Treatment Response of Prolactin Neutralizing Monoclonal Antibodies for Ovarian Cancer

Abstract

Prolactin is a pituitary gland secreted hormone in the response of various vital biological processes like ovulation and nursing. The accumulation of prolactin in cells can contribute to tumorigenesis of cancers. There is an initial evidence that prolactin signalling may be an important factor in ovarian and endometrial cancers. It was found to phosphorylate stat5, m-TOR, and ERK in ovarian cancer cells. However, prolactin receptor (PRLR) inhibitors are poorly used as targeting therapy and their mechanisms are inadequately understood, moreover, no human trials using prolactin peptide antagonists have been reported.

Here we hypothesize that the treatment response of prolactin neutralizing monoclonal antibodies applied on ovarian cancer organoids will lead a severe decrease in cancerous cells within the studied organoids. It appears that the prolactin receptor neutralizing mAb binds to the receptor in a non-competitive manner showing downstream signalling inhibition. The results demonstrate how neutralizing monoclonal antibody would bind to PRLR to inhibit the transducing signalling which as a result leads the cells to apoptosis.

This study is hopefully a starting point to applying different types of prolactin receptor targeted therapy and immunotherapy in organoids with ovarian cancerous cells niche simulation.

Literature Review 

  A woman’s risk of developing ovarian cancer in her life time is 1 of 75, and her chance of dying as a result is 1%. The disease usually occurs at late stage, since symptoms are mainly silent. Sadly, recurrence after 5 years would lead to the death in 71% of the cases. Ovarian cancer has 5 different histotypes that differ in almost all aspects, such as origination, pathogenesis, molecular alterations, risk factors, and prognosis (7). Ovarian cancer has various subtypes distributed on two types. Type 1 Carcinoma which includes Endometrioid Carcinoma and Clear Cell Carcinoma which are believed to arise from endometriosis of the ovary, and Mucinous Carcinoma, a subset of mucinous carcinomas is believed to progress in association with ovarian benign teratomas; although, the majority of mucinous carcinomas do not illustrate any teratomatous components. Lastly, Low-Grade Serous Carcinoma are very rare tumors. They are genetically stable and are characterized by their low number of genetic mutations. Type 2 Carcinoma includes High-Grade Serous Carcinoma which account for 68% of ovarian cancer and have the worst prognosis, as they are high-grade clinically aggressive neoplasms that are usually diagnosed at an advanced stage (21).

   Ovarian cancer has poor prognosis in patients who relapse after chemotherapy in most cases, due to acquired resistance of the cancer cells, which raise the need for different therapeutic approaches that include signaling pathways as potential targets in cancer treatment. Considerable efforts have been made to produce therapeutic agents targeting cancer signal transduction pathways (8). Ovarian cancer has numerous, complicated and redundant signalling pathways that could be divided into major and minor pathways. The main group is summarized as follows: First, Notch-3 pathway which is essential for the proliferation and survivor of the tumors. Additionally, overexpression of 2 target genes of the Wnt path, namely, Axin2 and FGF9, could promote the development of these carcinomas. Hedgehog pathway is active without the involvement of Smo membrane protein, although sonic hedgehog mRNA is only moderately (25% of the tumors) expressed in ovarian cancer cells. The PTEN-PI3K/AKT/mTOR pathway activation is implicated to cell cycle progression, decreased apoptosis, and increased metastatic potential.  NF-κB pathway is related to the carcinogenesis of more than half of ovarian tumors. Finally, mitogen-activated protein kinase signaling is involved in the pathogenesis as well. The minor pathways were termed minor in order to include signaling pathways that are either less important in different processes of ovarian cancer or not well studied as the major group, and as a result have less known information about them. These pathways could be associated

with the epithelial mesenchymal transition (EMT) which is correlated to ovarian carcinogenesis. These pathways are involved more in motility, invasion and apoptosis (18).

  Recently, Prolactin (PRL) is thought to play a tumorigenic role in different cancer types including ovarian cancer and it signaling pathway was approached as a therapy target in breast cancer (1). The PRL gene encoding is positioned on chromosome 6. PRL is a protein hormone which made of 197–199 amino acids and contain six cysteines forming three intramolecular disulfide bonds (Cys 4–11, 58–174, and 191–199 in PR) (19). PRL is a versatile and effective molecule that has more than 300 functions in the human body (2). Prolactin can affect Water and electrolyte balance, growth and development, endocrinology and metabolism, brain and behavior, reproduction, immunoregulation and protection and actions associated with pathological disease states (19). Its structure is similar to growth hormone and plays a key role in arbitrating communication between the nervous, immune and endocrine systems. Although prolactin is secreted mainly by lactotrophic cells in the anterior pituitary gland, it is known to be produced by other cell types (1). Its gene expression could be found in decidua, myometrium, lacrimal gland, thymus, spleen, circulating lymphocytes, and lymphoid cells of bone marrow, mammary epithelial cells and tumors, skin fibroblasts, and sweat glands and as a consequence it could exist in different body fluids such as sweat, serum, milk, tears and cerebrospinal fluid. The PRL that is produced by several cell types can perform in a more direct manner, i.e., as a growth factor, neurotransmitter, or immunomodulator, in an autocrine or paracrine style. Thus, locally produced PRL can act on adjacent cells (paracrine) or on the PRL-secreting cell itself (autocrine). Using paracrine or autocrine pathways, it is likely to activate many of the actions associated with PRL without ever affecting the serum concentration of the hormone (19).

   Evidence had shown that PRL, is a hormone/cytokine, that plays a role in different cancer types via local production or accumulation (2). It is one of four analytes used as biomarkers found in blood serum to discriminate between people with ovarian cancer and disease free (4). Studies showed that the overall survival of ovarian cancer patients from transcriptome analysis was lower when plethora PRL and prolactin receptors (PRLR) were expressed (9). Moreover, a study about the interaction between PRL and BRCA1 in ovarian cancer concluded that PRL inhibits a major tumor-suppressive function of BRCA1 by interfering with BRCA1’s upregulation of expression of p21, the cell cycle inhibitor (10). Within the last 20 years studies revealed that prolactin has a leading function in ovarian cancer progression, however not much data exists on this role in this particular cancer type (1).

PRL and prolactin receptor PRLR have a great role as growth factors in progression and growth of tumors and cancerous cells (6). Prolactin is known to affect different signalling pathways in the cells including Janus Kinase-2 (Jak2), Signal Transducer and Activator of Transcription-5 (Stat5), Ras and Mitogen Activated Protein Kinase (MAPK), Phosphatidylinositol 3-Kinase (PI3K) and AKT kinase. These signalling pathways are essential in differentiation, proliferation, survival, and motility of the cells.  Binding prolactin to its receptor acts as a firing signal for a rapid phosphorylation of the extracellular signal-regulated kinase (ERK1and 2), mitogen-activated protein kinase/ERK kinase1, signal transducer and activator of transcription 3, CREB, ATF-2, and p53 and activation of 37 transcription factors in ovarian carcinoma cells. PRL was found to phosphorylate STAT5, m-TOR and ERK in ovarian cancer cells (9). Ovarian cancer is known for its high rates of loss of tumor-suppressor gene BECN1 which offers new therapeutic opportunities by inducing sustained autophagy (6) fig. Research results have indicated the presence of mRNA for prolactin and overexpression of prolactin receptor PRLR in ovarian tumors (2). The prolactin was able to induce ovarian cancerous cell proliferation and to transform epithelial cells to cancerous tissue. Ras pathway activation was the way prolactin used to transform these cells (3). The importance of prolactin in the development of ovarian cancer is highlighted as an autocrine molecule which its blockade would result in autophagy mediated programmed cell death (6).  

  Prolactin receptor (PRLR) has been widely studied. It is known to be a cytokine type 1 receptor, which involves non-tyrosine kinase, single-pass transmembrane chains (20). It is composed of three different main domains spatially. Each domain is existing in a specific part throughout the extra, trans and intramembrane regions (5). The gene encoding PRLR is located on chromosome 5 and includes at least 10 exons for an overall length exceeding 100 kb. Differing from PRL, for which a single transcript encodes a distinctive mature protein, multiple isoforms of membrane-bound PRLR resulting from alternative splicing of the primary transcript (19). Prolactin receptor signaling is activated by the formation of a heterotrimeric complex including one ligand bound to two identical receptor moieties forming a homodimer. This ternary complex involves 3 intermolecular interactions termed sites 1 and 2 (between PRL and each receptor) and site 3 (between the two receptors). The functioning complex then triggers various intracellular signaling cascades including the canonical Jak2/STAT5 pathway, MAPK, AKT and SRC cascades (20).

  PRLRs and PRL are expressed by the ovarian cancer cell lines (13) Fig.1 Over the past years, a number of PRLR antagonists have been developed, which can be divided into two groups, PRLR analogue and anti-PRLR antibody (12). PRLR antibodies as a result were good candidates for detection, localization and analysis for the receptor in immunological experiments (5). They are also great targets for therapeutic upregulation of macro autophagy in cancer cells offering an alternative mechanism for cell apoptosis. For example, PRL antagonists are thought to induce the accumulation of redundant autolysosomes in 3D cancer spheroids, causing type II programmed cell death. It was noted that G129R, an antagonist peptide of prolactin (PRL), blocked the tumoral PRL/PRLR axis, resulting in inhibition of tumor growth in orthotopic models of human ovarian cancer. Prolonged treatment with G129R caused redundant autolysosomes in cancer spheroids, resulting in type II programmed cell death, or inducible autophagy. When testing the levels of prolactin in clinical samples of ovarian cancer patients, it was found that lower levels of tumor PRL/PRLR were related to longer patient survival (6). Tissue microarray analysis revealed that >98% of ovarian cancer express the PRLR (11). Ovarian Cancer cell lines and xenografts derived directly from primary human tumors have proven very valuable in fundamental cancer research and anticancer drug discovery (13).

  Ovarian cancer cell lines and patient derived xenografts (PDX) have been developed at each point in the disease path and diagnosed histology. The cell lines were studied in vitro, under standard cell culture conditions. PDX have alterable success and have been recognised in subcutaneous or intraperitoneal locations. Identification of elements connected with disease subtypes and clinical outcome in ovarian cancer cell lines and xenografts has led pathway-targeted therapeutic progresses (14). Another development recently on the same track is the use of organoids (15).   Organoids are ex vivo multicellular fragments that include the major cell types of a particular organ and approximate its in vivo organization. They are typically produced by culturing multipotent or pluripotent stem cells in a three-dimensional (3D) matrix (most often Matrigel) under circumstances that permit or promote self-organization of the cells. These conditions are determined experimentally but are often informed by prior knowledge of the signals that drive development or regeneration (22) Tumor organoids are innovative pre-clinical model system in oncology that allows ex vivo proliferation of tumors from individual patients Fig. 2. They are 3D cultures of cancer cells that can be originated from an individual patient tissue with a high success rate (15). They present the in vitro xenograft of a cancerous tissue. The use of tumor organoids in cancer research, drug development, and personalized medicine is still in its beginning, and studies to evaluate their potential as a model system is in growth.

   Cancer research had shown that organoids have the best success rate of initiation among cell lines and xenografts. They expand very well, suitable representor of cancer spectrum, suitable 3D growth, biologically stable and is suitable as conferment of drug resistant (13). A study reported the successful culture of 20 compared healthy and tumor organoids derived from treatment free surgical resections with a ∼90% success rate (15).

Hypothesis/Goals

The hypothesis suggests that PRLR monoclonal antibodies would play a key role in blocking prolactin receptor in ovarian cancer organoids derived from cancerous tissue. The goals are first, to study the effect of the anti-PRLR antibodies on the molecular level at the cells in limiting the proliferating, the growing, and the survival of the organoids compared to 2D cell lines, and to healthy cells. Second, to find out whether treatment responses of organoids predict treatment responses of patients.

Experimental Approaches

Cell lines and organoids culture:

After the approval of the study by ethical committees and obtaining patient consents. Cell lines of different ovarian cancer subtypes: PA-1, Caov-3, SW 626, SK-OV-3 will be taken from 10 ovarian cancer patients treatment free and cultured according to protocols.

To create organoids, patients derived tumor cell lines and human ovarian surface epithelial cells were seeded onto growth factor reduced, phenol red‐free Matrigel (Corning, > 9 mg/ml protein) using CSC medium supplemented with 2% (v/v) Matrigel to a density of 5,000–12,500 cells/cm². Organoids were grown for up to 10 days, and medium was renewed every 3–4 days to CSC medium without Matrigel.

Drug screens:

G129R-hPRL will be a gift from X lab, and 9 other different synthetic mouse, rabbit and horse PRL-antibodies will be purchased from (X) company. Drugs will be applied in different concentrations to cell lines and organoids. In each drug assay, cells will be exposed maximally to 1% DMSO or 1% ethanol in the highest drug concentrations and corresponding controls will be included in the assay. Cell lines and organoids will be stained with propidium iodide (PI) for dead cells and analysed at consecutive time points by IncuCyte® Cytotoxicity Assay For the fluorescent quantification of cell death. IncuCyte ZOOM™ Scratch Wound Processing Overview will be used to image cell lines after antibodies addition for 48 hours.

IncuCyte S3 Spheroid Viability Assay will be used to image organoids after antibodies addition for 6-10 days.

Images, videos and graphs, IC50 (inhibitor concentration) and LD50 (lethal dose) curves will be generated according to IncuCyte readings in both cell lines and organoids. 

Expected Results

  Sensitivity to drugs will be 48% of the different cell lines and 85% in tumor organoids in the total data set. The data set of single agent treatment will be analyzed individually. Single agent treatment lead to sensitivity in 34% of the cell lines and 63% of tumor organoids treated with antibodies other than G129-hPRL, in G129-hPRL assay prediction is 55% response in cell lines and 75% in tumor organoids.

Conclusion, Significance and Future Perspective

  We conclude that PRLR antagonist G129-hprl is an effective factor in decreasing cancer cells aggressiveness and invasion via blocking JAK2 and STAT5 signal pathways in ovarian cancer cells. Treatment response prediction could have more accurate prediction when using tumor organoids compared to 2D cell lines. Drug development in cancer is hindered by the lack of representative model systems that can recapitulate all critical components of the patient’s tumor. With the addition of a new technique, tumor organoid culture, to our inventory of pre-clinical cancer models, it is important to evaluate the translational potential of this new model system. This model system has the potential to improve drug development by better discriminating, in an early stage, which drugs are effective, and for which indications, also serving as a selection assay in precision medicine. It is recommended to develop organoids with more specific microenvironment element to simulate cells environment in the patient body and use them in drug assay in different types of cancers and various drug types. Prolactin seems to be a key player in ovarian cancer molecular system due to its resemblance to growth hormones, further studies on this hormone may reveal a lot of unknown facts that could be used for further precise drug development. 

 Fig. 1

Fig. 2: Ovarian cancer organoid culture. Watters et al., 2018

Fig. 3: Correlation between G129R-hPRL concentration and percentage of cell death (X-axis is G129R-hPRL concentration in micromolar.

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