CYFIP1 and PKM2 Synergistically Regulate ROS and Glycolysis in Colorectal Cancer Through the AKT/mTOR/HIF-1α Cascad
Conditions: Colorectal Cancer (CRC), Specimens Are to be Collected From Subjects Who Are Having or Have Had a Fluid or Tissue Removed, a Biopsy or a Blood or Bone Marrow Draw
Sponsor: hui hou

Title：CYFIP1 and PKM2 synergistically regulate ROS and glycolysis
in colorectal cancer through the AKT/mTOR/HIF-1α cascade
Serial Number：280211
Date：2025.09.19

Research plan
1.1 CYFIP1 is highly expressed in colorectal cancer
(1) Bioinformatics + clinical information + clinical tissue evidence show that CYFIP1 is
highly expressed in colon cancer: Based on the GEPIA system (http://gepia.cancer-pku.c
n/detail.php?gene=CYFIP1###), the expression level of CYFIP1 in colorectal cancer tissues
was analyzed to be higher than that in benign colorectal tissues; Detection of the exp
ression of CYFIP1 in human colorectal tissue specimens was conducted to further verify
the cell line data. We collected 64 colorectal cancer patients and adjacent tissues for
RT-qPCR and IHC verification; Analysis of the clinical pathological characteristics and sur
vival curves of the collected colorectal cancer patients proved the relationship between
the expression of CYFIP1 and tumor size, tumor stage, lymph node metastasis, and di
sease-free survival period.
(2) Determine the colorectal cancer cell lines to be used in subsequent experiments:
We conducted Western blot analysis of CYFIP1 protein expression in common colorectal
cancer cell lines HT29, SW480, SW620, HCT116, and normal colon cells NCM460.
1.2 CYFIP1 Affects Oxygen Metabolism and Glycolysis in Colorectal Cancer
(1) Whether CYFIP1 alteration affects oxygen metabolism in colorectal cancer: By infecti
ng the CYFIP1 colon cancer cell line SW480 with a slow virus, and conducting subsequ
ent sequencing analysis, we found that after knockdown of CYFIP1, there was a signific
ant enrichment in the HIF pathway, and the oxygen metabolism function of the cells c
hanged significantly; through Western blot, RT-qPCR, etc. verification of the infected SW
480 and HT29 cells, it was confirmed that the transcription and translation levels of HI
F-1α and related oxygen metabolism proteins were changed; further through experime
nts such as cell ROS and electron microscopy, it was confirmed that the damage to mi
tochondria and the generation of reactive oxygen species were affected; using Seahorse
XF to measure the oxygen consumption rate of the cells; using kits to measure the A
TP and pyruvate contents of colorectal cancer cells transfected with CYFIP1.
(2) Does the alteration of CYFIP1 affect the glycolysis of colorectal cancer and whether it
directly binds to PKM2: When measuring the common metabolic substrates of oxygen

metabolism and sugar metabolism such as ATP, glucose, and lactate, we found significant
changes in the products related to glycolysis. Based on this, after conducting metabolic group
sequencing analysis, we discovered obvious abnormalities in metabolites such as pyruvate, and
the glycolysis pathway was significantly enriched. Through Western blot and RT-qPCR, we
confirmed that the expression of PKM2 decreased but its transcription was not affected. We
verified the relationship between CYFIP1 and PKM2 using methods such as CH-IP, mass
spectrometry, and immunofluorescence, and explored whether the interaction between the
two affected the degradation of PKM2 and thereby influenced the glycolysis of colorectal
cancer; Using PKM2-IN-1 (a PKM2 inhibitor) to inhibit the overexpressed CYFIP1-infected
SW480 and HT29 cell lines, we measured the glycolytic metabolic products again and
conducted reverse functional verification.
1.3 The specific mechanism by which CYFIP1 affects oxygen metabolism in colorectal cancer
and the interaction between oxygen metabolism and glycolysis
(1) Determine the mechanism by which CYFIP1 affects oxygen metabolism in colorectal
cancer through the AKT/MTOR/4EBP1/HIF-1 α
pathway: Based on sequencing results and
literature review, the AKT/MTOR/4EBP1/HIF-1α pathway was the focus of the study; after
overexpression and interference of CYFIP1 expression in colon cancer cells, Western blot was
used to verify the protein of the entire AKT/MTOR/4EBP1/HIF-1 α
pathway, verifying the
completeness and consistency of the entire pathway; in the overexpressed colon cancer cell
line, rapamycin (a MTOR inhibitor) was used to verify the changes in ROS, ATP, and related
pathway proteins;
(2) Verify that CYFIP1 affects HIF-1α through PKM2 nuclear translocation and influencing
MTOR expression, thereby causing the intersection of glycolysis and oxygen metabolism: To
verify the mechanism by which CYFIP1 causes simultaneous changes in oxygen metabolism and
glycolysis after modification, immunofluorescence was used to observe the changes in the
nuclear transfer of PKM2 in overexpressed and interfered SW480 and HT29 cell lines (the
nuclear transfer of PKM2 will affect the transcription of HIF-1α, thereby causing an interaction
between glycolysis and oxygen metabolism); in the colon cancer cell line overexpressing CYFIP1,
PKM2-IN-1 was used to verify the changes in AKT/MTOR/4EBP1/HIF-1α pathway proteins,

ROS, ATP, and mitochondrial function, and rapamycin was used to observe the changes in
PKM2 expression;
(3) Verify that the lactic acid produced by the change in PKM2 caused by CYFIP1 leads to the
lactylation of HIF-1α and stabilizes HIF-1α, reducing its degradation: Overexpress CYFIP1 in
HT29 and SW480 cells, and use the L-lactic acid kit to prove that the expression of L-lactic acid
increased; in the colon cancer cell line with knocked-down CYFIP1, L-lactic acid was added, and
the expression of HIF-1α protein was determined, proving that L-lactic acid can rescue the
decrease in HIF-1 α ; WB was used to verify the expression levels of Pan-Kla and Pan-UB
proteins, proving that L-lactic acid increased overall lactylation and simultaneously reduced
overall ubiquitination; normal colon cancer cells were added with L-lactic acid, and the protein
amount of HIF-1α was measured at different times, proving that L-lactic acid can change the
half-life of HIF-1α; LC-MS/MS was used to reveal the lactylation sites of HIF-1α, molecular
docking was used to simulate the binding conformation of HIF-1α and L-lactic acid, indicating
that L-lactic acid binds at which sites; in summary, it is indicated that after the change of CYFIP1,
PKM2 undergoes corresponding changes, and the lactylation of HIF-1α mediated by L-lactic
acid plays a direct stabilizing role in HIF-1α expression.
1.4 CYFIP1 affects the immune microenvironment and chemotherapy resistance of colorectal
cancer through metabolic reprogramming of oxygen metabolism and glycolysis.
(1) CYFIP1 affects the immune microenvironment of colorectal cancer through metabolic
reprogramming: The measurement of lactic acid and HIF-1α lactylation indicated that lactic
acidification occurred in colorectal cancer after CYFIP1 modification, and it was found from
literature that the production of lactic acid would affect the immune microenvironment of the
tumor; THP1 cells were co-cultured with CYFIP1-modified colorectal cancer cells and the
following experiments were completed: WB verified the changes in MCT1 and MCT4, which
verified the changes in the ability of colorectal cancer cells and THP1 cells to take up and
excrete lactic acid; WB verified the changes in PD-L1 and PD-1 proteins; Flow cytometry
verified the expression levels of CD80, CD86, CD163, and CD206 in THP1 cells, proving the
changes in the proportion of M1 and M2 cells in immune cells after CYFIP1 modification. In the
co-culture environment of CYFIP1-knocked-down colorectal cancer cell lines and THP1 cells,

L-lactic acid was added, and in the co-culture environment of overexpressed CYFIP1 colorectal
cancer cell lines and THP1 cells, PKM2-IN-1 and rapamycin were added to repeat the above
experiments again to complete the functional rescue experiments; Finally, the CSCs change was
detected through tumor microsphere formation test;
(2) CYFIP1 affects the chemotherapy resistance of colorectal cancer through metabolic
reprogramming: SW480 cells were continuously stimulated with cisplantin chemotherapy for 6
months to obtain stable drug-resistant SW480 cells, namely SW480/DDP (hereinafter referred
to as DDP); CCK8 was used to detect the cell viability of DDP and SW480 cells at different drug
concentrations, and the IC50 values were compared; Stable knockdown of CYFIP1 and stable
high expression of CYFIP1 in SW480, HT29 cells, and stable knockdown of CYFIP1 in DDP cells
were constructed by cell transfection technology, and their cell viability at different drug
concentrations was detected by CCK8 technology; The tumor stem cell situation was verified
through tumor microsphere formation test; The lymphocyte separation technique was used to
simulate the in vivo microenvironment, and the changes were detected subsequently, while
comparing the responses of immune microenvironment after CYFIP1 modification in resistant
cells and normal cells; The immunohistochemical staining technique was used to verify the
CYFIP1, KI-67, AKT/MTOR/HIF-1α pathways and PKM2-related proteins in the tumor.
1.5 CYFIP1 affects the proliferation and apoptosis of colorectal cancer through metaboli
c reprogramming of oxygen metabolism and glycolysis.
(1) CYFIP1 affects the proliferation of colorectal cancer through metabolic reprogrammin
g: After knockdown and overexpression of SW480 and HT29 cells, CCK8, TRANSWELL, sc
ratch, clone and other experiments were conducted to verify the effect of CYFIP1 modi
fication on the functions of colorectal cancer cell lines; in the overexpressed cell lines
of SW480 and HT29, using PKM2-IN-1 and rapamycin respectively, the differences in CC
K8 and TRANSWELL disappeared, and after 8 hours of hypoxia in the knockdown cell li
nes of SW480 and HT29, the differences disappeared again, and the differences appear
ed again after reoxygenation; the in vitro animal experiments were divided into four gr
oups: knockdown, overexpression, overexpression + PKM2-IN-1, overexpression + rapamy
cin, control group, control + PKM2-IN-1, control + rapamycin; after knockdown and over
expression, there were differences in subcutaneous tumor formation, and after adding
PKM2-IN-1 and rapamycin injections, the differences disappeared; for the tumor tissues
of animals, HE staining, CYFIP1, Ki67, PKM2, HIF-1α and other protein immunohistoch
emical staining were performed, and CYFIP1 and PKM2 were double-stained by immuno
fluorescence.

(2) CYFIP1 affects the apoptosis of colorectal cancer through metabolic reprogramming:
The results of bioinformatics prediction of the correlation between CYFIP1 and apoptosi
s proteins were obtained; after knockdown of CYFIP1 in SW480 and HT29 cells, flow c
ytometry was used to verify whether apoptosis increased; the results of related apopto
sis proteins such as BCL2, caspase3, c-caspase3 were obtained, and PKM2-IN-1 and rap
amycin were used in the overexpressed cell lines of SW480 and HT29 again for verifica
tion; the expression of apoptosis proteins was verified again in the animal tumor tissu
es using Western blot and immunohistochemistry methods.
Acknowledgements
On behalf of all authors, we would like to thank the Graduate Research and Practice I
nnovation Project of Anhui Medical University (YJS20230196); Translational Medicine Res
earch of Anhui Province (2022zhyx-C39); Supported by the Natural Science Research Pro
ject of Anhui Provincial Universities (KJ2021A0317).
Ethics approval and consent to participate
The study was ratified by the Ethics Committee of the Second Affiliated Hospital of An
hui Medical University and conducted in compliance with the Declaration of Helsinki, a
nd the ethics number was (YX2023-183). Written informed consent form was obtained
from each eligible patient. The animal study protocol was ratified by the Animal Ethics
Committee of the institute, and all procedures were performed in accordance with th
e Guide for the Care and Use of Laboratory Animals (NIH, Bethesda, Maryland, USA).