on the aberrant activity of dysregulated transcriptional programs.
Therefore, disrupting these reprogramming pathways is generally
sufficient to induce growth arrest and reprogram malignant
cancer cells toward benign phenotype.
RCP belongs to a member of the class I Rab11 family of
interacting proteins (Rab11-FIP1) [5]. RAB11FIP1 is located within a
genomic region (8p11-12), which is usually amplified in breast
cancer [6]. RCP is upregulated in estrogen receptor (ER) positive
cancer and function as an oncoprotein to enhance tumor
progression [7]. Except that, the elevated level of RCP expression
was reported in head and neck squamous cell carcinoma [8], colon
cancers [9] and non-small cell lung carcinoma (NSCLC) [10].
promoting multiple tumor-associated processes [11–16]. RCP has
been reported to drive integrin β1 [17–19], EGFR [20, 21],
N-cadherin [22] and TfR1 [23] to transmit oncogenic signals.
However, the oncogenic properties of RCP in lung cancer remains
unclear.
Lung cancer development mainly depends on genetic abnorm-
alities. As one of the most important tumor suppressor genes,
TP53 is frequently mutated in lung cancer. A recent study
identified that 90% of small cell lung cancer cases containing
TP53 mutation [24]. p53 has been reported to regulate cell cycle,
cell division, DNA damage repair, and apoptosis [25]. As a
canonical tumor suppressor, p53 is often inactivated because of
missense mutations [26]. These missense mutations can lose p53’s
tumor suppressor functions that exhibits loss-of-function (LOF)
activities [27]. However, some of them can generate oncogenic
mutant p53 proteins to promote tumor malignancy that exhibits
gain-of-function (GOF) activities [28–30]. Moreover, mutant p53
can cause the amplification of oncogenes [31, 32]. For instance,
mutant p53 can promote tumor progression by binding to and
upregulating chromatin regulatory genes, like MLL1, MLL2, leading
to genome-wide increases of histones methylation and acetylation
[33]. Moreover, mutant p53 can accelerate the recycling of integrin
beta1 and EGFR to exert its oncogenic function, which is RCP-
dependent [21]. Previous data also suggest that mutant p53
exhibits GOF activities in an RCP/WIP/YAP signaling axis [34]. In
addition, RCP was reported to promote epithelial-mesenchymal
transition in a mutant p53 dependent manner [35]. These reports

1
Beijing Institute of Tropical Medicine, Beijing Friendship Hospital, Capital Medical University, Beijing 100050, China. 2School of Life Sciences, Tsinghua University, Beijing 100084,
China. 3The National Engineering Research Center for Protein Technology, Tsinghua University, Beijing 100084, China. 4Beijing Key Laboratory for Protein Therapeutics, Tsinghua
University, Beijing 100084, China. 5Department of Etiology and Carcinogenesis, National Cancer Center/Cancer Hospital, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100021, China. 6These authors contributed equally: Caihong Wang, Shaosen Zhang. ✉email: yluo@tsinghua.edu.cn

Received: 14 November 2021 Revised: 11 February 2022 Accepted: 18 February 2022

Published online: 7 March 2022
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Fig. 1 RCP is highly expressed in human lung cancer. A RCP protein levels in lung normal epithelial cells (BEAS-2B), lung normal fibroblast
(MRC-5) and lung cancer cells (A549, H1299, H157, H358, H441, H460, H520) were analyzed by western blot (WB). The amount was quantified
and normalized to the BEAS-2B cells. Representative results from three independent experiments are presented. B Representative images
(top) and relative intensity (bottom) showing RCP protein levels in lung normal tissues and lung cancer tissues. Scale bars, 500 μm. Raw RCP
immunohistochemistry image panels from The Human Protein Atlas (https://www.proteinatlas.org/ENSG00000156675 RAB11FIP1/pathology/
lung+cancer#img). C Representative images showing RCP protein levels in healthy people, benign and malignant lung cancer samples, and
correlation analysis of RCP immunohistochemistry scores with tumor malignancy (chi-square test). Scale bars, 500 μm (top) and 100 μm
(bottom). Data are represented as mean ± SD. ***p < 0.001. A One-way analysis of variance (ANOVA) followed by Dunnett’s multiple
comparisons. B Two-tailed Student’s t-tests. C Chi-square test.
Oncogene (2022) 41:2357 – 2371

suggest the critical role of RCP in the GOF of mutant p53.
However, whether p53 involved in regulation of RCP expression is
still unclear.
One of our previous works has demonstrated that mutant p53
drives cancer metastasis via RCP mediated exosome associated
Hsp90alpha secretion [13]. In the current study, we determined
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the expression and roles of RCP in lung cancer at first. DNA probe
pulldown assays and mass spectrometry profiling were done to
identify the upstream regulators of RCP. We further explore the
underlying molecular mechanisms by which p53 involving in
regulation of RCP expression through chromatin immunoprecipi-
tation, immunofluorescence and co-immunoprecipitation
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Fig. 2 RCP Drives Lung Cancer Cell Proliferation and Invasion in vitro. A Levels of cell surface EGFR and integrin β1 upon plasma membrane
(PM) from H1299 and H460 cells with or without RCP expression were detected by WB. RCP was knocked out by CRISPR/cas9 system, and
recovered by exogenous RCP overexpression vectors. The amount of EGFR and integrin β1 upon PM was quantified and normalized to the NC
group. Representative results from three independent experiments are presented. B The cell proliferation of H1299 and H460 with or without
RCP expression was measured using the cell counting kit 8 (CCk-8) at 24 h and 48 h. C Colony formation ability of H1299 and H460 cells with or
without RCP expression. Representative images (top) and quantitative numbers of colonies in six-well plates (bottom). Representative results
from three independent experiments are presented. Scale bar, 1 cm. D Invasion capability of H1299 and H460 cells with or without RCP
expression. Representative images (top) and quantitative numbers of invaded cancer cells per view (bottom). Scale bars, 200 μm. E Levels of
EGFR and integrin β1 upon plasma membrane (PM) from RCP overexpressed in BEAS-2B cells were analyzed by WB. F The cell proliferation of
BEAS-2B with RCP overexpression or empty vector control was measured using the cell counting kit 8 (CCk-8) at 24 h and 48 h. G, H Colony
formation (G) and invasion (H) capability of BEAS-2B cells with RCP overexpression or empty vector control. Quantitative numbers of colonies
(G, top) and representative images (G, bottom) in six-well plates. Scale bar, 1 cm. Quantitative numbers of invaded cells per view (H, left) and
representative images (H, right), representative results from three independent experiments are presented. Scale bars, 200 μm. Data are
represented as mean ± SD calculated from three independent experiments and each had four technical replicates. *p < 0.05; **p < 0.01; ***p <
0.001; ****p < 0.0001 and ns, not significant. Two-tailed Student’s t-tests (F, G, and H), one-way analysis of variance (ANOVA) followed by
Dunnett’s multiple comparisons (B, C, and D).

experiments. Our results showed that high expression of RCP
enhanced lung cancer progression. Sp1, Sp3 and Stat3 are
identified as transcription activators of RCP. And mutations in
TP53 upregulate RCP expression in different manners, which are
Sp1/3 dependent. Collectively, these findings verify that mutant
p53 transcriptional upregulated RCP to drive aggressive lung
cancer behavior, which provide new insights for the biological
networks underlying lung cancer pathogenesis.
RESULTS
RCP is highly expressed in human lung cancer
To explore the roles of RCP in lung cancer, we first detected the
relative RCP levels in human lung cancer cell lines (A549, H1299,
H157, H358, H441, H460 and H520) and noncancerous cell lines
(BEAS-2B and MRC-5) from lung tissue. Compared with non-
cancerous cells, the expression of RCP was significantly increased
in all lung cancer cell lines (Fig. 1A). In clinically, RCP protein levels
in tumor tissue sections are higher than in peritumoral and normal
lung tissues (The Human Protein Atlas, RCP expression in lung
tissue) (Fig. 1B). Moreover, immunohistochemistry (IHC) stain of
RCP was performed on a commercial lung cancer patient tissue
microarray. A higher RCP expression score in advanced and
metastatic stage was observed, and a chi-square test showed that
there are significant differences both between normal and tumor
tissues (p < 0.0001) and between early and late stages of tumor
tissues (p = 0.001459) (Fig. 1C, Table S1). Taken altogether, these
results show that the expression levels of RCP are significantly
upregulated in lung cancer and positive associated with lung
cancer progression.
RCP drives lung cancer cell proliferation and invasion in vitro
RCP had been reported to maintain the abundance of
membrane proteins from recycling vesicles to boost oncogenic
signals [6]. To gain the molecular functions of RCP in lung
cancer, we knocked out RCP in H1299 cells and extracted the
total plasma membrane protein for mass spectrometry profiling
(Fig. S1A). We observed that RCP depletion caused a decrease in
the abundance of membrane-associated proteins, the top ten
decreased proteins were listed (Table S2). Among them,
regulation of membrane-associated integrin β1 and EGFR’s
abundance by RCP in lung cancer cell lines was further
confirmed by immunoblotting and immunofluorescence (Fig.
2A, S1B, C). RCP deletion dramatically downregulated the levels
of membrane-associated integrin β1 and EGFR. On the contrary,
the abundance of integrin β1 and EGFR on membrane were
reversed after re-expression of exogenous RCP in RCP-knockout
H1299 cells. Furthermore, it was clearly observed that high-level
of RCP promote the aggregation of integrin β1 and EGFR
toward the pseudopodium sites of cell migration (Fig. S1B).
These results indicate that the sustained expression of RCP is
required to maintain high levels of membrane-associated
integrin β1 and EGFR.
Next, we speculated that upregulated RCP might promote the
proliferation and invasion of lung cancer cells. As shown in Fig. 2B,
C, S1D, E, RCP knockout or knockdown significantly inhibited the
proliferation rate and growth of H1299, H460 and A549 cells. Re-
expression of RCP rescues the proliferation and cell growth ability
in these cells (Fig. 2B, C). Similarly, downregulation of RCP
dramatically inhibited lung cancer cell invasion (Fig. 2D and S1F),
while the invasion capability was significantly enhanced with
upregulated RCP. Except that, overexpression of RCP in normal
human lung epithelial BEAS-2B cells resulted in acquisition of
tumorigenic properties such as enhanced cell growth and invasion
(Fig. 2E, H). Taken together, our observations suggest that RCP
drives lung cancer cell proliferation and invasion.
RCP drives lung cancer tumor growth and metastasis in vivo
Our experimental observations strongly suggested that upregu-
lated RCP drives cell proliferation and enhances cell invasion
in vitro. Next, we further investigated the role of RCP on growth
and local invasion of graft tumors with a subcutaneous
transplantation tumor model. As shown in Fig. 3A, B, RCP-
deletion significantly decreased tumor volume and mass, while
RCP overexpression re-increased tumor volume and mass. Except
that, tumor cells showed a form of invasion into the surrounding
normal tissues in the control and RCP overexpression groups.
While the invasion ability of RCP knockout tumors was inhibited
(Fig. 3C). These results show that RCP play an important role in
promoting lung cancer tumor growth and invasion.
Given that the influence of primary graft tumor burden on
metastatic ability of cancer cells in transplantation tumor model,
experimental metastasis model through intravenous inoculation
of lung cancer cells (H1299 and H460) was also conducted.
Compared with empty vector control, RCP knockout H460 cells
significantly suppressed tumor colonization and metastasis in
distant organs (Fig. 3D, I). In addition, RCP re-expression in RCP-
deletion cancer cells rescued the inhibited metastatic ability. We
found the similar results in H1299 experimental metastasis model
(Fig. S2A, F). Collectively, high expression of RCP in lung cancer
plays a critical role in promoting tumor growth, invasion,
colonization, and metastasis.
Sp1, Sp3, and Stat3 are identified as transcriptional activators
of RAB11FIP1
To explore the upstream factors that regulate the expression of
RCP, we first extract the total RNA and genomic DNA of lung
cancer and normal cells. We observed that the mRNA expression

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of RCP significantly increased in lung cancer cells compared with
normal lung epithelial BEAS-2B cells and lung fibroblast MRC-5
cells (Fig. 4A). On the contrary, the copy numbers of RAB11FIP1 of
these cell lines had no difference, indicating that there is no
amplification of RAB11FIP1 in these lung cancer cells (Fig. 4B). In
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addition, we observed the same results from human breast cancer
cell lines and noncancerous human breast epithelial MCF10A cells
(Fig. S3A, C). Taken together, these findings support the notion
that RAB11FIP1 maintains high transcription levels to enhance RCP
expression in cancer cells.
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Fig. 3 RCP drives lung cancer tumor growth and metastasis in vivo. A–C Tumor progression generated by subcutaneous injection of H460
cells with or without RCP expression into nude mice. A Quantification of tumor weight (left) and representative images (right) of injected
subcutaneous graft tumors. Scale bar, 1 cm. B Representative immunohistochemistry images showing RCP levels in H460 tumors with or
without RCP expression. Scale bar, 250 μm. C Representative H&E images of tumor local invasion. Scale bars, 250 μm (left) and 100 μm (right).
D–I Tumor metastasis generated by tail vein injection of H460 tumor cells with or without RCP expression into nude mice. D Representative
immunohistochemistry images showing RCP levels in macrometastases in lung and liver tissues. Scale bar, 250 μm (top) and 100 μm (bottom).
E Quantification (left) of visible metastatic tumor nodules and representative images (right) of lungs. (F and G) Representative histopathology
(H&E staining) of lung sections (F). The metastatic foci are indicated by the arrows. The rectangles in the top row indicate the enlarged areas
shown at the bottom. Scale bars, 2 mm (top) and 250 μm (bottom). G Numbers of metastatic foci per lung and the percentage (right) of the
lung metastasis area relative to lung section area (F). H, I Representative histopathology (H&E staining) (H) of the liver sections. I The number
(left) of metastatic foci per field of view and the percentage (right) of the liver metastasis area relative to the total liver section area. The
dashed lines indicate the border between macrometastases and normal liver tissue. Scale bar, 100 μm. Data are represented as mean ± SD. *p
< 0.05, **p < 0.001, ***p < 0.0001. Statistical analysis for multiple group comparisons were performed using one-way ANOVA with Dunnett’s
test (A, E, G, and I).

Until now, the transcriptional factors of RCP remain unknown.
To identify potential transcription factors, we biotinylated the
promoter region of RAB11FIP1 (GRCh38.p13: 37899439 to
37899977, 539 bp) to precipitate the DNA probe-bound proteins
from H1299 nuclear extracts and analyzed the interacting proteins
by mass spectrometry (Fig. S1D, E). Molecular functional analysis
identified seven potential transcription factors in the eluted
solution by peptide mass fingerprinting as “DNA-binding tran-
scription factor activity” (Table S3). By using a siRNA-based
functional screening, we found that Stat3, Sp1 and Sp3 knock-
down significantly decreased the transcription level of RCP (Fig.
4C, S3F). Furthermore, Sp1, Sp3 and Stat3 were all significantly
upregulated in cancer cells than in corresponding normal cells
(Fig. 4D and S3G). Next, we validated the binding of Sp1, Sp3 and
Stat3 with promoter of RAB11FIP1 by DNA competitive binding
assays. We found that the interaction of Sp1, Sp3, and Stat3 with
biotinylated DNA probes derived from promoter sequence of
RAB11FIP1 was blocked competitively by the unlabeled probes
(Fig. 4E). Moreover, the binding of Sp1, Sp3 and Stat3 with
promoter of RAB11FIP1 were confirmed by chromatin immuno-
precipitation (ChIP) (Fig. 4F, G).
By analyzing the promoter sequence of RAB11FIP1, we found
seven GC-rich motifs and one Stat3 binding site locate upstream
of the TATA box that act as potential Sp1/3 response elements and
Stat3 response element, respectively. To further evaluate the
contribution of these GC-rich motifs and Stat3 binding site to RCP
transcriptional regulation, we subcloned DNA fragments contain-
ing wild-type response elements, truncated response elements
lacking different GC-rich motifs into the promoter region of a
firefly luciferase reporter plasmid. We found only deletion of the
#2 or #3 GC-rich motifs significantly suppressed luciferase
expression (Fig. 4H). Subsequently, we performed ChIP assays to
determine whether endogenous Sp1 and Sp3 bind to these two
GC-rich motifs and detected an enrichment in RAB11FIP1 of 18-
fold (anti-Sp1) or 18.7-fold (anti-Sp3) relative to the amount bound
by isotype IgG (Fig. 4I). ChIP assay confirmed Sp1 and Sp3 binding
to #2 and #3 GC-rich motifs in the promoter of RAB11FIP1
(Fig. 4I, L). Moreover, deletion of the Stat3 binding site down-
regulated luciferase expression as well (Fig. 4J), and ChIP results
also confirmed this specific binding location of Stat3 (Fig. 4K, L).
Similarly, we identified two conserved GC-rich motifs and one
Stat3 binding site in murine RAB11FIP1 (Fig. S3H). Collectively, we
confirmed Sp1, Sp3, and Stat3 are transcriptional activators of
RAB11FIP1.
Sp1, Sp3, and Stat3 regulate RAB11FIP1 to increase tumor
invasion
To study the regulation of Sp1, Sp3 and Stat3 on RCP
transcription, we first treated H1299 cells with mithramycin (a
small molecule capable of occupying GC-rich motifs to inhibit
the transcription function of Sp1 and Sp3) and Stattic (a small
molecule inhibits the phosphorylation of Stat3 and thereby
inhibits the transcription ability of Stat3). Both inhibitors
caused a dose-dependent and time-dependent reduction in
RCP mRNA and protein levels (Fig. 5A, B). These results indicate
that Sp1, Sp3 and Stat3 are important for transcriptional
upregulation of RCP in cancer cells. Furthermore, knockdown
of either Sp1 or Sp3 downregulated RCP expression (Fig. 5C).
Co-suppression of both genes substantially reduced RCP
protein expression in a synergistic manner. In addition,
overexpression of either Sp1 or Sp3 markedly upregulated
RCP expression (Fig. 5C). Similarly, knockdown of Stat3
decreased the levels of RCP in H1299 cells (Fig. 5D). As
expected, overexpressing of Stat3 enhanced the transcription
of RCP (Fig. 5D). Therefore, Sp1, Sp3 and Stat3 are the
transcriptional activators that enhance RCP expression by
binding to the conserved GC-rich motifs and Stat3 binding
site in RAB11FIP1.
Next, we speculated that Sp1, Sp3 and Stat3 could affect the
distribution of membrane-associated EGFR and integrin β1
through regulating RCP expression. H1299 cells was treated with
mithrmycin and stattic, respectively. RCP expression was signifi-
cantly downregulated in H1299 cells with Sp1/3 or Stat3 inhibitors
(Fig. 5E). Additionally, EGFR and integrin β1 in the plasma
membrane distribution was decreased, especially around the
pseudopod where tumor cells migrate (Fig. 5F). Conversely, the
suppression by inhibitors treatment were slightly restored by
overexpression of RCP exogenously in H1299 cells (Fig. 5E, F),
while the mRNA levels of EGFR and ITGB1 was not affect by these
two inhibitors (Fig. S4A). Finally, the proliferation and invasion
ability of H1299 cells were significantly suppressed by the
inhibition of Sp1/3 or Stat3 (Fig. 5G, H). On the contrary,
exogenous overexpression of RCP can reverse the suppressed
cell proliferation and invasion of H1299 cells. Similarly, the amount
of EGFR and integrin β1 in the cell membrane, and the
proliferation and invasion ability of H1299 cells were significantly
suppressed by knocking down of Sp1/3 or Stat3 (Fig. S4B, C, D).
Conversely, the suppressed cell proliferation and invasion were
restored by upregulation of RCP. Overall, these data show that RCP
is transcriptionally upregulated by Sp1, Sp3, and Stat3, which
maintains the abundance of membrane-associated proteins (like
EGFR and integrin β1), thereby promoting cancer cell proliferation
and invasion.
Wild-type p53 suppressed RCP transcription in an Sp1 and
Sp3 dependent manner
Our prior study showed that RCP is a key effector downstream of
mutant p53 [13]. However, the molecular mechanism of p53/RCP
axis has not yet been established. So, we sought to determine
whether p53 functions in the regulation of RCP expression. First,
we evaluated the correlation between RCP expression level with
TP53 alterations in The Cancer Genome Atlas (TCGA) lung cancer

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dataset. Results showed that high levels of RCP are significantly
associated with TP53 alterations (Fig. 6A). Furthermore, lung
cancer cells with TP53 mutations showed higher levels of RCP
transcripts compared with wild-type p53 cells (Fig. S5A). Moreover,
wild-type p53 depletion increased both the protein and mRNA
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levels of RCP (Fig. 6B, C and S5B). Re-expression of wild-type
p53 significantly downregulated the protein and mRNA levels of
RCP (Fig. 6B, C and S5B). A luciferase reporter driven by the human
RAB11FIP1 promoter (GRCh38.p13: 37899439 to 37899977) can be
suppressed by wild-type p53 (Fig. 6D), which further confirmed
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Fig. 4 Sp1, Sp3 and Stat3 are identified as transcriptional activators of RAB11FIP1. A mRNA levels of RCP were analyzed by qRT-PCR and
normalized to β-actin mRNA levels in normal lung cells (BEAS-2B and MRC-5) and lung cancer cells (A549, H157, H358, H441, H460 and H520).
B RAB11FIP1 copy number were analyzed by qPCR and normalized to RPPH1 in normal lung cells (BEAS-2B and MRC-5) and lung cancer cells
(A549, H157, H358, H441, H460 and H520). C mRNA levels of RCP were analyzed by qRT-PCR and normalized to β-actin mRNA levels in H1299
cells (siCtrl, siARNTL, siCLOCK, siHLTF, siL3MBTL3, siSP1, siSP3, siSTAT3). All group transfected by siRNAs targeting indicated transcription factor
relative to nontargeting control siRNAs (siCtrl) (top). Similarly, protein levels of RCP were accessed by WB (bottom). D Sp1, Sp3, Stat3 and RCP
protein levels were assessed by immunoblotting (left) in BEAS-2B, A549, and H1299 cells, and quantified (right) and normalized to the
corresponding BEAS-2B cells. E Precipitates obtained by DNA pulldown assays in the absence or presence of unlabeled RAB11FIP1 genomic
DNA were examined by WB. F, G The binding ability of Sp1 (F), Sp3 (F) and Stat3 (G) with the RAB11FIP1 promoter region were detected by
chromatin immunoprecipitation (ChIP) in H1299 cells. H Luciferase assay of human RAB11FIP1 promoter constructs with deletion of different
GC-rich motifs in H1299 cells. I ChIP qPCR analysis of Sp1 and Sp3 binding to the validated sequence (which containing #2 and #3 GC-rich
motifs) locates at human RAB11FIP1 promoter in H1299 cells. J Luciferase assay of human RAB11FIP1 promoter constructs with deletion of
Stat3-binding site in H1299 cells. K ChIP analysis of Stat3 binding to the human RAB11FIP1 promoter in H1299 cells. L Promoter region of
human RAB11FIP1. Sp1/3 response elements (GC-rich motifs) and Stat3 binding site are indicated in red and highlighted. The capital letters
indicate the transcription start site, and the translation start site is indicated in green. Data are represented as mean ± SD. *p < 0.05, **p <
0.001, ***p < 0.0001. Statistical analysis for multiple group comparisons were performed using one-way ANOVA analysis with Dunnett’s test (A,
B, C, D, H, I, and J), two-tailed Student’s t-tests (K).

that wild-type p53 exerts its suppression activity through the
promoter of RAB11FIP1. Early studies reported that p53 can repress
mRNA transcription by directly binding to p53 response elements
in target promoters [36]. To assess the molecular basis underlying
p53-mediated RCP repression, we firstly analyzed the genomic
sequences of human and murine RAB11FIP1 through the JASPAR
and TRANSFAC databases. However, no putative p53 response
element was identified in RAB11FIP1. Therefore, we wonder
whether wild-type p53 can repress transcription by antagonizing
other transcriptional activators of RAB11FIP1.
Previous studies have reported that the interaction between
wild-type p53 and Sp1/3 impaired the transcriptional capabilities
of Sp1/3, leading to repression of several Sp1/3-inducible genes
involved in embryonic development and angiogenesis [37] as well
as tumorigenesis [38]. In this study, we found knockdown of Sp1/3
inhibit the effect of wild-type p53 on RCP expression (Fig. 6E, F).
Nevertheless, knockdown of Stat3 did not affect the suppression
activity of p53 on RCP expression. Ultimately, these observations
reinforce the idea that p53 indirectly modulates RCP expression
through Sp1/3. Furthermore, overexpression of wild-type p53
caused Sp1 and Sp3 downregulated compared with vector control
(p53 null), leading to substantial decrease in the RCP mRNA and
protein levels in H1299 cells (Fig. 6G). Consistently, levels of Sp1
and Sp3 in the nucleus were significantly reduced by introduction
of wild-type p53 (Fig. 6H and S5E). Contrary to wild-type p53,
expression of mutant p53 did not decrease RCP expression (Fig.
6G). Intriguingly, the introduction of “DNA contact” mutants
(R248W, R273H and R280K) robustly increased the transcripts and
protein levels of RCP, whereas “conformational” mutants (R175H
and Y220C) showed no effect compared to those in p53-null cells.
Consistently, RCP expression remains unaffected by knockout of
“conformational” p53 mutant R175H (Fig. S5C). Only “DNA
contact” p53 mutant (R273H and R280K) knockdown significantly
reduced RCP expression (Fig. S5D). Surprisingly, the total protein
levels of Sp1 and Sp3 had no difference among control, “DNA
contact” mutant and “conformational” mutant groups (Fig. 6G, H,
S5E, F). Notably, immunofluorescence showed that mutant p53
R273H displayed predominantly nuclear distribution (Fig. 6H). In
contrast, mutant p53 R175H exhibited reduced nuclear staining
with a compensatory increase in cytoplasmic staining, while Sp1/
3 showed a homogenous nuclear distribution. More importantly,
p53 R273H showed a higher colocalization efficiency with Sp1/3 in
nuclei than p53 R175H (Fig. 6H). Collectively, wild-type p53
decrease Sp1/3 expression, leading to downregulation of RCP in
an Sp1/3-dependent manner.
DNA contact mutants of p53 enhance Sp1 and Sp3
transcriptional activity to further drive RCP expression
To study the different mechanisms of “DNA contact” mutants and
“conformational” mutants upregulating RCP, ChIP assays were
performed. We used all three pairs of ChIP primers to test whether
different p53 binding to RAB111FIP1 promoter region via Sp1/3
(two GC-rich motifs) or Stat3 (Stat3 binding site). “DNA contact”
p53 mutant R273H binds to the promoter region of RAB11FIP1
together with Sp1/3 (Fig. 7A). Although “conformational” p53
mutant R175H form a complex with Sp1/3, but this protein
complex did not associate with promoter of RAB11FIP1. Wild-type
p53 cannot bind to the RAB11FIP1 promoter region (Fig. S6G).
Furthermore, a luciferase reporter driven by the human RAB11FIP1
promoter can be promoted by “DNA contact” p53 mutant R273H
in an Sp1/3-dependent manner (Fig. 7B). The co-IP results showed
that wild-type p53 and all forms of mutant p53 retained their
interaction with Sp1/3 in nuclei (Fig. 7C), which is consistent with
previous reports [39]. Interestingly, the levels of interaction
between “DNA contact” mutants and Sp1/3 were higher than
that between either wild-type p53 or “conformational” mutants
and Sp1/3. Although the ratio of nuclear p53 to total p53 was
different among the different p53 mutants and wild-type p53,
endogenous Sp1/3 was consistently localized mainly in the
nucleus (Fig. 6H and S5E). Lastly, we performed ChIP assays to
investigate whether the p53-Sp1/3 complex is involved in
regulating the DNA binding efficiency of Sp1/3. Consistent with
the role of wild-type p53 in diminishing Sp1/3 levels and
subsequently decreasing RCP expression, forced expression of
wild-type p53 triggered a significant decrease in the binding
efficiency of Sp1/3 to the RAB11FIP1 promoter (Fig. 7D).
Additionally, the ability of Sp1/3 binding to RAB11FIP1 was
enhanced by the “DNA contact” mutants not the “conformational”
mutants.
Taken together, these results confirm that wild-type p53
decrease Sp1/3 levels, leading to the suppression of RCP
transcription. Notably, loss of wild-type p53 or expression of
mutant p53 maintains Sp1/3 expression (LOF) to upregulate RCP
transcription. Specifically, only the “DNA contact” mutants of p53
enhance the DNA binding efficiency of Sp1/3 to further drive RCP
transcription (GOF) (Fig. 7E).
DISCUSSION
Most studies on oncogenic properties of RCP mainly focus on
breast cancer before, the global influence of RCP on lung cancer
has not yet been fully elucidated. In this study, we demonstrated
that the high expression of RCP enhanced lung cancer
progression. We first identified that Sp1/3 and Stat3 are the
transcription activators of RCP. Our previous work had demon-
strated that mutant p53 drives metastasis via RCP mediated
Hsp90alpha secretion in breast cancer, lung cancer and
pancreatic cancer [13]. As a transcriptional regulator, p53 is
involved in modulating RCP expression in an Sp1/3 dependent
manner. In contrast to wild-type p53 suppression of RCP
transcription by decreasing Sp1/3 levels, TP53 mutations are
associated with increased expression of Sp1/3 as well as

Oncogene (2022) 41:2357 – 2371


enhanced binding capability of Sp1/3 to RAB11FP1. Furthermore,
our work suggests a gain-of-function activity of “DNA contact”
mutant of p53 by which it increases the expression of RCP,
thereby promoting tumor progression. Collectively, these
findings imply that mutations in TP53 upregulates the transcrip-
tion of RCP in an Sp1/3 dependent manner to promote lung
Oncogene (2022) 41:2357 – 2371
C. Wang et al.
2365
cancer progression, providing important therapeutic implica-
tions for p53 mutant lung cancer.
Highly expressed RCP is critical for lung cancer progression
As a heterogeneous disease, lung cancer is difficult to treat. The
functional associations between oncogenes that drive lung cancer
 C. Wang et al.
2366
Fig. 5 Sp1, Sp3 and Stat3 regulate RAB11FIP1 to increase tumor invasion. A mRNA levels of RCP were analyzed by qRT-PCR in H1299 cells
after treatment with the indicated concentration of Mithramycin and Stattic for 48 h. B RCP protein levels in H1299 cells after treatment with
the indicated concentration of Mithramycin and Stattic for 24 h and 48 h. C RCP mRNA (top) and protein (bottom) levels in H1299 cells with
Sp1 and Sp3 knockdown (left) and Sp1 and Sp3 overexpression (right). D RCP mRNA (top) and protein (bottom) levels in H1299 cells with Stat3
knockdown (left) and Stat3 overexpression (right). E Abundance of EGFR and integrin β1 in cell membrane components were detected by
immunoblotting in H1299 cells with RCP overexpression, treated with Mithramycin or Stattic. The relative EGFR and integrin β1 intensity were
normalized to Na, K-ATPase α1. F Abundance of EGFR and integrin β1 in cell membrane components were detected by immunofluorescence
in H1299 cells with RCP overexpression, treated with Mithramycin and Stattic. Scale bar, 10 μm. G Colony formation ability of H1299 cells with
or without RCP expression, treated with Mithramycin or Stattic. Quantitative numbers of colonies in six-well plates (left) representative images
(right). Representative results from three independent experiments are presented. H Invasion capability of H1299 cells with or without RCP
expression, treated with Mithramycin or Stattic. Quantitative numbers of invaded cancer cells per view (left) and representative images (right).
Representative results from three independent experiments are presented. Data are represented as mean ± SD. *p < 0.05, **p < 0.001, ***p <
0.0001. Statistical analysis for multiple group comparisons were performed using one-way ANOVA with Dunnett’s test (A, C, D, G, and H).

progression has been wildly defined [40, 41]. Recently, some
molecular mechanisms of lung carcinogenesis and progression
have been elucidated, and molecularly targeted approaches for
lung cancer patients have now reached the clinical arena [42–44].
Even though, lung cancer is still the leading cause of death in
global [1]. Emerging evidences indicate that RCP is highly
expressed and associated with poor clinical outcomes in various
types of cancer [8, 9]. Consistent with that, the present study
shows that RCP is highly expressed in lung cancer and elucidates
its clinical correlation in cancer patients. Until recently, the
function of RCP had been found in esophageal cancer and head
and neck squamous cell carcinoma [11, 12, 14]. Herein, we
discovered high expression of RCP facilitated lung cancer
progression. More than that, invasive tumor cells with RCP
deletion showed a reduction in tumor growth and metastasis.
While RCP overexpression can recover tumor progression. There-
fore, blocking RCP can be a potential therapeutic target for lung
cancer.
Sp1, Sp3 and Stat3 are transcriptional regulators of RAB11FIP1
Though RCP had been reported as an oncogene to promote
cancer progression [6, 11–15, 45]. Our current study had also
demonstrated RCP can promote lung cancer progression. How-
ever, no one has reported the upstream regulators that enhance
the expression of RCP. In this study, we first identified Sp1, Sp3
and Stat3 are transcriptional regulators of RAB11FIP1. As previous
studies show that Sp1 and Stat3 functionally synergize to induce
RhoU transcription in breast cancer [46], another study demon-
strated physical interaction of Stat3 and Sp1/Sp3, their recruitment
to the Sp cis-elements in a Stat3 DNA-binding domain-dependent
fashion to increase NHE3 transcription [47]. Based on our results
that knockdown Sp1/3 or Stat3 (as well as Sp1/3 or Stat3
inhibitors) leads to repressed RCP transcription, co-suppression of
these TFs reduced RCP expression in a synergistic manner,
suggesting that stat3 and Sp1/3 may cooperate to induce high
RCP expression. Previous studies have found that the expression
levels of Sp1, Sp3 and Stat3 proteins and their transcriptional
activity are positively correlated with the malignancy of tumors,
and they all play a role in promoting the occurrence and
development of tumors [48]). Sp1 also been reported to interact
with many other nuclear factors to regulate downstream gene
expression. Moreover, Sp1 usually interacts with GC and GT
oligonucleotide sequences in the promoter region of target gene.
Consistent with that, our study further found that Sp1 and Sp3
binding to #2 and #3 GC-rich motifs in the promoter of RAB11FIP1.
For the precise molecular mechanism underlying Sp1, Sp3 and
Stat3 transcriptional regulation of RAB11FIP1 need further
investigation.
GOF p53 regulate RCP expression dependent on Sp1/3
Mutation of TP53 is one of the most frequent genetic lesions in
human cancers. Mutant p53 exhibits various gain-of-function
activities that contribute to tumor development and progres-
sion [49]. It had been found that GOF p53 inactivate other p53
family members, in particular the tumor proteins p63 and p73
[50, 51]. Other evidences suggest that these pro-oncogenic
functions of mutant p53 proteins are mediated by affecting the
transcription of various genes, as well as by protein-protein
interactions with transcription factors and other effectors.
Remarkably, our previous study indicated that the “DNA
contact” mutp53 (R273H and R280K) had GOF effects by
upregulating RCP expression to facilitate the secretion of
eHsp90alpha [13]. In addition, other groups had proved that
GOF p53 facilitated tumor progression in RCP-dependent
manner [13, 34, 52].
The molecular mechanism of the mutant p53/RCP axis has
been established in the present study. We observed that p53
regulated RCP expression dependent on Sp1/3. We demon-
strated that mutant p53 transcriptionally upregulates RCP
expression. Our increasing understanding of the mechanisms
by which mutant p53 mediates these oncogenic activities has
been derived from exploring the consequences of the physical
interaction between mutant p53 proteins and other transcrip-
tion factors. Our results demonstrated that wild-type p53
decrease Sp1 and Sp3 levels, thus resulting in suppressed
transcription RCP. One possible mechanism is that wild-type
p53 binds to Sp1 and Sp3 to induce their degradation [38].
There is a general agreement that mutations in p53 abrogate its
ability to repress Sp1/3 target genes [37]. Consistent with this
observation, we found that either “conformational” mutants or
“DNA contact” mutants can induce RCP expression via main-
taining Sp1/3 levels (LOF). However, whether all p53 mutants
are defective in binding to Sp1/3 remains debated. Lowe
laboratory found that only the introduced wild-type p53 bound
to Sp1, whereas the endogenous p53 Y220C mutant did not
[38]. The effects of “conformational” and “DNA contact”
mutations in p53 on its cellular localization have been studied
in depth [53]. Given the diverse intracellular localizations of p53
and nuclear localization of Sp1/3, we performed co-IP with
either Sp1 or Sp3 and confirmed that both “conformational”
and “DNA contact” mutants retain the ability to interact with
Sp1/3. Our results show that the “DNA contact” mutants form a
tight complex with Sp1/3 in nuclei, whereas the “conforma-
tional” mutants participate in a weak and transient protein-
protein interaction with Sp1/3 because of their retention in the
cytoplasm. Importantly, through ChIP assays, we identified that
only the “DNA contact” mutants had the GOF effects by
enhancing Sp1/3 bind to the GC-rich motifs in RAB11FIP1. Thus,
GOF mutant p53 aids RCP in oncogenesis dependent on Sp1/3,
raising the possibility of targeting GOF mutant p53 with Sp1/3
inhibitors.
In conclusion, we first identified the function of RCP on lung
cancer progression, and the transcriptional regulatory mechan-
ism of RCP. Furthermore, we elaborated the regulation

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 C. Wang et al.
2367

mechanism of different p53 on RCP, respectively. Wild-type p53 their retention in the cytoplasm. Different with “conforma-
decreases Sp1 and Sp3 levels to suppress RCP expression. While tional” mutants of p53, “DNA contact” mutants have GOF effects
the “conformational” mutants participate in a weak and by enhancing the interaction between p53 and Sp1/3 to induce
transient protein-protein interaction with Sp1/3 because of RCP expression. Our results support the notion that different

Oncogene (2022) 41:2357 – 2371

 C. Wang et al.
2368
Fig. 6 Wild-type p53 Suppressed RCP Transcription in an Sp1 and Sp3 Dependent Manner. A RAB11FIP1 mRNA levels in p53 wild-type and
p53 alterations Lung cancer patients. Data from TCGA lung cancer database (ID:LUAD). B RCP mRNA levels (top) and protein levels (bottom)
were detected in H460 and H1299 cells with or without wild type p53. C RCP mRNA levels (top) and protein levels (bottom) were detected in
A549 cells with or without wild type p53. D Luciferase reporter assay of RAB11FIP1 transcription levels in H1299 cells transfected with wild-type
p53. E Luciferase reporter assay detected the RAB11FIP1 transcription levels in wild type and p53 knockout H460 cells with Stat3 and Sp1/3
knockdown. F RCP mRNA levels were detected in H1299 p53 null cells and p53 overexpression cells with Stat3 and Sp1/3 knockdown. G RCP
mRNA levels analyzed by qRT-PCR and protein levels analyzed by immunoblotting in H1299 cells transfected with wild type p53 and different
mutant p53. The quantities were normalized to the ctrl vec group. H Representative images (left) and quantified results (right) of co-
localization between p53 and Sp1/3. Immunostaining for p53 (red) and Sp1(top) or Sp3 (bottom) (both in yellow) in H1299 cells transfected
with Ctrl vec, wild-type p53, p53 R175H or p53 R273H. Cells were subjected to F-actin staining with Alexa Fluor 488 Phalloidin and nuclear
staining with DAPI (blue). Scale bar, 10 μm. Colocalization efficiency of Sp1 (top) or Sp3 (bottom) and mutant p53 was quantified. Data are
represented as mean ± SD. *p < 0.05, **p < 0.001, ***p < 0.0001. Statistical analysis for multiple group comparisons were performed using one-
way ANOVA with Dunnett’s test (B, C, E, F, and G), Student’s t-test (A, B, D, and H).

p53 mutants utilize distinct mechanisms to acquire unique
oncogenic potential during cancer progression.
MATERIALS AND METHODS
Cell proliferation assays and colony formation assays
Cell proliferation assays and colony formation assays were performed as
we described previously with modifications [54]. Cells were seeded in 96-
well plates (2 × 103 cells/well for H1299 and A549 cells, 2 × 103 cells/well for
H460 cells, 3 × 103 cells/well for BEAS-2B cells) and cell viability was
measured by using the Cell Counting Kit-8 (Dojindo lab, Cat:CK04-500) at
24 h and 48 h. Cells were seeded in 6-well plates (5 × 102 cells/well for
H1299 and A549 cells, 5 × 102 cells/well for H460 cells, 1 × 103 cells/well for
BEAS-2B cells) and cultured in completed medium for 1 week. Colonies
were fixed with 4% paraformaldehyde and stained with 0.5% crystal violet
overnight. Colonies were quantified by counting and images were
obtained.
tumor sizes and body weights of animals were monitored. Tumor volume
was measured with calipers every 3 days and calculated as the formula v =
0.5 × length × (width)2. At protocol-defined endpoints, all mice were
sacrificed, tumors were isolated and weighed. For intravenous injections
of H460 and H1299 cells, 1 × 106 cells per injection, n = 6 mice per group.
The lungs were dissected and fixed in Bouin’s solution to quantify the
visible metastatic tumor nodules. The livers were removed and fixed in 4%
(wt/vol) PFA.
Histological examination
Histological examination assays are described previously [13]. The fixed
mouse lung and liver tissues were dehydrated and embedded in
paraffin and were cut into 6-μm sections. The sections were counter-
stained with haematoxylin and eosin (H&E). Olympus IX71 optical
microscope was used to obtain the liver and lung metastasis areas. The
metastatic lesions were analyzed with Image-Pro Plus 6.0 (Image-Pro
Plus, RRID:SCR_007369).

Coimmunoprecipitation assay Biotin labeling of RAB11FIP1 and DNA pulldown assay

Co-IP assays are described as previously [13]. Briefly, cells were lysed in
NP40 buffer with complete protease inhibitor mix at 4 °C. Protein G
Sepharose was used to preclear samples. And equal amounts of total
protein were immunoprecipitated with corresponding antibodies or IgG at
4 °C with constant rotation. The complexes were precipitated with protein
G beads. Antibodies were used as followed: anti-p53 (Santa Cruz
Biotechnology Cat# sc-126) for p53, anti-Sp1 (Millipore Cat# 07-645,
RRID:AB_11213103)) and anti-Sp3 (D20, Santa Cruz, sc-644) and control IgG
(Cell Signaling Technology Cat# 3900, RRID:AB_1550038).
Invasion assay
24-well transwell inserts (Millipore, Billerica, MA) were lined with Matrigel
(Corning, New York, USA). The lower chambers were filled with media
containing 10% FBS and the chemoattractant EGF (25 ng/ml), the upper
chambers filled with media containing 2% FBS. The cells invaded into
Matrigel plugs for 24 h, and were then stained with 0.5% crystal violet
overnight. Invaded cells were quantified by counting and images were
obtained.
Mouse studies
Female BALB/c nude mice ages 4-6 weeks (Beijing, Vital River) were housed
in pathogen-free barrier facilities and maintained in a 12 h light/dark cycle
in isolated ventilated cages, 22-26 °C with sterile pellet food and water
supply ad libitum. The laboratory animal facility has been accredited by
AAALAC (Association for Assessment and Accreditation of Laboratory
Animal Care International) and the IACUC (Institutional Animal Care and
Use Committee) of Tsinghua University approved all procedures in this
study (Approval numbers: 16-LYZ4 and 18-LYZ2) in accordance with the
Guide for the Care and Use of Animals. The investigators were not blinded
to the group allocation during the experiments.
For the subcutaneous tumor implantation assays, we established H460
cell lines with knockout of endogenous RCP (CRISPR RCP, RCP-deletion
cells), and further exogenous overexpression of RCP (RCP OE) in
RCP-deletion cells, cells were suspended in 50 μl of RPMI-1640 medium
and were subcutaneously inoculated in six-week-old female Balb/c-nu
mice. 1 × 106 H460 cells per injection, n = 6 mice per group. Primary
tumors were quantified twice per week via caliper measurement. The
The biotinylated RAB11FIP1 DNA probes (GRCh38.p13: 37899439 to
37899977) were generated by using PCR with the oligonucleotide primers
modified at their 5’ends. The sequences of RAB11FIP1 DNA probes were
shown in Table S9. Nuclear extracts from H1299 cells were prepared using
a Nuclear Complex co-IP Kit with complete protease inhibitor mix. Nuclear
extracts were incubated with DNA probes overnight at 4 °C with constant
rotation. The complexes were precipitated with the Streptavidin Magnetic
Beads (NEB, UK), and washed four times with PBS buffer with complete
protease inhibitor mix and then resolved on 10% SDS-PAGE gel.
Precipitates were analyzed by Western blot. Proteins were immunoblotted
with the following antibodies: anti-Sp1 (D4C3, Cell Signaling, #9389) and
anti-Sp3 (D20, Santa Cruz, sc-644). An aliquot of each lysate was used as
input control.
RAB11FIP1 luciferase reporter assay
The pGL3-basic vector (RRID:Addgene_48743) and pRL-CMV plasmid
were gifts from Dr. P. Jiang. To evaluate the contribution of each GC-rich
motif to RCP transcriptional regulation, the genomic fragments of
RAB11FIP1 containing all GC-rich motifs (pGL3-RAB11FIP1 with wild-type
response elements) or truncated response elements lacking different
GC-rich motifs were cloned into pGL3-basic vector. The reporter
plasmids were then transfected to H1299 cells together with a Renilla
luciferase plasmid. For examining the roles of wild-type p53 and mutant
p53 in regulation of RCP transcription, pGL3 -RAB11FIP1 and pRL-MCV
were cotransfected in H1299 cells expressing wild-type p53 or mutant
p53 (R175H, Y220C, R248W, R173H or R280K). 24 h after transfection, the
luciferase activity was determined using a dual Luciferase Assay System
(Promega, Madison, WI). Transfection efficiency was normalized on the
basis of the Renilla luciferase activity.
Chromatin immunoprecipitation and ChIP qPCR
The DNA fragments were prepared using a ChIP Assay Kit (Millipore,
Billerica, MA). Briefly, H1299 cells were crosslinked with 1% (wt/vol)
formaldehyde solution for 10 min at 37°C. The crosslinking reaction was
stopped by the addition of 125 mM glycine and then lysed in 1 ml SDS
lysis buffer with complete protease inhibitor mix for 10 min on ice.
Lysates were then sonicated to generate DNA fragments with the

Oncogene (2022) 41:2357 – 2371

 C. Wang et al.
2369

Fig. 7 Mutant p53 GOF regulate RCP expression dependent on Sp1/3. A The binding ability of conformational mutant p53 (R175H) and
DNA contact mutant p53 (R273H) with the RAB11FIP1 promoter region were detected by chromatin immunoprecipitation (ChIP). B Luciferase
reporter assay of RAB11FIP1 transcription levels in H1299 cells (p53 R175H and p53 R273H expression) with STAT3 and SP1/3 knockdown. C p53
and Sp1/3 were detected in precipitates by co-IP with Sp1/3 in H1299 cells stably expressing p53 wild type or p53 mutants (top). The co-IP
efficacy was normalized to p53 wild type (bottom). D ChIP assays for the binding efficiency of Sp1 (left) or Sp3 (right) to the RAB11FIP1
promoter in H1299 cells transfected with Ctrl vec, wild-type p53 or mutant p53. E Schematic summarizing the different mechanisms of the
multiple actions of wild-type p53 and mutant p53 in modulating RCP expression. Data are represented as mean ± SD. *p < 0.05, **p < 0.001,
***p < 0.0001. Statistical analysis for multiple group comparisons were performed using one-way ANOVA with Dunnett’s test (B, C, and D).
Comparisons between two groups were performed using two-tailed, unpaired Student’s t-tests (B).

Oncogene (2022) 41:2357 – 2371

 C. Wang et al.
2370
average size approximately 200–1000 bp. Sonicated samples were spun
down and subjected to overnight immunoprecipitation with antibodies:
anti-Sp1 (Millipore Cat# 07-645, RRID:AB_11213103)) and anti-Sp3 (D20,
Santa Cruz, sc-644) or control IgG at 4 °C with constant rotation, 4 μg of
specific antibodies or IgG were added per 2 ml of diluted cell lysates.
The next day, bounded DNA fragments were eluted using low salt
immune complex buffer, high salt immune complex buffer, and TE
buffer sequentially. The DNA fragments were recovered by phenol/
chloroform extraction and ethanol precipitation. DNA pellets were
resuspended and amplified by quantitative PCR as described above. The
ChIP primer pairs were listed in the Table S8.
Quantification and statistical analysis
Data are presented as means ± SD. Immunoblots, co-IP, pull-down,
invasion and qRT-PCR assays were conducted more than 3 times to
ensure reproducibility. All data were plotted and analyzed with GraphPad
Prism 6 Software (GraphPad Prism, RRID:SCR_002798). All statistical tests
were two-sided, and differences were considered statistically when the P
value was less than 0.05, with *p < 0.05, **p < 0.01, ***p < 0.001. Statistical
analysis for multiple group comparisons were performed using one-way
analysis of variance (ANOVA) with Dunnett’s test or two-way ANOVA with
Tukey’s test. Comparisons between two groups were performed using two-
tailed Student’s t-tests. Longitudinal models for analyzing the repeated
measures of tumor volume.
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ACKNOWLEDGEMENTS
This work was supported by the National Natural Science Foundation of China
(No. 82103071 to CW) and the National Major Scientific and Technological Special
Project for “Significant New Drugs Development” (No. 20181821569 to YL).
AUTHOR CONTRIBUTIONS
CW and SZ performed all experiments with the assistance from BM and YF. SZ and
CW conceived and designed the research. YL supervised the research. C.W and SZ
wrote and edited the paper. All authors commented on the paper.
COMPETING INTERESTS
The authors declare no competing interests.
ADDITIONAL INFORMATION
Supplementary information The online version contains supplementary material
available at https://doi.org/10.1038/s41388-022-02260-7.
Correspondence and requests for materials should be addressed to Yongzhang Luo.
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