Bradley J. Monk, MD, discusses his insight on the potential of immunotherapy in ovarian cancer, as well as what promise it might hold in other gynecologic malignancies.
Bradley J. Monk, MD
Bradley J. Monk, MD
Results of the phase III JAVELIN 200 Ovarian trial have the potential to make a practice-changing impact on the field of ovarian cancer, according to Bradley J. Monk, MD, as immunotherapy could finally make its mark on management of this disease.
The randomized phase III study is comparing the PD-L1 inhibitor avelumab (Bavencio) alone or combined with pegylated liposomal doxorubicin versus pegylated liposomal doxorubicin alone in patients with platinum-resistant/refractory ovarian cancer (NCT02580058). The coprimary endpoints of the trial, which is estimated to be completed in March 2018, are progression-free survival (PFS) and overall survival (OS).
Earlier findings of a phase Ib study, which were presented during the 2016 American Society of Clinical Oncology Annual Meeting, showed that the objective response rate for avelumab was 9.7%, with partial responses observed in 12 of the 124 patients on the trial. Additionally, the disease-control rate was 54%, the median PFS was 11.3 weeks, and the median OS was 10.8 months.
Monk, professor and director of the Division of Gynecologic Oncology at Creighton University School of Medicine at St. Joseph’s Hospital and Medical Center in Phoenix, and a gynecologic oncologist with Arizona Oncology, shared his insight on the potential of immunotherapy in ovarian cancer, as well as what promise it might hold in other gynecologic malignancies.
TARGETED ONCOLOGY:Please provide an overview of your discussion of immunotherapy in ovarian cancer.
Immunotherapy is an emerging treatment in all solid tumors and is approved in many: head and neck, bladder, melanoma, lung, and kidney, but not ovarian cancer. Five randomized phase III trials are ongoing of avelumab and atezolizumab (Tecentriq), combining checkpoint inhibitors with chemotherapy either in the frontline setting or in platinum-resistant patients, or in the relapse of platinum-sensitive patients. We are excited about these trials.
We combine immunotherapy agents with chemotherapy for 2 reasons. Tumor-infiltrating lymphocytes (TILs) are not present in about 40% of newly diagnosed epithelial ovarian cancers, and without TILs, checkpoint inhibitors are not likely to work. PD-L1 expression is also not there in about 30% of patients. We are hoping that by adding chemotherapy to checkpoint inhibition, we will be able to cause immunogenic cell death. As the cells die as a result of the chemotherapy, it will auto-immunize the patient, and then checkpoint inhibitors will help the patient’s immune system fight the cancer.
TARGETED ONCOLOGY:How far along are some of these trials?
We have a number of early phase I and phase II studies, but we are probably going to need a randomized trial to gain FDA approval of a checkpoint inhibitor in ovarian cancer. There are some early signals that may appear in some of these early trials; for example, the rate of pathologic complete response after neoadjuvant chemotherapy would be 1 potential accelerated approval mechanism.
The first randomized phase III study to report will be the JAVELIN 200 Ovarian trial, which is avelumab versus pegylated liposomal doxorubicin versus the combination of pegylated liposomal doxorubicin and avelumab. It’s a 3-arm trial; the study has targeted to enroll 550 patients and it is almost at 500. Again, these are platinum-resistant patients who recur within 6 months of platinum-based therapy. The endpoint, which is a coprimary one of PFS and OS, could report out relatively soon. We are excited about it.
TARGETED ONCOLOGY:Could immunotherapy agents have similar potential in other gynecologic cancers?
Immunotherapy is not only important in ovarian cancer, but also in cervical and endometrial cancer. In cervical cancer, there is a randomized phase III trial of a checkpoint inhibitor getting ready to start. I can’t tell you the details quite yet, but it’s imminent.
We are most excited about the use of checkpoint inhibitors, particularly pembrolizumab (Keytruda) in microsatellite instability (MSI)-high recurrent endometrial cancer. This is for second-line endometrial cancer. Loss of expression of these mismatched-repair proteins that have MSI-high mutational burden have shown exceptional responses to single-agent pembrolizumab. There's a Prescription Drug User Fee Act date for pembrolizumab, as reported by Merck, in early June 2017. This could be the first checkpoint inhibitor approved in a gynecologic cancer.
Disis ML, Patel MR, Pant S, et al. Avelumab (MSB0010718C; anti-PD-L1) in patients with recurrent/refractory ovarian cancer from the JAVELIN Solid Tumor phase Ib trial: Safety and clinical activity.J Clin Oncol34, 2016 (suppl; abstr 5533).


New research shows that long-term endocrine therapy can reduce the risk of breast cancer recurrence in the long term. But side effects keep some women from taking it.
Like many breast cancer survivors, Julie Barthels wonders if the disease will return.
“My breast cancer came silently, without my knowledge. How do I know what is silently growing inside of me now?” she asked.
According to new research, taking endocrine therapy for five years can drastically reduce the recurrence.
Taking it longer might offer continued protection.
About two of every three breast cancers are hormone receptor (HR) positive.
These are the people who can benefit from long-term endocrine therapy, such as tamoxifen.
Tamoxifen blocks the effects of estrogen.
The researchers conducted a meta-analysis of 88 trials involving 62,923 women. All had estrogen receptor (ER) positive breast cancer.
All were disease-free after five years of prescribed endocrine therapy.
The rate of recurrence was steady during a 5-year to 20-year period.
Distant recurrence was found to have a strong correlation with original tumor size and lymph node status.
Women with larger tumors and more lymph node involvement had a higher risk of recurrence, ranging from 10 to 41 percent.
The study, conducted by an international team of researchers, is published in The New England Journal of Medicine.
Doctors put it in perspective
Dr. Paula Klein is director of cancer clinical trials at Mount Sinai Downtown-Chelsea Center as well as an associate professor of hematology and medical oncology at the Icahn School of Medicine at Mount Sinai in New York.
Asked about the research, Klein told Healthline there are some caveats.
“This is a meta-analysis. They were trials of women scheduled to receive five years of therapy, but we don’t know if they completed their therapy. We know there’s not an insignificant number of patients who are noncompliant,” she said.
Klein observed that the analysis was of patients diagnosed before the year 2000.
“This paper is really not completely relevant to today’s population. We have better screening and diagnostic methods. We’re better at staging, surgery, radiation, and systemic therapy. The mortality rates of breast cancer have gone down over several decades. Patients don’t have to be extra worried,” she said.
Klein explained that patients can have one of three variations of HR-positive breast cancer.
It can be ER-positive and progesterone receptor (PR) negative, ER-negative and PR-positive, or ER-positive and PR-positive.
All three groups of HR-positive patients are treated with the same anti-estrogen therapies.
Dr. Sarah P. Cate, director of the Special Surveillance and Breast Program at Mount Sinai Downtown-Chelsea Center, told Healthline that this study won’t change current practices.
“Most practice-changing types of studies are those that are randomized and prospective. While this study is important, I don’t know that it’s presenting much different data than already presented in prior studies done in a randomized fashion,” she said.
Who needs endocrine therapy
Eileen Phillips of Colorado received her diagnosis in 1998.
She had two lumpectomies, chemotherapy, and radiation treatment.
In 2000, while still taking tamoxifen, the cancer recurred in the other breast.
This time, she had a double mastectomy but wasn’t prescribed tamoxifen.
After her diagnosis in 2010, Barthels had surgery and chemotherapy before starting on tamoxifen.
Klein said until the past few years, the standard prescription was five years of tamoxifen or an aromatase inhibitor (AI).
AIs are usually used in postmenopausal women whose ovaries no longer produce estrogen. The drugs halt production of estrogen in an enzyme called aromatase.
Klein explained that recent studies show that it’s safe and effective to take these medications for 10 years.
Who should do so?
High-risk patients who are tolerant, motivated, and compliant, according to Klein.
Who should not?
“Certainly anyone who does not have ER-positive or PR-positive breast cancer,” she said. “Or extraordinarily low-risk women who have serious contraindications to the medications.”
It’s something that must be decided on an individual basis.
“We know that 30 percent of early stage ER-positive breast cancers recur, usually somewhere other than in a breast. The question of how to identify which patients will benefit from extended treatment with estrogen is still up in the air. In general, if you have an ER-positive tumor, there’s tremendous benefit from endocrine treatment,” said Cate.
“In younger women with more aggressive disease, we always give 10 years of tamoxifen. For younger patients, the data supports tamoxifen, but we can also do ovarian suppression with drugs or ovary removal, then AI,” she added.
“Another study found there’s very little benefit in giving older patients 10 years of anti-estrogen drugs, and there’s a higher risk of complications. Older post-menopausal patients have many other options in terms of endocrine treatment with different side effect profiles,” said Cate.
Why some women quit endocrine therapy
Compliance is a problem when a drug has to be taken for many years, according to Cate.
And there are troublesome side effects.
Barthels’ side effects include weight gain, joint swelling, fatigue, and depression.
“All of these side effects have been difficult because I had a very active lifestyle before the cancer,” she told Healthline.
Barthels’ follow-up practices are more extensive than most. Though she hasn’t had a breast cancer recurrence, she’s since been treated for skin and renal cancer.
None of that has stopped her from taking tamoxifen, which she’ll continue until 2021, a full 10 years.
Frances Hathaway of New York was diagnosed with stage 3 breast cancer in 1998.
She had surgery and chemotherapy. But she couldn’t tolerate the side effects of tamoxifen.
“The mental effect was not good so I stopped taking it. I went to a very dark place and had thoughts of suicide, which is not my personality or nature. Uterine cancer is another risk, so what’s the point?” said Hathaway.
Working around side effects
When it comes to better compliance, Klein said two things must be considered: quality-of-life complaints and real long-term toxicities.
“For the nagging quality-of-life issues there are non-hormonal remedies for many of them. You need to first establish that the complaints are related to the medicine. They may be age-related,” she explained.
Klein said that postmenopausal women who can’t tolerate one AI may do better with a different one. And premenopausal women who can’t tolerate tamoxifen have other options as well.
“The most serious side effects of tamoxifen are higher risk of uterine cancer and blood clots. AIs can cause accelerated bone loss. Both share all the quality of life issues: vaginal dryness, hot flashes, night sweats, and changes to mood, weight, and sexual desire. Change of life stuff,” said Klein.
Fear of recurrence
Barthels’ multiple cancer diagnoses certainly warrant concern.
“I am in a place of awareness about recurrence and it can make me fearful at times,” she said.
“This can be hard for people without cancer to understand, as they may feel like treatment is over and let’s move on. It is more complicated than that. It really is about acceptance for me, and that is a process I practice on a regular basis,” she continued.
Since having a double mastectomy, Phillips said thoughts of recurrence don’t weigh too heavily.
“I’m coming up on 20 years as a survivor,” she said. “There is hope.”
For Hathaway, a nagging feeling led her to discover a lump 19 years after her first bout with breast cancer.
She had more surgery and radiation treatments, but she isn’t taking tamoxifen.
Now a patient of Klein, Hathaway is currently enrolled in a clinical trial at Mount Sinai. In the trial, patients with breast cancer are using yoga and meditation to relieve chemotherapy symptoms.
Promising new research
Researchers at Mount Sinai have identified a protein (PTK6) that promotes cell growth and survival in a number of cancers, including ER-positive breast cancer.
That includes those who are resistant to tamoxifen.
The discovery could be a stepping stone to new targeted therapies.
Dr. Hanna Irie is an assistant professor of medicine (hematology and medical oncology) and oncological sciences at The Tisch Cancer Institute, Icahn School of Medicine at Mount Sinai, and senior author of the study.
“Endocrine therapies are still the most effective medical therapy for this subtype of breast cancer, and the end goal is to inhibit growth and/or kill ER-positive breast cancer cells. However, some breast cancer patients still develop metastatic ER-positive disease despite these common endocrine therapies, so newer treatments are very important and necessary to kill endocrine therapy-resistant cancers,” she said in a press release.
This study was published November 17 in NPJ Breast Cancer.
What women need to know
Cate recommends discussing your options with your physician, but if you’re younger than 50, have lymph node involvement or late-stage disease, you should “most definitely consider taking endocrine therapy for 10 years.”
And for small tumors in postmenopausal women, it’s a case-by-case decision.
“Patients are calling now and asking if they should get another 5 years, but they’re 10 or 15 years out from diagnosis. We don’t know the answer, but it’s not standard of care because all the studies have been about continuous therapy,” said Cate.
Klein wants breast cancer survivors to know that the outlook is much brighter now.
“Regardless of what you hear, we’re doing better,” said Klein
Barbara Jacoby is an award winning blogger that has contributed her writings to multiple online publications that have touched readers worldwide.


polyostotic fibrous dysplasia...fibrous dysplasia who exhibit café au lait (pale brown) spots on the skin and an endocrine imbalance leading to precocious puberty, especially in girls, have a form of the disorder called McCune-Albright syndrome. Sometimes these patients also have symptoms of hyperthyroidism or acromegaly. Patients with McCune-Albright syndrome have somatic mutations (mutations in body cells as...
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We provide creative activities to aid wellbeing, stimulate cognitive function and provide important tools for self-expression. Taking part in creative activities can also help patients to manage long-term conditions better, and cope with anxiety and depression.
These take place in both inpatient and outpatient departments, at patient bedsides and within the community.
Some current and previous projects include:
Creative Drawing Sessions for Staff
Our artist-in-residence Liz Atkin runs Creative Drawing workshops at both our hospital sites and for community staff. As well as sessions running after work, Liz also visits staff rooms on the wards so clinicians can have a creative break from their work.
The workshops aim to channel attention through simple yet inspiring drawing activities, designed to refocus anxiety and alleviate work-related stress.
Royal Horticultural Society (RHS) Wellbeing Garden
Our wellbeing garden at University Hospital Lewisham (UHL) is a site for numerous arts workshops as well as providing inspiration for other projects across the Trust.
Working closely with the RHS Community Development team we have developed a programme of arts activities for the garden space, including drawing workshops, live music performances, mosaic-making workshops and a staff photography competition inspired by the benefits that getting outdoors brings.
Inpatient 'creative activity packs'
We provide arts and crafts pack for inpatients to use, these contain all the materials needed to undertake some drawing, painting, colouring in and crafts. At the end of their hospital stay patients can take their creations home with them. This is couple of with creative iPad sessions where volunteers work with patients to use creative apps and games.
Singing for Lung Health
Delivered in partnership with Trinity Laban Conservatoire of Music and Dance, our Singing for Lung Health group was designed to help people living with lung conditions to manage feelings of breathlessness whilst taking part in a fun and social activity.
Research has proven that singing can help people with chronic lung conditions by improving a sense of control over the breath, improving posture and teaching you to breathe more slowly and deeply.
Victoria Rance, artist-in-residence
The ‘I Wish’ artist residency was led by local artist Victoria Rance, from APT Studios in Deptford. Victoria worked with patients, parents and carers on the children’s inpatients ward at Queen Elizabeth Hospital who communicated their wishes to the artist. These were then realised as small sculptures and gifted back to the participants.
Photographs of the ‘wishes’ now traverse the corridor spaces between the children’s wards helping to connect these different departments.


- Primary research
- Open Access
Effect of nicastrin on hepatocellular carcinoma proliferation and apoptosis through PI3K/AKT signalling pathway modulation
Cancer Cell International volume 20, Article number: 91 (2020)
Increasing evidence has proven that the γ-secretase complex plays significant roles in the carcinogenesis of malignancies. However, the independent effect of nicastrin (NCSTN), the largest constituent of the γ-secretase complex, on the progression of hepatocellular carcinoma (HCC) remains to be discovered.
In our study, we used open online databases, including the Oncomine database, GEPIA and KMPlotter, to analyse the expression of 4 genes and their correlation with prognosis in HCC. NCSTN expression in 60 HCC patients from our centre was determined by immunohistochemical staining and qRT-PCR. The clinical and prognostic significance of NCSTN expression were analysed statistically. Stable Sk-hep1 cell lines with NCSTN overexpression were established using lentivirus-based vectors, and RNAi technology was used to transiently downregulate NCSTN expression in HepG2 cell lines. Cell growth and apoptosis were assessed by using EdU, clone formation, flow cytometry and Western blotting assays.
Bioinformatics analysis showed that NCSTN mRNA expression was generally higher in HCC tissues than in normal tissues according to a meta-analysis of 9 HCC datasets, excluding PS-1, PEN-2 and APH-1. Moreover, NCSTN was associated with a poor prognosis in HCC patients from The Cancer Genome Atlas (TCGA). Although the relationship between NCSTN levels and the clinicopathological features of HCC patients was not significant, a survival analysis of HCC patients from TCGA indicated that overall and disease-free survival were significantly associated with NCSTN expression. NCSTN expression in HCC cell lines regulated cell growth and apoptosis in vitro. NCSTN downregulation in HepG2 cells inhibited tumour growth ability in vivo. In addition, NCSTN downregulation in HepG2 cell lines decreased p-PI3K and p-Akt expression, and IGF1, a PI3K/Akt activator, neutralized the effects on PI3K and Akt phosphorylation. Moreover, NCSTN overexpression in Sk-hep1 cells activated the PI3K/Akt signalling pathway, and MK-2206, a PI3K/Akt inhibitor, reversed this activation according to Western blotting analysis.
We suggest that NCSTN serves as an oncogene in HCC by promoting growth and inhibiting apoptosis via the PI3K/Akt pathway, providing a potential novel therapeutic target for HCC treatment.
Hepatocellular carcinoma (HCC) is one of the most common malignant cancers and is characterized by high mortality and a low 5-year survival rate . Although curative therapies such as liver transplantation, radiofrequency ablation and radioembolization are rapidly developing and have improved early-stage HCC outcomes , the overall prognosis remains unsatisfactory because most patients are at an advanced stage when first diagnosed. Therefore, there is an unmet need for more effective treatments for advanced HCC. Accumulating evidence has demonstrated that the abnormal expression and mutation of genes are involved in the carcinogenesis and progression of HCC . In this regard, therapies targeting a specific oncogenic molecule expressed in cancer tissue are a reliable approach for treating HCC. However, it is necessary to identify a potential therapeutic target to improve the clinical treatment outcome of HCC patients.
The γ-secretase complex (GS), composed of presenilin-1 (PS-1), anterior-pharynx defective-1 (APH-1), PSEN enhancer 2 (PEN-2) and nicastrin (NCSTN), can regulate endocytosis, lysosomal acidification, and autophagy [4, 5]. Dysfunction and abnormal activation of these substrates often leads to tumourigenesis, metastasis, and drug resistance in existing treatment regimens . In recent years, the γ-secretase complex has been increasingly recognized as a vital target in human malignancies. NCSTN, one of the main constituents and the gatekeeper of the γ-secretase complex, modulates the composition of this complex, maintains enzyme stability and executes GS substrate recognition . Moreover, NCSTN has a GS-independent function in the process of controlling cell death through the Akt pathway . Consistent with the biological correlation of NCSTN in malignant diseases, studies have shown that NCSTN is upregulated in patients with colon cancer who underwent chemotherapy  and that stable knockdown of NCSTN enhances the antitumour effect of EGFR inhibitors by blocking the Notch and Akt signalling pathways . Intriguingly, several studies have proven that genome-wide liver carcinoma screening identified NCSTN as one of the 50 potential driver genes , and NCSTN is abnormally upregulated and frequently amplified in liver cancer . However, the precise relationship between NCSTN expression and HCC prognosis remains unknown. Together, these aforementioned studies prompted us to explore the unequivocal role of NCSTN in HCC progression and to confirm whether NCSTN could be a novel therapeutic target against HCC.
In this study, we hypothesized that NCSTN could activate the PI3K/Akt pathway to promote cell proliferation and inhibit cell apoptosis in HCC. To test this hypothesis, we used bioinformatics analyses to analyse the underlying function of NCSTN in malignant tumours. Gain-of-function and loss-of-function studies were utilized to validate the effects of NCSTN expression modulation in vitro and in vivo. Our work may be useful for developing a new therapeutic target for HCC.
Cell lines and culture
Seven HCC cell lines (Huh-7, Hep-3B, HepG2, PLC/PRF/5, Sk-hep1, 97H and SMMC-7721) were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL, Grand Island, NY, USA) supplemented with 10% foetal bovine serum (FBS; Sijiqing, Zhejiang, P. R. China), 100 U/ml penicillin and 100 U/ml streptomycin. The HepG2, 97H, Sk-hep1, and SMMC-7721 cell lines were purchased from the American Type Culture Collection (Manassas, VA, USA). Huh-7, Hep-3B, and PLC/PRF/5 cell lines were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, P. R. China). All cells were maintained in a humidified chamber with 5% CO2 at 37 °C.
Three short hairpin (sh) RNAs targeting the CDS of human NCSTN mRNA were cloned into a lentiviral vector (PLKO.1), and the NCSTN open reading frame was cloned into a lentiviral vector (GENEray, Shanghai, China). HepG2 cells were first transfected with shNCSTN 1, shNCSTN 2 and shNCSTN 3 (40 nM) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) and were then infected with a lentivirus delivering shNCSTN 1 (sequence: CCGGGAGCATGCTAAAGCCTATAAACTCGAGTTTATAGGCTTTAGCATGCTCTTTTTG). A control plasmid (shNC) was used as a negative control. Meanwhile, lentiviruses carrying vector-NC or vector-NCSTN were produced from 293T cells and used to infect Sk-hep1 cells. After 72 h of lentiviral infection, puromycin (2 μg/ml) was used to select cells with stable NCSTN expression for 2 weeks. Selective NCSTN silencing in HepG2 cells and stable NCSTN expression in Sk-hep1 cells were detected by qRT-PCR and Western blotting analysis.
Reagents and antibodies
Primary antibodies for proliferating cell nuclear antigen (PCNA) (#13110), p/t-PI3K (#4228/#4292), p/t-Akt (#4060/#4691), Bax/Bcl-2 (#5023/#4223), and caspase 3/cleaved caspase 3 (#9665) were purchased from Cell Signaling Technology (Danvers, MA, USA). An anti-NCSTN antibody (#14071-1-AP) was purchased from Proteintech (Chicago, USA). Antibodies against glyceraldehyde 3-phosphate dehydrogenase (GAPDH; #CW0100) and horseradish peroxidase (HRP)-labelled anti-rabbit secondary antibodies (#CW0102/#CW0103) were obtained from CWBIO (Beijing, P. R. China). MK-2206 (#SF2712) and recombinant human IGF-1 (#P5502) were purchased from Beyotime (Shanghai, China).
Real-time polymerase chain reaction (qRT-PCR)
Total RNA was isolated from cells using TRIzol® reagent (#15596-018; Invitrogen). Reverse transcription was performed using a PrimeScript RT reagent kit (#RR037; Takara) according to the manufacturer’s instructions. qRT-PCR was performed to evaluate NCSTN expression in different cell lines using the primers shown in Table 1. Relative gene expression levels were calculated using the comparative Ct (ΔΔCt) method, where the relative expression was calculated as 2-ΔΔCt, with Ct representing the threshold cycle.
After fixing with 4% formalin and embedding in paraffin wax, tissues were cut into 5-μm sections using a microtome. The sections were deparaffinized in xylene and rehydrated with graded concentrations of alcohol. Subsequently, the slides were treated with 3% H2O2 to block endogenous peroxidase activity, and antigen retrieval was performed by boiling in 1 mM EDTA (pH 8.0) for 10 to 15 min. To reduce nonspecific binding, 10% normal goat serum was applied. Then, the slides were incubated with a primary rabbit anti-human NCSTN antibody at 4 °C overnight. After washing with PBS, the sections were incubated with rabbit conjugate (#GK500705; Dako Corporation, Carpinteria, CA, USA) for approximately 1 h at 37 °C. The colour reaction was performed with DAB-positive substrate, and the slides were counterstained with haematoxylin. The staining intensity was classified into 4 grades: 0 (negative), 1 (weak), 2 (moderate), or 3 (strong). The percentage of NCSTN-positive cells was scored as 0 (0%), 1 (1–10%), 2 (11–50%), 3 (51–80%) and 4 (81–100%). The overall score was calculated using the following formula: overall score = intensity score x percentage score. Total scores of 0–4, 5–8, and 9–12 were defined as weak positive, moderate positive, and strong positive, respectively .
Patients and specimens
HCC specimens and adjacent normal tissues were collected from 60 HCC patients at Sun Yat-sen Memorial Hospital of Sun Yat-sen University (Guangzhou, China) from 2016 to 2019. The specimens were immediately frozen in liquid nitrogen after resection from HCC patients and then stored at − 80 °C. The clinical characteristics of the HCC patients involved in the study are shown in Additional file 1: Table S2. The study was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital. All patients provided informed consent for all treatments performed and to have their data used for research purposes.
Western blotting analysis
To examine protein expression after shRNA and vector-NCSTN lentiviral transfection, HepG2 and Sk-hep1 cells were collected. Total protein was extracted from cells lysed with RIPA lysis buffer (#CW2333; CWBIO), phosphatase inhibitors (#04906845001; Roche) and protease inhibitors (#05892791001; Roche). Cells were lysed in 4× SDS sample buffer. Protein samples were then separated by SDS-PAGE (#P0012A; Beyotime) and transferred to polyvinylidene difluoride membranes (#ISEQ 00010; Millipore, Billerica, MA, USA). After blocking with 5% bovine serum albumin (BSA), the membranes were exposed to antibodies against NCSTN, PCNA, p/t-PI3K, p/t-Akt, Bax/Bcl-2, caspase 3/cleaved caspase 3, and GAPDH with continuous agitation overnight at 4 °C, followed by incubation with an anti-rabbit HRP-linked secondary antibody at room temperature for 1 h. The bands were visualized using an enhanced chemiluminescence detection kit (#WBKLS0500; Millipore).
Cell viability assay
HepG2 cells were first transfected with shNCSTN 1, shNCSTN 2 and shNCSTN 3 (40 nM) using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) Then, HepG2 cells were seeded into a 96-well plate at densities of approximately 1 × 104 and 0.5 × 104 cells/well. Next, the cells were cultured with medium containing 10% FBS for 48 h. Following the manufacturer’s protocol, a Cell Counting Kit-8 (CCK-8; #C0040; Beyotime) assay was utilized to quantify the viable cells. Absorbance (450 nm) was measured using a microplate reader (Dynex, Chantilly, VA, USA), and the absorbance value obtained from the responding control group was normalized as 100%.
Cells were seeded in serum-free media 6 h prior to treatment to allow for cell cycle synchronization. After 48 h of shRNA interference or vector-NCSTN plasmid transfection, the cells were pulsed with 5-ethynyl-2′-deoxyuridine (BeyoClickTM EdU Cell Proliferation Kit with Alexa Fluor 488; #C0071; Beyotime) for 2 h before fixation in 4% paraformaldehyde (PFA) for 15 min and subsequent EdU detection per the manufacturer’s protocol. The results were imaged under an inverted fluorescence microscope. Quantification of EdU+ HCC cells was carried out using Adobe Photoshop CC 2018. The percentage of EdU+ cells for each field of view captured was recorded and analysed.
Cells treated under different conditions for 48 h were detached from 6-well culture plates, washed twice with PBS, and pelleted by centrifugation at 1000 rpm for 5 min. The cell pellets were resuspended in binding buffer with Annexin V-FITC (AV) and propidium iodide (PI). The apoptosis rate was measured by flow cytometry.
Colony formation assay
The effect of NCSTN knockdown on the colony-forming ability of HepG2 and NCSTN-overexpressing Sk-hep1 cells was analysed by colony formation assay. The infected cells were seeded into 6-well plates at a concentration of 400 cells per well. After incubation for 7 days, most single colonies contained more than 50 cells. The adherent cells were washed twice with PBS and then stained with crystal violet. Finally, the cells were rinsed with water, and pictures were taken.
The cancer genome atlas (TCGA) database
The TCGA database contains gene information from large-scale genomic sequencing. Data including the gene expression profiles and clinical information of HCC patients were retrieved from the Genomic Data Commons Data Portal within the TCGA data portal (https://portal.gdc.cancer.gov/). The clinical information is shown in Additional file 2: Table S1, and the prognosis associated with NCSTN was calculated by several online databases, such as Oncomine (https://www.oncomine.org) , GEPIA (http://gepia.cancer-pku.cn) , KMPlotter (http://www.kmplot.com), CBioPortal (https://www.cbioportal.org) [16, 17], gene set enrichment analysis (GSEA) software  and String (https://string-db.org) , to obtain a more comprehensive understanding of the γ-secretase complex.
Female BALB/c nude mice, 4–7 weeks old, were purchased from the Laboratory Animal Center of Sun Yat-sen University (Guangzhou, China). The mice were maintained in a specific pathogen-free environment with laminar air-flow conditions at 22–25 °C. All animals had free access to standard laboratory mouse food and water. Animal experiments were approved by the Bioethics Committee of Sun Yat-sen University and were performed according to the established guidelines.
Xenograft tumour growth
A total of 1 × 107 HepG2 cells were suspended in 100 μL serum-free DMEM and injected subcutaneously into the nude mice. The mice were then randomized into two groups (n = 3/group). The tumour volume was estimated based upon the following formula: V = (L*W*W)/2, where “L” and “W” represent the larger and smaller dimensions obtained from each measurement, respectively. The nude mice were sacrificed after 1 month, and the solid tumours were weighed and fixed in formaldehyde.
All data are represented as the mean ± SD from three independent experiments. The median expression level of NCSTN was used as the cutoff for the high NCSTN group and the low NCSTN group. Student’s t test was performed for statistical analysis, and P < 0.05 was considered significant.
Bioinformatics analysis indicated that NCSTN was pivotal in promoting HCC progression
We tried to determine whether NCSTN was the key gene in the progression of HCC patients by using bioinformatics analysis. As shown in Fig. 1a, a meta-analysis of 9 HCC datasets vividly showed that only NCSTN expression was higher in tumour specimens than in normal liver tissues over 9 datasets. However, only 7 datasets showed that APH-1 expression was slightly higher in tumour specimens. Moreover, 1 dataset revealed that PEN-2 expression was higher in tumour specimens, while 3 datasets showed that PS-1 had similar results. Moreover, these results were further confirmed in GEPIA, another online visualization site based on TCGA and GTEx data. The box plots shown in Fig. 1b reveal that NCSTN expression was markedly higher in liver hepatocellular carcinoma (LIHC) (P < 0.01). However, the other 3 members of the γ-secretase complex, excluding NCSTN, showed no obvious significance when comparing their expression between HCC and normal liver tissues (Fig. 1b). Importantly, the overall survival (OS) and disease-free survival (DFS) curves of LIHC patients with high or low NCSTN expression are shown in Fig. 1c. The results showed that not only the OS rate but also the DFS rate was significantly lower for patients with high NCSTN expression than for patients with low NCSTN expression. Furthermore, high NCSTN expression, but not the other 3 genes of the γ-secretase complex, indicated poor prognosis in 364 TCGA HCC patients, as analysed in the open online database KMPlotter (Fig. 1c). For genetic alterations, the CBioPortal database was used to analyse the changes in these four members using the mutation and CNA data of TCGA-LIHC patients. As shown in Fig. 1d, NCSTN was altered the most (13%), and the main type was amplification. In summary, bioinformatics analysis showed that high NCSTN expression represented a poor prognosis and may exert some underlying functions as an oncogene, independent of the γ-secretase complex.
The NCSTN expression and prognosis of 60 HCC patients in our centre were significantly related
To analyse the mRNA expression levels of NCSTN in 60 HCC patients, we analysed matched tumour and normal tissues by using qRT-PCR. Overall, NCSTN expression was significantly upregulated in HCC. Compared with the normal tissues, 53 (88.3%) HCC patient tissues were characterized by higher NCSTN expression (Fig. 2a). Consistent with the bioinformatics analysis results, only 32 (53%) patients had high PS-1 expression, 38 (63.3%) patients had high PEN-2 expression, and 35 (58.3%) patients had high APH-1 expression (Additional file 3: Figure S1). In addition, by calculating the OS and DFS curves, we found that high NCSTN expression was related to poor prognosis in HCC patients (Fig. 2b, c). On the other hand, the IHC results of 60 HCC patients further confirmed that NCSTN expression was higher in tumour specimens than in matched normal tissues (Fig. 2d, e). Taken together, these results showed that high NCSTN expression was correlated with poor prognosis in HCC patients.
Oncogenic function validated by NCSTN upregulation in Sk-hep1 cells and NCSTN downregulation in HepG2 cells
To confirm the lowest and highest expression levels of NCSTN in 7 different HCC cell lines, we used Western blotting and qRT-PCR to detect the protein and mRNA expression levels of NCSTN, respectively. Figure 3a shows that NCSTN protein expression was lowest in Sk-hep1 cells and highest in HepG2 cells among the 7 HCC cell lines. Similar results were obtained from qRT-PCR (Fig. 3b). However, not all mRNA and protein expression levels were consistent among the 7 HCC cell lines. We found that Sk-hep1 cells expressed lower NCSTN than the other 6 cell lines, while HepG2 cells expressed higher NCSTN than the other cells (Fig. 3b). To determine the most efficient RNAi sequence in HepG2 cells, we measured NCSTN mRNA expression after knocking down the NCSTN gene with 3 different shRNA sequences (Fig. 3c). This result showed that all 3 shRNA sequences could knock down NCSTN mRNA expression, but shNCSTN 1 dramatically inhibited NCSTN expression in HepG2 cells. We also conducted a cell growth experiment to estimate which shRNA sequences could affect HCC cell viability. As shown in Fig. 3d, shNCSTN 1 could reduce many more viable cells than the other two shRNA sequences. To further verify the knockdown efficiency of shNCSTN 1, we measured the protein expression level of NCSTN using Western blotting (Fig. 3e). The shNCSTN 1 sequence markedly downregulated NCSTN protein expression in HepG2 cells. Thus, these results suggested that the RNAi efficiency was adequate. On the other hand, for gain-of-function analyses, we overexpressed NCSTN in Sk-hep1 cells, which have relatively low NCSTN expression. The NCSTN expression level was significantly increased in NCSTN-transfected Sk-hep1 cells, as assessed by qRT-PCR and Western blotting (Fig. 3c, e).
NCSTN downregulation inhibited HepG2 cell proliferation, and NCSTN-transfected Sk-hep1 cells showed higher proliferative ability
Provided that the correlation between NCSTN and cell proliferation was ambiguous, Western blotting analysis was adopted to identify PCNA expression. As shown in Fig. 4a, after NCSTN silencing in HepG2 cells, the shNCSTN 1 group exhibited markedly decreased PCNA expression compared with the shNC group. This result indicated that NCSTN knockdown had an inhibitory effect on PCNA, which could promote the process of leading strand synthesis during DNA replication. Since NCSTN expression was related to PCNA expression, we then determined proliferation in HepG2 cells with NCSTN knockdown by using EdU thymidine analogue incorporation and clone formation assays (Fig. 4b, c). Consistent with the Western blotting results, the EdU-positive cell ratio was much lower in the shNCSTN 1 group than in the other groups, and the number of colonies was lower in the shNCSTN 1 group than in the shNC group.
On the other hand, relatively contrary results were obtained in the gain-of-function assay. These data suggested that NCSTN regulated proliferation in both HepG2 and Sk-hep1 cells.
NCSTN expression affected apoptosis in both HepG2 and Sk-hep1 cells
Based on the GSEA results for TCGA data, we found that 54 signalling pathways, including cancer-related pathways and apoptosis pathways (Fig. 5a, b), were enriched in the high NCSTN expression group (Additional file 4: Table S3). Considering the significance of NCSTN in regulating cell proliferation, we further investigated the antiapoptotic effect of NCSTN. Western blotting analysis revealed that the protein expression of cleaved caspase 3 and Bax was upregulated, while caspase 3 and Bcl-2 were downregulated in the shNCSTN 1 group. Comparably, cleaved caspase 3 and Bax were downregulated, and caspase 3 and Bcl-2 were upregulated the vector-NCSTN group (Fig. 5d, e). Moreover, Annexin V staining and flow cytometry were used to further study whether NCSTN and apoptosis were strongly related. As shown in Fig. 5c, the rate of apoptotic HCC cells in the shNCSTN 1 group was significantly different from that in the control group. A much higher early apoptosis rate appeared in the vector-NC group than in the vector-NCSTN group. Therefore, we concluded that HCC cell line apoptosis was strongly related to NCSTN expression.
NCSTN regulated cell proliferation and apoptosis through the PI3K/Akt pathway
In general, most oncogenes promote cancer progression via specific signalling pathways. As exhibited in Fig. 6a, KEGG results for GSEA contained two PI3K-related pathways: “INOSITOL_PHOSPHATE_METABOLISM” and “PHOSPHATIDYLINOSITOL_SIGNALING_SYSTEM”. PIK3CA, PIK3R1, PIK3CB and AKT2 all participated in the “PATHWAYS_IN_CANCER” exhibited in the PPI network (Fig. 6b, Additional file 5: Table S4). Therefore, we further investigated the phosphorylation levels of PI3K and Akt in HCC cell lines treated with specific NCSTN-targeting shRNAs or NCSTN plasmids. As a result, p-PI3K and p-Akt levels were markedly decreased in the shNCSTN 1 group compared with the shNC group, while IGF1, an agonist of the PI3K/Akt pathway, reversed the effect that resulted from knocking down NCSTN. On the other hand, NCSTN overexpression significantly increased the phosphorylation levels of PI3K and Akt. MK-2206, an inhibitor of the PI3K/Akt pathway, suppressed the high expression of p-PI3K and p-Akt caused by upregulating NCSTN in Sk-hep1 cells (Fig. 6c). Therefore, we found that NCSTN might activate the PI3K/Akt pathway to regulate cell proliferation and inhibit apoptosis in HCC.
Lentivirus-mediated NCSTN silencing in HepG2 cells inhibited tumour proliferation in vivo
Evidence from the cell lines and human tissues mentioned above indicated that NCSTN is implicated in HCC tumourigenesis. To further confirm the hepatocarcinogenesis of NCSTN in vivo, xenograft tumour mouse models were injected with HepG2 cells. Compared with that in the shNC group treated with vector-NC plasmid, tumour growth in the group treated with vector-NCSTN plasmid was significantly inhibited (Fig. 7a, b). In addition, the tumour weight in the shNCSTN group was 57.01% of that of the shNC group at the end of the study (Fig. 7c). In summary, we concluded that NCSTN also possessed antitumour effects in vivo.
In our present study, we found that NCSTN promotes cell growth and negatively regulates apoptosis via the PI3K/Akt signalling pathway. Indeed, 53 out of 60 HCC patients in our centre had higher NCSTN expression in tumour specimens than in paired normal tissues. Furthermore, by regulating the PI3K/Akt pathway, NCSTN depletion in HepG2 cells and NCSTN overexpression in Sk-hep1 cells both had obviously different effects on cell proliferation and antiapoptosis processes, hallmarks of carcinogenesis and programmed cell death.
Previous studies have shown that the γ-secretase complex, comprised of PS-1, APH-1, PEN-2 and NCSTN, is recognized as a therapeutic option for treating HCC [10, 20,21,22]. However, inhibitors of the γ-secretase complex are well known to have serious adverse effects, as they are not cell selective or system specific . Targeted therapy strategies have been developed and are now widely used in clinics. To date, several monoclonal antibodies against NCSTN have been developed and have shown great efficacy in inhibiting cancer cell proliferation in vitro and in vivo . However, whether NCSTN monoclonal antibodies could effectively act on liver cancer cells remains unclear. Therefore, it is pivotal to determine the specific function of NCSTN in HCC.
NCSTN, the largest constituent of the γ-secretase complex , has a single transmembrane domain and a large extracellular domain with functional regions . Intriguingly, it has been reported that NCSTN could exert its function alone rather than participate in the γ-secretase complex in mouse muscle , which indicates that NCSTN is likely to exert some functions independently. Similarly, in light of our bioinformatics analysis results, we found that NCSTN is overexpressed in HCC over 9 microarray datasets from the Oncomine database. However, the expression levels of PS-1, APH-1 and PEN-2 are slightly higher in HCC tissues than in normal tissues in only several microarray datasets. According to the analysis of TCGA data with GEPIA, we further confirmed that the mRNA expression of PS-1, APH-1 and PEN-2 in HCC patients from TCGA was not significantly different between tumour specimens and normal tissue specimens. On the other hand, we also found that among TCGA data for HCC patients analysed by KMPlotter, only NCSTN expression was closely related to survival time, not PS-1, APH-1 and PEN-2. We also found that NCSTN was altered most among these four genes in TCGA-LIHC patients according to the corresponding CNV and mutation data analysed by the CBioPortal database. Together, we hypothesized that the tumourigenesis of NCSTN can also play a role without the involvement of other members of the γ-secretase complex. The bioinformatics analysis results prompted us to explore the independent function of NCSTN in HCC patients.
Next, we investigated the mRNA and protein expression of NCSTN in patients at our centre by using qRT-PCR and IHC staining. The IHC score results for 60 HCC patients showed high degrees of consistency with those obtained from qRT-PCR. It turned out that high NCSTN expression and poor prognosis are closely related. Likewise, Lee et al.  also identified NCSTN as an oncogene that is upregulated and frequently amplified in human HCC. In all, although its correlation with the clinicopathological features of HCC patients failed to have great significance for both TCGA data (Additional file 2: Table S1) and 60 patients from our centre (Additional file 1: Table S2), NCSTN expression was closely related to the clinical prognosis of HCC patients. These observations led us to test the role of NCSTN in tumour cell proliferation and apoptosis. Therefore, we analysed the impact of NCSTN inhibition in HepG2 cells using transient shRNA oligos targeting NCSTN mRNA and NCSTN overexpression in Sk-hep1 cells using an NCSTN plasmid.
Other than its requirement for the γ-secretase complex, NCSTN is likely to regulate cell death through the PI3K/Akt and P53/caspase3 pathways in fibroblasts, human cells and neurons . Furthermore, Lombardo et al.  reported that NCSTN regulates Akt activation in breast cancer cells and that NCSTN inhibition in malignant breast cells can inhibit breast tumour formation in vivo. Studies have also demonstrated that anti-NCSTN monoclonal antibodies exert antitumour effects in invasive breast cancer cells . Notably, these results are consistent with not only the KEGG pathway results for GSEA based on TCGA data but also our observation that NCSTN depletion could induce apoptosis and reduce proliferation in HCC cell lines, partly through the PI3K/Akt pathway.
However, these conclusions are based on the responses of only 2 HCC cell lines and several nude mice, so they might not reflect the process of intact organisms in the human body. The precise oncogenic mechanism of NCSTN needs to be further elucidated in detail.
Collectively, our findings support the hypothesis that NCSTN modulates proliferation and apoptosis through the PI3K/Akt pathway, thereby causing tumourigenesis in HCC. Our present work provides a better understanding of the potentially oncogenic mechanism of NCSTN in silico, in vitro and in vivo. We proposed that NCSTN, independent of the GS complex, may be a promising target for HCC treatment development.
Availability of data and materials
- GS complex:
Gene set enrichment analysis
Copy number variation
PSEN enhancer 2
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The authors express sincere thanks to the Natural Science Foundation of Guangdong Province of China (No. 2020A1515010944), the Science and Technology Program of Guangzhou, China (STPG; No. 2016201604030054), and the National Natural Science Foundation of China (No. 81800560).
Ethics approval and consent to participate
This study was approved by the Ethics Committee of Sun Yat-sen Memorial Hospital. All patients provided informed consent for all treatments performed and to have their data utilized for research.
The animal experiments were approved by the Bioethics Committee of Sun Yat-sen University and were conducted based upon acknowledged guidelines.
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The authors declare that they have no competing interests.
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Additional file 1: Table S2.
Correlation between NCSTN expression and clinicopathological characteristics of 60 HCC patients in our center.
Additional file 2: Table S1.
Relationship between NCSTN expression and clinicopathological characteristics in 370 HCC patients from TCGA database.
Additional file 3: Figure S1.
mRNA Expression level of APH-1, PEN-2 and PS-1 in 60 patients.
Additional file 4: Table S3.
Gene sets enriched in phenotype high.
Additional file 5: Table S4.
The enriched genes of cancer-related pathways.
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Wang, X., Wang, X., Xu, Y. et al. Effect of nicastrin on hepatocellular carcinoma proliferation and apoptosis through PI3K/AKT signalling pathway modulation. Cancer Cell Int 20, 91 (2020). https://doi.org/10.1186/s12935-020-01172-4
- Hepatocellular carcinoma