The Expression Proles and Clinical Signicance of PKM2 and Notch1 in Colorectal Cancer

Background Studies have shown that pyruvate kinase M2 (PKM2) and Notch1 are highly expressed in colorectal cancer (CRC) tissues and have a certain relationship with disease occurrence and development. The expression levels of PKM2 and Notch1 are also related to the effect of chemotherapy and radiotherapy, which seriously influence the prognosis of CRC patients. Thus, both PKM2 and Notch1 have been identified as key targets of CRC treatment and research. However, correlations between PKM2 and Notch1 have not yet been established. group were detected with the CCK-8 and wound healing assays. Results The Immunohistochemical analysis showed that PKM2 and Notch1 were highly expressed in CRC and related to tumor stage and lymph node metastasis. The qRT-PCR and western blot results showed that PKM2 and Notch1 were notably upregulated in CRC cells both at the mRNA and protein levels. PKM2 and Notch1 form a positive feedback loop to promote the occurrence and development of CRC, and inhibition of PKM2 and Notch1 has a synergistic effect on the proliferative and invasive capabilities of CRC cells. Conclusion The combination of the PKM2 inhibitor compound 3K and the Notch1 inhibitor TGN presents a novel and effective strategy for treatment of CRC.


Introduction
Colorectal cancer (CRC) is the third most commonly diagnosed malignancy and the fourth leading cause of cancer-related death worldwide [1]. Without treatment, patients with inoperable or metastatic CRC have a median life expectancy of about 8 months [2]. Since the early stage of CRC is often asymptomatic, most patients are diagnosed in the mid to late stages of disease and, thus, miss the opportunity for surgery. Even if surgery is performed early, some patients will develop postoperative recurrence and metastasis. Therefore, chemotherapy is the primary treatment option for such patients. However, CRC cells are highly resistant to various chemotherapeutic drugs. Therefore, it is very important to develop novel drugs for the treatment of CRC.
Pyruvate kinase (PK), which is a key enzyme in glycolysis, catalyzes phosphoenolpyruvate into pyruvate and releases energy. Mammalian cells produce four isoforms of PK: L, R, M1, and M2 [3]. PKM2 is mainly expressed in embryonic cells, adult stem cells, and actively proliferating tissue cells. As the embryo develops, PKM2 is gradually replaced by several other isoenzymes. However, during tumorigenesis, PKM2 expression is increased and gradually replaces other isozyme types [4]. High expression of PKM2 in CRC tissues is predictive of a poor outcome [5]. Targeting of PKM2 with compound 3K inhibits the development of CRC by suppressing glycolysis [6].
The Notch signaling pathway is a highly conserved intercellular signal transduction system that consists of the Notch receptor, Notch ligand, and transcription factor CSL. The Notch receptors Notch1-4 are transmembrane glycoproteins. Notch1 expression is closely correlated with poor overall survival of CRC patients and, thus, is a potential prognostic biomarker [7].
The aim of the current study was to investigate the relationship between the key lactate metabolism enzyme PKM2 and the Notch signaling pathway, and to determine whether simultaneous inhibition of PKM2 and Notch1 with compound 3K and tangeretin (TGN), respectively, exerts synergistic effects against CRC cells. The results suggest that this approach presents a novel strategy for treatment of CRC.

Study approval and patient consent
The study protocol was approved by the Ethics Committee of Lanzhou University Second Hospital (Lanzhou, China) and conducted in accordance with the ethical principles for medical research involving human subjects as described in the Declaration of Helsinki. Written informed consent was obtained from each patient prior to surgery.

Tissue samples
CRC tissues (n = 66) were collected from patients (age range, 34-80 years; mean age, 60.3 years) who underwent surgery in Lanzhou University Second Hospital between March 2018 and February 2019. A diagnosis of CRC was confirmed by an experienced pathologist. None of the patients received chemotherapy or radiation therapy before resection.

Immunohistochemical analysis
The CRC tissues were fixed, dehydrated, embedded in paraffin, and then cut into 4 µm-thick slices, which were stained with hematoxylin. Prior to analysis, the tissue specimens were deparaffinized using a graded series of xylene and rehydrated with a graded series of ethanol. Antigen retrieval was performed using sodium citrate and blocking was performed using H2O2. The sections were then incubated with primary antibodies against PKM2 and Notch1 for 16 h at 4℃ and then with horseradish peroxidase-conjugated secondary antibody (Beijing Zhongshan Jinqiao Biological Technology Co., Ltd., Beijing, China) for 2 h at room temperature. The presence of the secondary antibody was detected using a Cell and Tissue Staining Horseradish Peroxidase-Diaminobenzidine Kit (Beijing Zhongshan Jinqiao Biological Technology Co., Ltd.). An orthophoto microscope was used to obtain images of the stained tissue specimens.
All of the stained tissue specimens were graded by two qualified pathologists using a two-level scoring method. The proportion of positively stained cancer cells of each section was counted and the degree of staining was quantified. The grades of the stained tissue specimens are shown in Table 1. Finally, the results of the two scores were multiplied. A score < 3 indicated negative protein expression, while a score of ≥3 indicated positive expression.

Cell Counting Kit-8 (CCK-8) assay
The RKO and HT29 cells were plated in the wells 96-well plates at a density of

Statistical analyses
All statistical analyses were performed using IBM SPSS Statistics for Windows, version 26.0. (IBM Corporation, Armonk, NY, USA). Differences among groups were evaluated using one-way analysis of variance. A probability (p) value of < 0.05 was considered statistically significant.

PKM2 and Notch1 were upregulated in CRC tissues
The result showed that PKM2 and Notch1 were highly expressed in 71.2% (47/66) and 68.2% (45/66) of CRC tissues, respectively ( Figure 1 and Table 2), and were clearly co-expressed (Table 3, R = 0.356, p < 0.01). Notably, there were significant differences between PKM2 and sex, age, lymph node metastasis, and tumor stage, and between Notch1 and lymph node metastasis and tumor stage.

The relationship between PKM2 and Notch1 in CRC cells
The results showed that suppression of PKM2 decreased the mRNA and protein

Discussion
CRC is one of the most common cancers and among the leading causes of cancerrelated mortality worldwide [8]. In addition, CRC is a highly proliferative and aggressive malignancy, resulting in poor patient prognosis. Excision and chemotherapy are the main treatment strategies for CRC, although the benefits are limited due to side effects and recurrence [9]. Therefore, there is an urgent need to understand the mechanisms underlying the carcinogenesis of CRC and to develop new therapeutic strategies.
PKM2, which is upregulated in a various types of cancer, including CRC, catalyzes the final step of glycolysis and readjusts glycolytic flux to meet the specific metabolic needs of proliferating cells [10,11]. PKM2 promotes the mobility of gastric cancer cells via regulation of the PI3K/AKT pathway [12]. In prostate cancer, high expression of PKM2 during hypoxia leads to resistance to an mTOR inhibitor [13]. In CRC patients, high expression of PKM2 is predictive of a poor 5-year survival rate [5]. The Notch signaling pathway plays key roles in determining cell fate in multicellular organisms [14]. Abnormal expression of Notch1 has been attributed to the severity of CRC [15] and high Notch1 protein expression was correlated with a poor outcome in CRC [7]. Notch1 is also a prognostic biomarker of poorer survival of CRC patients [16]. Therefore, the aim of the present study was to determine the relationship between PKM2 and Notch1 in CRC.
Immunohistochemical analysis revealed high protein co-expression of PKM2 and Notch1 in CRC tissues. Moreover, as compared to NCM460 cells, the mRNA, and protein levels of PKM2 and Notch1 are highly expressed in RKO and HT29 cells.
Targeting of PKM2 or Notch1 by gene silencing or pharmacological methods is an effective anti-cancer strategy. For example, the PKM2 inhibitor shikonin is especially effective against bladder cancer via the induction of necroptosis [17]. Another PKM2 inhibitor, compound 3K, has anti-proliferation effects in liver and colon cancers [18,19]. TGN, a Notch1-specific inhibitor, is effective against gastric cancer cells [20].
In the present study, the PKM2 inhibitor compound 3K and the Notch1 inhibitor TGN were employed to determine the feasibility of simultaneous inhibition of PKM2

Availability of data and materials
All data can be obtained from the manuscript or from request to the author.