Endometrial Cancer Overview - Signaling Pathway. Diagnostics Marker. Targeted Therapy and Clinical Trials.

An Introduction to Endometrial Cancer

Endometrial cancer (EC) is the most common gynecologic malignancy. It is the fourth most common cancer in women in the United States after breast, lung, and colorectal cancers. Risk factors are related to excessive unopposed exposure of the endometrium to estrogen, including unopposed estrogen therapy, early menarche, late menopause, tamoxifen therapy, nulliparity, infertility or failure to ovulate, and polycystic ovary syndrome. Additional risk factors are increasing age, obesity, hypertension, diabetes mellitus, and hereditary nonpolyposis colorectal cancer. The American Cancer Society recommends that all women older than 65 years be informed of the risks and symptoms of endometrial cancer and advised to seek evaluation if symptoms occur. There is no evidence to support endometrial cancer screening in asymptomatic women. Evaluation of a patient with suspected disease should include a pregnancy test in women of childbearing age, complete blood count, and prothrombin time and partial thromboplastin time if bleeding is heavy. Most guidelines recommend either transvaginal ultrasonography or endometrial biopsy as the initial study. The mainstay of treatment for endometrial cancer is total hysterectomy with bilateral salpingo-oophorectomy. Therefore, in order to improve the benefit of screening and the survival rate of patients, it is very important to establish a system of early diagnosis and targeted therapy for endometrial cancer. At present, the main molecular signaling pathways of endometrial cancer including RTK signaling pathway, Wnt signaling pathway, Notch signaling pathway, as well as p53 and p16 signaling pathway.

1 Main Signaling Pathways in Endometrial Cancer Therapy

Fig.1 Endometrial cancer signaling pathway. Targeted agents (listed in orange boxes) include those in clinical use (colored in red) and those in preclinical or early phase development (colored in green) for the treatment of advanced stage endometrial cancer.

The endometrial cancer is the most common gynecologic malignancy that starts in the endometrium of women. Carcinogenesis of EC is associated with several critical regulatory molecules, which involve in different signaling pathways. A number of signaling pathways have been identified to be involved in the multiple-step development of EC, including PI3K/AKT/mTOR signaling pathway, WNT/β-catenin signal transduction cascades (including APC/β-catenin pathway), MAPK/ERK pathway, VEGF/VEGFR ligand receptor signaling pathway, ErbB signaling pathway, P53/P21 and P16INK4a/pRB signaling pathways. Based on the fundamental biological function of these signaling pathways and pathological features of EC, four capabilities of transforming endometrial cells are critical to the primary carcinogenesis and metastasis: 1) evading apoptosis, 2) enhancing cell proliferation, 3) blocking differentiation, 4) inducing angiogenesis. At the same time, above molecular mechanisms also provide multiple potential targets for the treatment of endometrial cancer.

1.1 RTK signaling pathway

RTK signaling pathway integrates both intracellular and extracellular signals and serves as a central regulator of basic biological processes of cells such as metabolism, growth, proliferation, survival and angiogenesis. mTOR pathway was identified to be activated during tumor formation and PTEN gene mutations, which plays a role in cellular signaling by inhibiting the MAPK kinase pathway, were associated with the histology, early stage, and favorable clinical behavior of endometrial cancers. MAPK regulates cell proliferation and differentiation via RAF-MEK-ERK signaling cascade. The ErbB receptors are mediators of cell proliferation, migration, differentiation, apoptosis, and cell motility by signaling transduction. Activation of AKT is closely associated with endometrial carcinogenesis and prognosis by controlling specific gene expression, thereafter occurs new hallmarks for cell transformation such as angiogenesis, evading apoptosis, enhancing proliferation and blocking differentiation. VEGFs and the angiopoietins are key factors in angiogenesis and play a significant role to myometrial invasion and lymph node metastasis.

1.2 Wnt signaling pathway

The Wnt signaling pathway is one of the most evolutionary-conserved signal transduction pathways. The Wnt signaling pathways include β-catenin dependent Wnt signaling pathway (i.e. canonical Wnt/β-catenin) and β-catenin independent Wnt signaling pathway (i.e. Non-canonical, such as Wnt/JNK pathway, Wnt/calcium pathway). Among these Wnt signaling pathways, the β-catenin dependent pathway has been associated with human endometrial cancer. For canonical Wnt signaling pathways, the major effector is the transcription factor β-catenin. Moreover, progesterone could inhibit Wnt signaling by induction of DKK1 and FOXO1 expression, the inhibition of Wnt signaling by progesterone was partly circumvented in Wnt activated Ishikawa cells. During the menstrual cycle, estradiol can enhance Wnt/β-catenin signaling, and constitutive activation of Wnt/β-catenin signaling will cause endometrial hyperplasia, which may develop further into endometrial cancer.

1.3 Notch signaling pathway

Notch signaling pathway is a highly conservative pathways, which have an important role in cell proliferation, differentiation and apoptosis. Notch signaling pathway starts in the form of through the ligand binding, then Notch1 receptor proteolytic cracking in the film, in order to release the Notch intracellular structure domain (NICD) activity. Then transferred to the nucleus and as transcription activator, in order to improve the target genes, such as HES1 expression. In addition, pathway can lead to tumorigenesis, related to duration, cell type and background. The research show Notch signaling pathway is a key factor regulating cell proliferation activity in endometrial cancer.

1.4 p53 and p16 signaling pathway

Apoptosis disorders are among the many mechanisms significant in carcinogenesis. One of the most extensively researched genes that takes part in apoptosis is the TP53 gene. Abnormal p53 protein production due to gene mutation leads to an inability to repair the damaged DNA and as a result, via complex mechanisms, to uncontrollable cancer cell growth and the presence of mutations in gene TP53, which is associated with statistically significant shorter patient survival, occurs in 17-61% of endometrioid cancers. The P16 gene is responsible for cell proliferation in the G1 phase. Its alterations and abnormal epigenetic modifications for are also play an important role during EC carcinogenesis.

2 Endometrial Cancer Diagnosis

The number of cases of endometrial cancer has shown a tendency to increase in recent years. Endometrial cancer originates from the endometrium and is classified, based on the development mechanism, into types 1 and 2, and have significantly different gene expression profiles. Studies of genes with abnormal expression in endometrial cancer have identified multiple oncogenes, tumor suppressors, mismatch repair genes, apoptosis-associated genes, levels of hormone receptors and DNA ploidy and aneuploidy as biomarkers of endometrial cancer. The use of these molecules and genes may facilitate accurate diagnosis and prognostic prediction and contribute to individualized treatment. Trials of drugs which target these biomarkers and searches for new biomarkers using cDNA microarrays and RT-qPCR are ongoing and it is likely that these findings can be translated to clinical use.

2.1 Molecular Markers for Endometrial cancer

Oncogenes that have been reported as biomarkers of endometrial cancer include K-ras, HER2/neu, EGFR, PI3KCA and FGFR2. These oncogenes are normally inactivated, and their activation causes cell division. K-ras is associated with signal transduction in growth and differentiation and has a high rate of mutation in pancreatic and colorectal cancer. The mutation of K-ras has also been found in 10–30% of cases of endometrioid cancer. HER-2/neu is a member of the receptor tyrosine kinase (RTK) family, EGFR serves as a trigger for cell proliferation through the Ras-Raf-MAP kinase pathway and PI3K and AKT are downstream serine/threonine kinases. The overexpression of HER-2/neu occurs in 9–30% of cases of endometrioid cancer and is frequently observed in non-endometrioid tumors. HER-2/neu amplification in endometrial cancer is relatively rare and immunohistochemical data are inconsistent. The mutation of PI3KCA occurs in 24–36% of cases of endometrioid cancer and a mutation in PTEN is simultaneously observed in 14–26% of these cases. A study concerning the copy numbers and expression levels of these genes in endometrial cancer indicated that the amplification of PI3KCA is associated with the activity of PI3K and suggested that PI3K is an influential target of anticancer drugs. In recent years, the mutation of FGFR2, another tyrosine kinase, has been found in 12% of cases of endometrial cancer. FGF is mitogenic for various cell types and is associated with regulation and tumor angiogenesis and metastasis. The mutation of FGFR2 causes carcinogenesis, which indicates that the inhibition of the activity of mutated FGFR2 may be a therapeutic approach and that FGFR2 is a potential candidate molecular target for the treatment of endometrial cancer. Tumor suppressor genes, which have been reported as biomarkers of endometrial cancer, include PTEN, p53, p21 and CDKN2A/p16. PTEN is the most frequently mutated gene in endometrial cancer, with a rate of 25–83% among all cases and a particularly high rate in endometrioid adenocarcinoma and tumors with microsatellite instability (MSI). In endometrial cancer, the overexpression of p53 occurs in 15–30% of cases and the mutation of p53 is found in 10–20% of cases and the loss of expression of p16 is observed in 14–74% of cases.

Table 1 Candidate molecular markers in endometrial cancer

2.2 Protein Markers for Endometrial cancer

In addition to the oncogenes and cancer suppressor genes described in the previous sections, several other proteins have been identified as biomarkers of endometrial cancer. CA125 is routinely tested as part of the diagnosis and treatment of endometrial cancer, and its determination before treatment can be valuable in the staging of endometrial cancer. However, the diagnostic sensitivity of CA125 in endometrial cancer is unsatisfactory, especially in early tumor stages. The clinical utility of other known tumor markers such as CA15.3 have proven to be unsatisfactory in patients with endometrial cancer. The human epididymis protein (HE4) is a potential biomarker useful in the diagnosis of ovarian cancer. Elevated levels of HE4 are also observed in patients with endometrial cancer. The serum concentration of HE4 has been shown to correlate with the depth of myometrial invasion and the stage of endometrial carcinoma. VEGF plays a crucial role in the initiation of physiological and pathological angiogenesis and overexpression of VEGF and its receptors are related to poor prognosis in patients with endometrial carcinomas. Some study results demonstrate a statistically significant relationship between Ki-67 positivity and tumor type of endometrioid endometrial carcinomas, advanced clinical stage of the disease, poor grade of differentiation, and deeper myometrial invasion (p < 0.05). Cyclooxygenase is an enzyme responsible for prostaglandin synthesis. Cyclooxygenase-2 (Cox-2) overexpression is correlated with neoangiogenesis, metastatic ability and poor prognosis, and the protein may play a critical role in carcinogenesis. It might be a new therapeutic target, but further studies are needed. In addition, E-cadherin and β-catenin are two basic adhesion molecules relevant to endometrial cancer. High expression of β-catenin is characteristic of endometrioid endometrial carcinoma (31-47%) in contrast to non-endometrioid endometrial carcinoma and low expression of E-cadherin and β-catenin correlates with poor prognosis.

3 Targeted Therapy for Endometrial Cancer

T Endometrial cancer is a common gynecological malignancy in Western countries. Additionally, the incidence of endometrial carcinoma is estimated to increase by 1–2% yearly. Most women are diagnosed at an early stage and have relatively good survival rates; however, women who are diagnosed with advanced-stage or recurrent disease have a poor prognosis. This background has led to studies on gene abnormalities related to the onset and progression of endometrial malignancy and the results of these studies have gradually revealed the developmental mechanism of endometrial cancer. The development of a malignant tumor generally requires an accumulation of genetic mutations through a process referred to as “multistep carcinogenesis”. In the endometrium, genetic and epigenetic abnormalities have been described at each step leading from a precursor lesion to invasive cancer. Several molecules associated with these abnormalities have been identified and novel treatment regimens for endometrial cancer are being developed based on drugs targeting these molecules, i.e., molecular-targeted drugs. In Table 2-5, selected clinical trials of novel therapeutic targets for the treatment of endometrial cancer are presented.

3.1 Endometrial cancer therapy for RTK signaling pathway

Treatment targeting the general characteristics of cancer includes antiangiogenic agents, such as bevacizumab, aflibercept and thalidomide. Bevacizumab is a humanized monoclonal antibody against vascular endothelial growth factor (VEGF)-A. Aflibercept (VEGF Trap-Eye) is a fusion protein that exhibits high-affinity binding to VEGF-A, VEGF-B and placental growth factor and thus has a unique mechanism of action as an inhibitor of angiogenesis. Thalidomide has a plurality of antitumor properties, including an anti-angiogenetic action, although the precise mechanism has not been elucidated. Targeting of molecules in the carcinogenic pathways of endometrial cancer includes the use of human epidermal growth factor receptor (EGFR) inhibitors, such as gefitinib, erlotinib and cetuximab. Gefitinib and erlotinib are low-molecular-weight tyrosine kinase inhibitors. Cetuximab is a monoclonal antibody against EGFR. Trastuzumab and lapatinib are human EGFR type 2 (HER2)-related inhibitors that affect signal transduction. The effects of PTEN mutations in type I endometrial cancer may be reduced by mammalian target of rapamycin (mTOR) inhibitors, such as temsirolimus and ridaforolimus, by blocking the phosphoinositide 3-kinase/AKT/mTOR pathway. Drugs with a plurality of targets include sunitinib, brivanib, sorafenib and imatinib. Sunitinib is a multi-kinase inhibitor that is currently under clinical trials to assess its effectiveness against recurrence or metastasis of endometrial cancer. Brivanib is a multi-targeted tyrosine kinase inhibitor and sorafenib is a tyrosine kinase inhibitor with an antiangiogenic action.

Table 2 Clinical trials of VEGF mAb Bevacizumab

Table 3 Clinical trials of HER2 mAb Trastuzumab

Table 4 Clinical trials of mTOR inhibitor Temsirolimus

According to statistics, a total of 12 Abiraterone acetate projects targeting endometrial cancer testosterone are currently in clinical stage, of which 5 are recruiting and 7 are not recruiting.

Table 5 Clinical trials of multi-kinase inhibitor Sunitinib

3.2 Endometrial cancer therapy for Wnt signaling pathway

Wnt signaling is one of the key cascades regulating development and stemness, and has also been tightly associated with cancer. Overcoming immune evasion by cancer cells is a promising therapeutic approach and immune checkpoint blockade was shown to be highly effective in the treatment of tumors. Therefore, inhibiting this pathway has been a recent focus for cancer research with multiple targetable candidates in development. Small molecule inhibitors, including IWPs and LGK974, were shown to selectively inhibit the acyl-transferase Porcupine and thus Wnt secretion, leading to a size reduction of MMTV-Wnt1-driven tumors. In addition, a novel orally available Porcupine inhibitor ETC-159 that was found to prevent growth of R-spondin-fusion positive CRC, is undergoing clinical testing since July 2015. Although anti-Wnt secretion therapeutics appear promising, the number and impact of potential side effects are currently unclear. OMP-54F28 is Fzd inhibitors that can reduce the size of tumor xenografts and overall tumor initiating cell number in mouse models. In addition, OMP18R5 is a monoclonal antibody targeting five of ten human Fzd receptors. It was shown to inhibit the growth of human tumor xenografts and to synergize with standard-of-care therapeutic agents. PRI-724 and the closely related compound ICG-001 specifically target the complex formation of β-catenin and CBP and inhibit the self-renewing downstream effects of β-catenin-CBP activity and leads to reduction of tumor burden. Tankyrase inhibitors such as XAV939, which stabilize Axin by blocking its PARsylation, have shown promising results as Wnt inhibitors.

3.3 Endometrial cancer therapy for Notch signaling pathway

Given the substantial roles of Notch signaling in cancer, CSC maintenance and angiogenesis, targeting Notch signaling with various investigational agents including small molecule inhibitors and large molecule mAbs has been moved from preclinical research into early clinical development. MK-0752 is a potent non-competitive oral γ-secretase inhibitor (GSI) and can induce G0/G1 arrest and apoptosis of T-ALL cell lines harboring Notch activating mutations. RO-4929097, a potent competitive oral GSI, was evaluated by Roche and the National Cancer Institute Cancer Treatment and Evaluation Program. There are several other GSIs currently in phase I clinical trials, such as PF-03084014, BMS-708163 and LY3039478. Another class of agents under development is the mAb either against Notch receptors or Notch ligands. OMP-21M18 is a humanized mAb against DLL4, which blocks the ligand to interact with Notch1 and Notch4 in the Notch signaling pathway. REGN421, a fully humanized mAb against DLL4 demonstrated a single agent activity in advanced solid tumors. A fully humanized monoclonal antibody OMP-59R5 selectively targeting Notch 2/3 receptors was tested in a phase I dose escalation study given as a single agent. OPM-52M51 is an anti-Notch1 antibody currently tested in a phase 1 clinical trial.

3.4 Endometrial cancer therapy for mutant p53

The majority of p53 mutations are missense mutations and result in the accumulation of dysfunctional p53 protein in tumors. These mutants frequently have oncogenic gain-of-function activities and exacerbate malignant properties of cancer cells, such as metastasis and drug resistance. CP-31398 (styrylquinazoline) was identified through a structure-based screening as a compound which could restore native wild-type p53 conformation from a denatured conformation. STIMA-1 induces mutant p53 (p53 R175H and p53 R273H)-dependent growth suppression and leads to mutant p53-dependent apoptosis. NSC652287 is known to inhibit tumor growth of renal cell carcinoma cells with DNA–protein cross-linking and upregulation of wild-type p53 and p21. NSC319726 facilitates the binding of mutant p53 to zinc and restores the proper folding and transcriptional activity of mutant p53. Other drugs and compounds that restore wild-type p53 activity also include PK7088, NSC87511, SCH529074 and WR-1065. In addition, there are other types of compounds that deplete mutant p53, such as Hsp90 inhibitors (17-AAG, Ganetespib), histone deacetylase inhibitors (SAHA, Depsipeptide), YK-3-237 and NSC59984.