CLIAMS: ,TagSPECI:TECHNICAL FIELD

The present disclosure generally relates to the field of cancer biology. More particularly, the present disclosure relates to the generation of an in vitro model for epithelial to mesenchymal transition using engineered c-myc, c-Met, twist and E-cadherin genes.

BACKGROUND

Cancer is a multi-faceted disease for which successful treatments have been sought for several decades. However, the rate of discovery of newer successful therapies is not proportional to the extensive efforts which have been made to study the disease. A large number of cancer patients relapse after anti-cancer therapy. Therefore, there is a need for novel drugs that prevent cancer relapse and metastases. Cells from the primary tumour can spread to different regions of the body during the process of metastasis by undergoing epithelial to mesenchymal transition (EMT). EMT is a natural phenomenon that occurs during early development. In adults, EMT is reactivated in cancer where it is involved in the initial steps of metastasis and in the formation of cancer stem cells. EMT is required to confer the property of motility on epithelial cells, to enable metastasis. Frequently, internal organs including the lungs, liver and bone are the target sites for metastases. Establishment of tumour in vital internal organs interferes with their proper functioning leading to organ failure and death, thus decreasing the probability of successful treatment of the patient.

Studies on the mechanism of EMT through in vitro EMT models can throw light on many aspects of metastasis leading to novel cancer therapies. Therapeutics targeting EMT will target the crucial steps of metastasis and the formation of secondary sites of cancer. Companies like Verastem Inc. and Boehringer Ingelheim have developed in vitro EMT platforms. The EMT model developed by Verastem Inc. uses SV40 viral oncogene for immortalising breast culture cells with overexpression of ras and inhibition of E-Cadherin. Boehringer Ingelheim has established EpH4 cell lines as an EMT platform by expressing ras/V12 ras, and bcl2 in a spontaneously immortalized breast cancer cell line. Use of viral regulators for immortalisation of cells frequently involves inactivation of tumor suppressor genes which might lead to transformation of cell lines. In such cases, there is a possibility that the basic properties of the cell lines might be altered which is not desirable.

In the light of the aforementioned discussion, there exists a need for an in vitro EMT model that is derived from healthy and normal tissue, to screen novel drugs which perturb and potentially inhibit EMT and also serve as a platform in which specific aspects of the mechanisms of EMT may be studied. It is an object of the present invention to develop an in vitro EMT model based on ‘normal’ control cells and thus physiologically more relevant. The present invention uses a cellular regulator, c-myc to effect immortalisation instead of a viral oncogene. It is another object of the present invention to create a novel in vitro EMT platform which can be used in the laboratory at several points in the drug discovery process such as target discovery or validation, screening for small and large molecule drugs alone or in combination with other drugs, and target deconvolution. It is another object of the present invention to identify novel biomarkers related to cancer and to carry out basic mechanistic studies of cancer progression. The present invention discloses a controlled generation of a platform of EMT using specific engineered genes which are known to be involved in the process of EMT.

BRIEF SUMMARY

The following presents a simplified summary of the disclosure in order to provide a basic understanding to the reader. This summary is not an extensive overview of the disclosure and it does not identify key/critical elements of the invention or delineate the scope of the invention. Its sole purpose is to present some concepts disclosed herein in a simplified form as a prelude to the more detailed description that is presented later.

Exemplary embodiments of the present disclosure are directed towards a regulated in vitro model for human EMT.
According to an exemplary aspect of the present subject matter, c-Myc immortalized primary human epithelial cells were engineered to express c-Met to serve as an inducer of EMT. Recombinant Twist served as an optional and additional inducer of EMT. Expression of engineered E-Cadherin (CDH) gene in such cells served as an assayable reporter of EMT.
Another exemplary aspect of the present subject matter is directed to assayable markers for induction and inhibition of EMT. EpCAM and cytokeratins serve as epithelial biomarkers and N-Cadherin and vimentin serve as mesenchymal cell biomarkers.

BRIEF DESCRIPTION OF DRAWINGS
Other objects and advantages of the present invention will become apparent to those skilled in the art upon reading the following detailed description of the preferred embodiments, in conjunction with the accompanying drawings, wherein like reference numerals have been used to designate like elements, and wherein:
Figure 1 represents one preferred embodiment of the in vitro EMT platform.
DETAILED DESCRIPTION

It is to be understood that the present disclosure is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The present disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, it is to be understood that the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting.

The use of “including”, “comprising” or “having” and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms “a” and “an” herein do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item.

Referring to Figure 1, it represents a preferred embodiment of the in vitro model of human EMT. Primary epithelial cells obtained from healthy breast tissue (1) were immortalized (2) with an engineered c-myc construct followed by the introduction of engineered cMet-RFP and Ecad-GFP constructs. Transition of the immortalized primary epithelial cells (2) into mesenchymal cells (4) occurs via intermediate phenotypes (3). An engineered construct of Twist-BFP is introduced into the cells, as required, to induce cells into EMT.

According to a non-limiting exemplary embodiment of the present disclosure, a regulated in vitro model of human EMT is disclosed. The proposed model has well defined steps with assayable markers with E-Cadherin reporter gene serving as an inbuilt reporter for phenotypic screening and characterisation. The proposed model is a platform for drug screening including phenotypic screening, for target deconvolution and target validation, and also for identification of new drug candidates and iterative SAR required for the creation of druggable molecules. In addition, the proposed model aids in discovery of biomarkers related to cancer, and basic mechanistic studies of cancer progression.

In accordance with a non-limiting exemplary embodiment of the present disclosure, primary epithelial cells are immortalized using the well-studied oncogene myc. Use of other genes like hTERT and Bmi1 for immortalization of cell lines are within the scope of the present invention. Subsequent expression of engineered c-MET-reporter gene construct is used to induce EMT. Optionally, a TWIST- reporter gene construct serves as an additional inducer of EMT. E-Cadherin-reporter gene construct served as an assayable marker for EMT induction. Other means of inducing EMT like silencing of E-cadherin, expression of ras, twist, zeb and snail genes are also within the scope of the present invention. The recombinant constructs were introduced into the epithelial cell cultures by transfection, though other means of introducing DNA into cells that are known in the art like electroporation, use of poly-ethylene imine and calcium phosphate are also within the scope of the present invention. Reporter genes, including those which are fusion proteins with green fluorescent protein (GFP), blue fluorescent protein (BFP) and red fluorescent protein (RFP) were used in the constructs. Other reporter genes that can be easily identified and measured to ensure that the recombinant constructs are taken up and/ or expressed by the cells are within the scope of the present invention. Preferably, different constructs may be fused with different fluorescent tags to yield unique reporter genes. .

In accordance with a non-limiting exemplary embodiment of the present disclosure, primary epithelial cells from healthy breast tissue are immortalized with c-myc and used for EMT induction. The epithelial cultures were transfected in 35mm culture dishes with a myc plasmid which also expressed the Neomycin (Neo) resistant gene using Lipofectamine (Life Technologies) when they are approximately 70% confluent. The cDNA of c-myc was cloned into the multiple cloning site (MCS) of the vector pCMV-XL5 (Origene) using restriction enzymes EcoRI and NotI. 10 micrograms of plasmid DNA expressing myc was incubated with up to 9 microliters of Lipofectamine and the complex incubated on cells for up to an hour. 48 hours post-transfection Neomycin was added to the culture medium to select for transfected cells. Colonies were allowed to form for up to 3 weeks, passaged, expanded, and weaned away from the antibiotic until a cell line which has reasonable number of passages is established.

According to one preferred embodiment, the neomycin resistant gene in pCMV-XL5 was replaced with Zeocin resistant gene by digesting with BstB1 and AvrII. This vector was then used for cloning of the c-myc oncogene.

In accordance with a non-limiting exemplary embodiment of the present subject matter, an expression plasmid of E-cadherin-GFP fusion protein was constructed and introduced into primary epithelial cells by transfection as described above to serve as a visual read-out for EMT. The CMV promoter of the parent plasmid pCMV-CDH-GFP Neo (Origene) was replaced by the E-cadherin promoter region. E-Cadherin promoter was amplified by PCR from control human breast tissue and by using the restriction enzymes Spe I and BglII, the CMV promoter was replaced by the E-Cadherin promoter.

According to one preferred embodiment, the Neo resistant gene of the parent plasmid pCMV-CDH-GFP Neo (Origene) was replaced by puromycin resistant gene using BstBI and AvrII.

In accordance with a non-limiting exemplary embodiment of the present subject matter, EMT was induced in the primary epithelial cells by the transfection and expression of the oncogene c-Met which is a fusion protein with td-Tomato (a red fluorescent protein). The cDNA of c-Met was cloned into the MCS of the vector ptd Tomato-N1 (Clontech) using restriction enzymes NheI and SacI.

According to a non-limiting exemplary embodiment of the present subject matter, Twist-1 is used as an additional inducer of EMT. The cDNA of Twist-1 was engineered as a fusion protein with BFP and was cloned into the MCS of the vector pCMV-AC-mBFP (Origene) using restriction enzymes AscI and NotI.

According to a non-limiting exemplary aspect of the present invention, assayable markers were used for studying the induction and inhibition of EMT.

In accordance with a non-limiting exemplary embodiment of the present subject matter, EpCAM and cytokeratins were used as epithelial markers and N-Cadherin and vimentin were used as mesenchymal cell markers.

The EMT platform disclosed in the present invention would help in the discovery of novel cancer biomarkers, screening of novel molecules which perturb EMT and metastasis, and to discover and validate novel cellular targets relevant to cancer and EMT.

Screening of novel molecules that perturb EMT and metastasis can be done by using the in vitro EMT model proposed in this disclosure. The mesenchymal cells that result because of the epithelial to mesenchymal transition (EMT) exhibit the properties of migration as well as invasion and quantification of these phenomena will permit screening of compounds that perturb or inhibit EMT. Migration assays measure the number of cells traversing a membrane, and invasive assays monitor cell movement through extracellular matrices. Cells are seeded in multi-well plates with appropriate media and chemo-attractant factors that cause migration and/or invasion. The migration and invasion of cells may be affected by the addition of compounds which increase or decrease these effects. Quantification of these effects are performed by microscopic methods, or with High Content Screening methods.