WO2020035745A1

WO2020035745A1 - A process for detecting genesis and monitoring disease status of cancer in a patient

Info

Publication number: WO2020035745A1
Application number: PCT/IB2019/051694
Authority: WO
Inventors: Prabhudas S. Patel
Original assignee: PANDYA, Shashank J.
Priority date: 2018-08-17
Filing date: 2019-03-02
Publication date: 2020-02-20

Abstract

The present invention provides a process for acquiring a new trait during cancer development and for detecting genesis of cancer and monitoring disease status in a patient. Particularly, the present invention relates to comparing detecting a level of sialylation and fucosylation within a biological with a level of sialylation and fucosylation in a control sample; identifying alterations in said level of sialylation and fucosylation detected within said biological sample from said level of sialylation and fucosylation in said control sample respectively for ascertaining aberrant glycosylation; determining type and status of cancer based on identified alterations; and determining one more drugs for targeting sialylation and fucosylation for remodelling glycome in cancerous cell and inhibiting determined type of cancer progression. The present invention provides a new hallmark of cancer which has significant clinical value for well being of cancer patient.

Description

A PROCESS FOR DETECTING GENESIS AND MONITORING DISEASE

STATUS OF CANCER IN A PATIENT

Field of Invention:

The present invention relates to a process for detecting genesis and monitoring disease status of cancer in a patient. As normal cell progress to neoplastic state, it acquires distinctive and complementary capabilities, which are called as the hallmark of cancer. Particularly, the present invention relates to a comparison of a level of sialylation and fucosylation; a major terminal glycosylation of the cells of a patient with a control level.

Background of Invention:

In 2000, an article entitled“The hallmarks of cancer” was published by Hanahan and Weinberg which proposed six hallmarks of cancer that provides a logical framework for understanding the biology of cancer. It included sustaining proliferative signaling, evading growth suppressors, activating invasion and metastasis, enabling replicative immortality, inducing angiogenesis and resisting cell death. (Figure: la) Almost after a decade they have reworked on the hallmarks of cancer and proposed another two emerging hallmarks:“Deregulating cellular energetics” and “Avoiding immune destruction” and two enabling characteristics: “Genome instability and mutation” and Tumour-promoting inflammation”. (Figure: lb) The evidences indicate that tumorigenesis in humans is a multistep process and that these steps reflect genetic alterations that drive the progressive transformation of normal human cells into highly malignant derivatives. The hallmarks of cancer are characterized by functional capabilities that allow cancer cells to survive, proliferate and disseminate during tumorigenesis. Further, they have also proposed that refinement of these organizing principles will surly come in the foreseeable future which will continue in the remarkable conceptual progress in the hallmarks of cancer.

Further, many researches have been done in the field of cancer and many patents have demonstrated the identification and treatment of cancer.

US20050170351A1 provides a number of genetic identifiers (genesets) which may be used as diagnostic tools to determine the presence or risk of breast cancer in a patient. The invention also provides genesets which may be used to classify a breast tumour cell as to its molecular subgroup. Each of the identified genesets may be used to product customised specific nucleic acid microarrays for use in diagnosis and classification of breast tumour cells.

WO2018067886A2 discloses omics signatures for stress-induced mutagenesis in cancer and methods related to same are presented. Advantageously, signatures can be characterized and a score can be calculated to provide useful guidance for therapy not otherwise obtainable from o ics data.

US20100151468A1 discloses a methods of detection, including early detection, for cancer or other diseases and normal physiologic processes mediated by global epigenetic changes, by using one or more of the following biomarkers: a global DNA methylation Index, a global histone H4 acetylation index and a global bistone H4 trimethylation index. These methods are useful for, among other things, assessing the effectiveness of treatment, monitoring relapse, and clinical staging of cancer and other chronic as well as acute diseases. These methods are also useful for among other tilings monitoring the effectiveness of strategies and therapies used to modify lifestyle and contextual effects to prevent disease, foster wellness and enable health promotion.

US20060094059A 1 discloses screening system utilizes dynamic measurements of pathway activity to detect the activities of drags within cellular pathways. The methods of the invention can be used to identity previously unknown drug activities and therapeutic uses, even for drags that have been well characterized with standard biochemical assays. It demonstrated the utility of tire invention by screening a portion of the known pharmacopeia. It identified dozens of drags, previously or currently marked for a variety of indications with surprising and previously- unsuspeeted activity against‘hallmark^’ cancer pathways. It also showed that over 20 of these drugs indeed have anti-proliferative activity in human tumor ceils, underscoring the utility and predictability of the screening system. The methodology disclosed therein proposed to extend the utility of the current pharmacopeia and provide die basis for de novo discovery of drugs with a broad range of therapeutic indications.

EP2322658A1 discloses a method for diagnosing aggressiveness and/or genetic instability of a breast cancer in a patient from a breast cancer sample of said patient, comprising: a) measuring in vitro the expression level of the POLQ gene and the expression level of a control gene in said patient breast cancer sample; b) calculating for said POLQ gene expression level ratio to the expression of the said control gene in said patient breast cancer sample; c) comparing the said POLQ expression level ratio to a corresponding threshold value and d) diagnosing breast cancer aggressiveness and genetic instability if the said POLQ expression level ratio is superior to a corresponding threshold values. Dedicated microarrays and kits are also described, as well as a method of selecting a suitable treatment.

The complexity of cancer is the main problem in the field of cancer. It can be reduced to a smaller number of underlying principles called“Hallmark of Cancef’. Recognition of these traits might affect the development of new ways to treat cancer.

There is a rising need for the identification of new trait to reorganize conceptual framework of cancer for early identification and better management of cancer. In the course of noteworthy progress in cancer research, newer observations have modified the original formulation of the hallmark capabilities. The present invention discloses aberrant glycosylation as a new hallmark of tumor proliferation, invasion, metastasis and angiogenesis. Protein glycosylation is the most widely observed and structurally diverse form of post-translational modifications after phosphorylation. It is the enzymatic process that produces glycosidic linkages of sachharides to other sachharides, proteins or lipids. Alterations in cell surface glycosylation particularly, terminal motifs may results in altered cell-cell adhesion, cell-matrix interactions, inter and intra- cellular signaling and cellular metabolism. The understanding of biologically relevant aberrant glycosylation can serve as clinically important biomarker for various cancers. Altered sialylation and fucosylation have been associated with early detection, staging, treatment monitoring and prognosis of cancer patients.

The different strategies targeting sialylation and fucosylation like the use of chemical inhibitors of ST and FUT and specific antisense oligodeoxynucleotides silencing ST and FUT gene expression have been developed to analyze the role of sialylation in cancer progression and metastases. It may serve as new potent antiinflammatory, immunosuppressive and anti-metastatic agents for future therapeutic applications. It has been also suggested that fluorinated analogs of sialic acid and fucose can be taken up and metabolized resulting in a global, family-wide shutdown of sialyl or fucosyltransferases and remodeling of cell surface glycans. Hence, there is much advancement in development of various glycan antagonists and inhibitors of ST and FUT. The progress made in the field of drugs targeting sialylation and fucosylation seems to remodel the glycome in cancerous cell, which can prove wonders in inhibiting cancer progression.

Summary of Invention:

Accordingly, the present invention provides a process for detecting genesis of cancer and monitoring disease status in a patient. The process comprises the steps of: providing a biological sample from said patient, wherein said biological sample includes at least one of Blood, Saliva, Tissue from said patient; detecting a level of sialylation within said biological sample; comparing said level of sialylation with a level of sialylation in a control sample; detecting a level of fucosylation within said biological sample; comparing said level of fucosylation with a level of fucosylation in said control sample; identifying alterations in said level of sialylation and fucosylation detected within said biological sample from level of sialylation and fucosylation in said control sample respectively for ascertaining aberrant glycosylation, wherein said identifying alterations includes identifying alterations in glycan biosynthesis pathways including altered expression of glycosyltransferases and glycosidases, altered expression of glycoconjugate acceptor together with availability and abundance of the sugar nucleotide donars, altered sugar nucleotide transporter activity and improper function of the golgi structure; determining type of cancer based on identified alterations; and determining one more drugs for targeting sialylation and fucosylation for remodelling glycome in cancerous cell and inhibiting determined type of cancer progression.

In one embodiment, the present invention provides that sialylation is governed by sialyltransferases and sialidases.

In one another embodiment, the present invention provides that sialyltransferases are selected from ST3Gal (a2, 3-ST), ST6Gal (a2, 6- ST), ST6GalNAc and ST8Sia (a2, 8-ST).

In still one embodiment, the present invention provides that sialidase activity is increased in patients with oral oprecancerous conditions (OPC) and oral cancer patients.

In yet one embodiment, the present invention provides that alterations in different subtypes NEU1, NEU2 and NEU3 and NEU4 are correlated with cancer progression in various cancer cell lines.

In one another embodiment, the present invention provides that NEU3 is up regulated in prostate cancer, colon cancer, head and neck squamous cell carcinoma (HNSCC) cell lines, and lymph node metastasis.

In still one embodiment, the present invention provides that altered enzyme activities of a-2, 3 and a-2, 6 STs in serum, saliva and tissue of patients with OPC and oral cancer patients is monitored.

In yet one embodiment, the present invention provides that fucosylation is catalyzed by fucosyltransferase enzymes (FTs) selected from FUT1 to 11, protein o-fucosyltransferase 1 (POFUT1) and protein o-fucosyltransferase 2 (POFUT2).

In another embodiment, the present invention provides that altered expression of various FTs selected from FUT3 , FUT4, FUT6, FUT7, FUT8 to mediate cancer cell migration and thereby metastasis.

In one embodiment, the present invention provides that a significant alterations in FUT3 and FUT5 mRNA expressions is observed in oral cancer patients.

In one another embodiment, the present invention provides that breast cancer, non-small cell Lung cancer, aggressive prostate cancer, proliferation and invasion in human hepatocarcinoma cell lines show high FUT8 protein expression and are correlated with lymphatic metastasis and stage status.

In yet one embodiment, the present invention provides that FUT4 is associated with the proliferation and metastasis of breast cancer, FUT3 mRNA over expression is responsible for increased SLe^x biosynthesis leading to metastasis in colon carcinoma cell line, and increased FUT7 levels is an indicator of poor prognosis.

To further clarify advantages and features of the present invention, a more particular description of the invention will be rendered by reference to specific embodiments thereof, which is illustrated in the appended drawings. It is appreciated that these drawings depict only typical embodiments of the invention and are therefore not to be considered limiting of its scope. The invention will be described and explained with additional specificity and detail with the accompanying drawings.

Brief Description of Drawings:

These and other features, aspects, and advantages of the present invention will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:

Figure: 1 illustrates Hallmarks of Cancer (reproduced from Hanahan and Weinberg, 20001 and 2011);

Figure 2 illustrates a flow chart for a process for detecting genesis of cancer and monitoring disease status in a patient;

Figure 3 illustrates role of mucins (MUCs) in tumor progression and interactions with several pathways;

Figure 4 illustrates role of altered glycosylation in tumor progression;

Figure 5 illustrates different alterations in glycosylation and correlation with other hallmarks of cancer;

Figure 6 illustrates levels of serum and salivary a-2, 3 and a-2, 6 ST in PT, CR and NR; and Figure 7 illustrates levels of a-L-fucosidase between the untreated/pre-treatment (PT) patients with OC and patients who achieved a CR or who had an NR.

Further, skilled artisans will appreciate that elements in the drawings are illustrated for simplicity and may not have been necessarily been drawn to scale. Furthermore, in terms of the construction of the device, one or more components of the device may have been represented in the drawings by conventional symbols, and the drawings may show only those specific details that are pertinent to understanding the embodiments of the present invention so as not to obscure the drawings with details that will be readily apparent to those of ordinary skill in the art having benefit of the description herein. Detailed Description:

For the purpose of promoting an understanding of the principles of the invention, reference will now be made to the embodiment illustrated in the drawings and specific language will be used to describe the same. It will nevertheless be understood that no limitation of the scope of the invention is thereby intended, such alterations and further modifications in the illustrated system, and such further applications of the principles of the invention as illustrated therein being contemplated as would normally occur to one skilled in the art to which the invention relates.

It will be understood by those skilled in the art that the foregoing general description and the following detailed description are exemplary and explanatory of the invention and are not intended to be restrictive thereof.

Reference throughout this specification to “an aspect”, “another aspect” or similar language means that a particular feature, stmcture, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrase“in an embodiment”,“in another embodiment” and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

The terms "comprises", "comprising", or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a process or method that comprises a list of steps does not include only those steps but may include other steps not expressly listed or inherent to such process or method. Similarly, one or more devices or sub-systems or elements or structures or components proceeded by "comprises...a" does not, without more constraints, preclude the existence of other devices or other sub-systems or other elements or other structures or other components or additional devices or additional sub-systems or additional elements or additional structures or additional components.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The system, methods, and examples provided herein are illustrative only and not intended to be limiting.

Embodiments of the present invention will be described below in detail with reference to the accompanying drawings.

The present invention provides a new trait of cancer cells which further simplify complexity of cancer development and aid in the development of process for detecting genesis of cancer and monitoring disease status in a patient, new ways to treat cancer. The level of sialylation and fucosylation is compared with respect to the control level.

Referring to Figure 2, a flow chart for a process for detecting genesis of cancer and monitoring disease status in a patient. The process 200 includes the steps of: step 202 of detecting a level of sialylation within a biological sample, wherein said biological sample includes at least one of Blood, Saliva, Tissue from said patient; step 204 of comparing said level of sialylation with a level of sialylation in a control sample; step 206 of detecting a level of fucosylation within said biological sample; step 208 of comparing said level of fucosylation with a level of fucosylation in said control sample; step 210 of identifying alterations in said level of sialylation and fucosylation detected within said biological sample from level of sialylation and fucosylation in said control sample respectively for ascertaining aberrant glycosylation, wherein said identifying alterations includes identifying alterations in glycan biosynthesis pathways including altered expression of glycosyltransferases and glycosidases, altered expression of glycoconjugate acceptor together with availability and abundance of the sugar nucleotide donars, altered sugar nucleotide transporter activity and improper function of the golgi structure; step 212 of determining type and status of cancer based on identified alterations; and step 214 of determining one more drugs for targeting sialylation and fucosylation for remodelling glycome in cancerous cell and inhibiting determined type of cancer progression.

In an embodiment, the process 200 further comprises at least one of: determining an increase in level of glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC) involved in loss of E- cadherin; coorelating glycosylation with tumor initiation, progression and metastasis.

In an embodiment, sialylation is governed by sialyltransferases and sialidases, wherein sialyltransferases are selected from ST3Gal (a2, 3-ST), ST6Gal (a2, 6- ST), ST6GalNAc and ST8Sia (a2, 8-ST).

In an embodiment, the process 200 further comprises determining alterations in different neuraminidases (NEU) subtypes NEU1, NEU2 and NEU3 and NEU4, wherein said NEU1, NEU2 and NEU3 are localized in the lysosomes, cytosol and plasma membranes, respectively and NEU4 is found in lysosomes or in mitochondria and endoplastic reticulumare; correlating said alterations in different neuraminidases (NEU) subtypes with one more cancer cell lines. In an embodiment, the process 200 further comprises determining alterations in enzyme activities of a-2, 3 and a-2, 6 STs in serum, saliva and tissue of patients with OPC and oral cancer patients.

In an embodiment, fucosylation is catalyzed by fucosyltransferase enzymes (FTs) selected from FUT1 to 11, protein o-fucosyltransferase 1 (POFUT1) and protein o -fucosyltransferase 2 (POFUT2).

In an embodiment, the process 200 further comprises selecting altered expression of various FTs from FUT3, FUT4, FUT6, FUT7, FUT8 to mediate cancer cell migration and thereby metastasis; determining and decrease in FUT3 and FUT5 mRNA expressions in oral cancer patients.

In an embodiment, FUT4 is associated with the proliferation and metastasis of breast cancer, FUT3 mRNA over expression is responsible for increased SLe^x biosynthesis leading to metastasis in colon carcinoma cell line, and increased FUT7 levels is an indicator of poor prognosis.

In an embodiment, the process 200 further comprises determining alterations in composition and decrease in quantity of cell surface fucosylation-associated molecules by fucosidases (FUCA) expression; and limiting invasiveness of cancer cells in early -stage breast tumors, wherein lower FUCA protein levels exhibit increased cell surface fucosylation, which enhances the malignant; an over expression of a-L-fucosidase 1 (FUCA1) exhibits suppressed growth of cancer cells and induced cell death by protein defucosylation.

Glycosylation as a hallmark of cancer

Glycosylation is the process of addition of glycans to glycoproteins and is the major posttranslational modification of proteins which play an important role in malignant transformation and metastasis. Glycosylation depends on the action of glycosyltransferases and glycosidases in different tissues or cells. Cancer cells continuously shed their surface components, which can be used as tumor markers for various malignancies in bodily fluids. Glycosylation is associated with the acquisition of hallmark capabilities. Alterations in glycan expression may be due to under or overexpression of glycosyltransferases. These glycosyltransferases are deregulated at the level of epigenetics, transcription, post-transcription, and/or chaperone, altered glycosidase activity, altered expression of glycoconjugate acceptor together with availability and abundance of the sugar nucleotide donars, altered sugar nucleotide trans- porter activity and improper function of the golgi structure where many of the glycosyltransferases are harboured. Glycans exist as membrane-bound glycoconjugates or as secreted molecules, which become integral parts of the extracellular matrix. Changes in these glycan structures are associated with many physiological and pathological events such as cell growth, migration and differentiation. Consequently, aberrant glycosylation occurring in cancer cells influence cell proliferation, adhesion and motility as well as angiogenesis and metastasis. Aberrant glycosylation interfere with almost all the steps involved in malignant transformation and therefore can be said as classic hallmark of cancer. Glycosylation is not a template based process such as DNA, RNA or protein synthesis but is rather based on the balance achieved by the expression and activity levels of the different enzymes involved in the glycosylation process such as glycosyltransferases and glycosidases and on the availability of the nutrient resources and expression of enzymes responsible for their synthesis and interconversion. The study of the changes in the enzymes associated with altered glycosylation provides new directions for understanding the molecular nature of cancer, cellular transformation and often new opportunities for identifying biomarkers of disease and developing interventional strategies for treatment of cancer. Increased sialylation and fucosylation of cell surface glycoconjugates is among the key molecular changes associated with malignant transformation and cancer progression. Therefore, the present invention provides a clinical significance of aberrant glycosylation via altered sialylation (sialidases and sialyltransferases) and fucosylation (fucosidases and fucosyltransferases) in various cancers. In particular, aberrant protein glycosylation as a new hallmark of cancer will be conversed in relation with various human cancers.

Association of glycosylation with various hall marks of cancer:

Alterations in glycoproteins on cell surface and alterations in MUCs leading to loss of E- cadherin play a key role in progression and metastasis of tumors (Figure 3). Matrix metalloproteinases (MMPs) are the enzymes which play an important role in metastasis and invasion by proteolytic degradation of extracellular matrix (ECM), disruption of cell-cell and cell matrix adhesion, migration and angiogenesis. Previous studies have depicted reduced E- cadherin and increased MMP-9 expression in squamous cell carcinoma of head and neck. Moreover, earlier studies have reported decreased E-cadherin expression in oral squamous cell carcinoma (OSCC) patients. Elevated expression of MMPs has been reported by earlier studies. We have also reported that MMP-2 and MMP-9 are key players in invasion and metastasis in OSCC and breast cancer. MMPs are a highly regulated super family of enzymes that degrade almost all ECM and basement membrane components, processes which are essential for invasion and subsequent metastasis. Resisting cell death is one of the hallmarks of cancer cells. Mutations and/or downregulation of molecules involved in the Fas receptor-Fas ligand (FasR- FasL) apoptotic pathway are well known mechanisms exploited by cancer cells to escape apoptosis. Earlier studies have discovered a novel strategy whereby tumor cells escape from Fas-mediated apoptosis. Hypersialylation of the Fas receptor has been shown to disable apoptosis induction in cancer cells. Aberrant silaylation of glycoproteins and glycolipids has been shown to be linked to increased immune cell evasion, chug evasion, drug resistance, tumor invasiveness and vascular dissemination leading to metastasis.

Further, reduction of E-cadherin results in upregulation of epidermal growth factor receptor (EGFR) transcriptionally in head and neck cancer. Down-regulation of E-cadherin expression results in a strong up-regulation of EGFR in keratinocytes, whereas E-cadherin transfection reversed this effect. Increased EGFR expression has been also observed in various cancers. Increased expression of pEGFR in malignant oral cancer tissues . Mutations in E- cadherin have been reported to cause increased EGFR activation and reduced E- cadherin- EGFR association. In contrast, formation of E- cadherin-mediated cell-cell adhesion has been shown to activate EGFR in various experimental settings. There are several classes of receptor tyrosine kinases, including EGFR, Her2-neu, insulin-like growth factor 1 receptor (IGF-1R) and c-Met, which can inhibit E- cadherin-dependent adhesion when they induce epithelial- mesenchymal transition (EMT). The regulation is bidirectional, as E-cadherin can in turn inhibit activation of EGFR, Her2-neu, IGF-1R and c-Met. Studies have depicted that loss of E-cadherin activates EGFR-MEK/ERK signaling, which promotes invasion via the ZEB l/MMP-2 axis in non-small cell lung cancer. Moreover, the ERK1/2 blockade prevents EMT in lung cancer cells and promotes sensitivity to EGFR inhibition.. Thus, alterations in glycosylation causes loss of E- cadherin which is further implicated in EGFR activation as depicted in Figure. 4.

There are simultaneous changes in expression of vascular endothelial growth factor (VEGF), MMP-9 and E-cadherin in process of metastasis. P53 and EGFR were shown to be overexpressed and associated with stage of disease. Earlier studies have shown interplay between EGFR overexpression, hTERT activation, p53 mutation and MMPs. Simultaneous association of p53, E-cadherin, COX-2 and EGFR protein was observed with overall survival. We have observed that these changes are associated/govemed by abbrent glycosylation.

In a nut shell, altered expression of sialic acid, ST, sialoproteins, sialidase, fucoproteins, fucose, FUT, fucosidase, galactose and mannose in various cancers are disclosed. Moreover, glycosylation is inter-correlated with other hallmarks (Figure 5) like invasion and metastasis, proliferation, angiogenesis, evasion of growth suppressors and apoptosis and replicative immortality. Therefore, the broadened view of glycosylation changes during all steps of tumor progression in various cancers and its correlation with other hallmarks suggests that, glycosylation can be considered as a novel hallmark of cancer.

Sialylation

Sialylation affects the half-lives of many circulating glycoproteins and plays a role in a variety of biologic processes such as cell-cell communication, cell matrix interaction, adhesion, and protein targeting. Alterations in total sialic acid (TSA), sialidase, sialyltransferase (ST) and mRNA subtypes expression and sialoproteins have been reported in various cancers. The transfer of sialic acids from CMP sialic acids to the acceptor carbohydrates is catalyzed by the sialyltransferase (ST) family. The amount and type of sialylation of tumor cell membrane depend on the activity of a number of different STs. Nowadays there are many recent advances made in inhibitors of ST. Aberrant sialylation in cancer cell is a characteristic feature associated with malignant properties including invasiveness and metastatic potentials. Sialic acid is linked either through a-2, 3 or a-2, 6 linkage to subterminal galactose or a-2, 8 linkage to another sialic acid forming poly sialic acid catalyzed by specific ST. The different STs can be distinguished on the basis of oligosaccharide sequence used as acceptors and anomeric linkage formed with the penultimate sugar residue. Sialylation is governed by sialyltransferases and sialidases. Sialic acids are transferred from a donor substrate to terminal positions of glycoprotein and glycolipid carbohydrate groups by STs. STs are categorized into four families on the basis of the carbohydrate side chain they synthesize, namely ST3Gal (a.2, 3 -ST), ST6Gal (a.2, 6- ST), ST6GalNAc and ST8Sia (a2, 8-ST). On the other hand, their removal from glycan chains is catalyzed by sialidases. Sialidases also called neuraminidases (NEU) are glycosidases catalyzing the removal of a-glycosidically linked sialic acid residues from carbohydrate groups of glycoproteins and glycolipids. They are classified according to their major intracellular locations as intra-lysosomal, cytosolic, lysosomal membrane and plasma membrane associated NEUs. NEU1, NEU2 and NEU3 are known to be localized predominantly in the lysosomes, cytosol and plasma membranes, respectively and NEU4 is found in lysosomes or in mitochondria and endoplastic reticulum. The amount and type of sialylation of tumor cell membrane depend on the activity of a number of different STs. The activity of these enzymes affects the conformation of glycoproteins and therefore contributes to either increased recognition or masking of biologically relevant sites in molecules and cells. Alterations in sialidase, STs and mRNA subtypes expression have been reported in various cancers.

An elevated sialidase activity in patients with OPC and oral cancer patients has been reported. Alterations in different subtypes NEU1, NEU2,NEU3 and NEU4 which was found to be correlated with cancer progression in various cancer cell lines. NEU3 was also found to be up regulated in prostate cancer which plays a role in tumor progression through androgen receptor signaling. In colon cancer, high expression of the sialidase NEU3 in cancer cells leads to protection against programmed cell death by modulation of gangliosides is documented. In addition, NEU3 also plays a major role in maintenance of self-renewal and tumorigenic potential of colon cancer cells. In various head and neck squamous cell carcinoma (HNSCC) cell lines, NEU3 has been reported to regulate the EGFR signaling through ganglioside modulation which is further associated with lymph node metastasis. In colorectal cancer, NEU4 is implicated as an important player in control of sialyl Lewis antigen (SLe) expression and its impairment. Further, a significant over expression of SLe^x in malignant tissues as compared to adjacent normal tissues was observed which is further associated with disease progression and poor prognosis of the patients.

Altered enzyme activities of a-2, 3 and a-2, 6 STs in serum, saliva and tissue of patients with OPC and oral cancer patients have significance in treatment monitoring. Figure 6 depicts levels of serum and salivary a-2, 3 and a-2, 6 ST in Pre-treatment (PT), Complete responders (CR) and Non-responders (NR) during post treatment follow-ups. It has been observed that levels of serum and salivary a-2, 6 ST along with salivary a-2, 3 ST were significantly decreased in CR (p=0.012, p=0.001 and p=0.010 respectively) as compared to PT levels. The levels of serum a-2, 6 ST were found to be significantly increased (p=0.024) in NR as compared to PT levels. The levels of serum and salivary a-2, 3 ST and a-2, 6 ST were also found to be increased in NR as compared to PT levels. During neoplastic transformation, the activity of the Golgi localized STs is usually increased and as a consequence, cancer cells express more heavily sialylated tumor associated carbohydrate antigen (TACA) at their surface. Various STs play role in formation of TACA in various cancers. ST3GAL1 plays role in formation of sT antigen, ST3GAL4 in sLeX formation, ST6GAL1 in CD75s and ST2H formation, ST6GALNAC1 in sTn antigen etc. The common glycan alterations observed in various cancers are increased SLe^x/a, increased Tn epitopes, increased sialyl Tn epitopes, increased sialyl T antigens and increased a-2, 6 sialylation. ST3GAL1 has been observed as an independent adverse prognostic factor for recurrence and survival in clear cell renal cell carcinoma patients. Further, it was observed that ST3GAL1 plays the major role in the T antigen sialylation, and its expression is associated with progression and recurrence in bladder cancer. In cervical cancer, loss of ST6GAL1 has been showed to promote cell apoptosis and to inhibit the invasive ability of cancer cells. Increased ST6GAL1 and subsequently elevated levels of cell-surface a 2, 6- linked sialic acids have found to be associated with metastasis and therapeutic failure in colorectal cancer. In hepatocellular carcinoma, ST6GAL1 and ST8SIA2 regulation has been shown to affect unusual properties of invasion and chemosensitivity by modulating the PI3K/Akt signaling pathway. Further, over expression of ST3GAL4 leads to SLe^x antigen expression in gastric cancer which in turn induces an increased invasive and aggressive phenotype. ST3GAL4 has also been reported as a bio marker for diagnosis and prognosis of multi drug resistance in acute myeloid leukemia. In addition, elevated mRNA level of ST6GAL1 and ST3GAL4 are found to be positively associated with the high risk of pediatric acute lymphoblastic leukemia. Hence, expression of STs are often found to be de regulated in various cancers like colorectal, liver, gliomas, gastric and oral cancer. However, despite of increased amounts of evidence showing the involvement of STs and aberrant sialylation in cancer progression, therapeutic strategies to reduce aberrant sialylation lag behind.

Fucosylation

Fucosylation is one of the most common modifications involving oligosaccharides on glycoproteins and glycolipids. Fucosylation consists of transfer of fucose residue from GDP to N- glycans, O-glycans and glycolipids. Fucosylation of glycoproteins is one of the most important features that mediate several specific biological functions. Fucosylation is catalyzed by a family of fucosyltransferase enzymes (FTs), consisting of 13 members, including FUT1 to 11, protein o- fucosyltransferase 1 (POFUT1) and protein o -fucosyltransferase 2 (POFUT2). FUTs promote attachment of fucose to N-, O- and lipid linked glycans through an a 1, 2- (by FUT 1 and FUT2), a 1, 3- (by FUT 3 to 7 and FUT 9 to 11), a 1, 4- (by FUT 3 and FUT5) and a 1, 6- (by FU8) linkage or directly link to the serine/ threonine residues of EGF-like or thrombospondin receptor (by POFUT 1 & 2). Increasing fucosylation of tumor cells contributes to several abnormal characteristic such as decreased adhesion and uncontrolled tumor growth. The changes in fucose, fucoproteins, a-L- fucosidase activity and different subtypes of FUT activity in various cancers are reported to have significant clinical utility. Fucosylated glycans can be generally divided into two subcategories, (i) core fucosylated and (ii) terminally fucosylated glycans.

Core Fucosylation: Core fucosylation is the addition of a 1-6 fucosyltransferases (encoded by FUT8). Up regulation of core fucosylation and the associated FUT8 gene has been observed in most cancers. Importantly, in most of cancers the presence of core fucosylated glycans on the cell surface is largely mirrored by their presence, thereby demonstrating the potential for further use of specific protein gly coforms for early cancer detection.

Terminal Fucosylation: Cell surface glycans frequently carry fucose residues in a 2-3 and/or a 2-4 linkage at the terminus of the N- and O- linked glycan structures, giving rise to the formation of specific Lewis blood group antigens, such as Lex/y and Lea/b. Several FUTs are involved in the formation of Lewis antigens including those coded by FUT 1-7 and FUT 9 with FUT 3-7 and FUT 9 gene products known to produce the Le^x structure. FUT 1-2 genes, on the other hand are involved in creating the precursor of H-antigen. Although fucosylation is essential for normal biological functions, alterations in fucosylation are strongly implicated in cancer and increasing metastatic potential. Alterations in fucosidase and fucosyltransferase expression have been reported in various cancers. a-L-fucosidase (EC: 3.2.1.51) is a lysosomal enzyme that catalyzes the hydrolytic cleavage of terminal fucose residue that is involved in maintaining the homeostasis of fucose metabolism. The presence of fucosidases (FUCAs) is necessary for rapid turnover of N-glycans (including fucose) followed by reglycosylation and reinsertion of the proteins in plasma membrane.

Serum a-L-fucosidase is useful marker for close monitoring of patients during post-treatment follow-up. Figure 7 illustrates a-level of L-fucosidase between the untreated/ PT patients with OC and patients who achieved a CR or who had an NR. (PT: Pre-treatment, CR: Complete responders, NR: No Responders). Further, a significantly higher serum and salivary a-L- fucosidase activity in oral cancer patients is compared to controls. It has been observed that high FUCA expression alters the composition and decrease the quantity of cell surface fucosylation- associated molecules, thereby limiting the invasiveness of cancer cells in early-stage breast tumors. Therefore, the tumor cells expressing lower FUCA protein levels exhibit increased cell surface fucosylation, which enhances the malignant potential of the tumor cells in triple-negative breast cancer. Over expression of a-L-fucosidase 1 (FUCA1) suppressed the growth of cancer cells and induced cell death by protein defucosylation which is further involved in tumor suppression in several cancers. In HNSCC, primary tumors exhibiting higher FUCA1 expression were found to be associated with significantly worse patient survival. In addition, down- regulation of FUCA1 is also correlated with increased aggressiveness of thyroid cancer.

It has been demonstrated that altered expression of various FTs such as FUT3 , FUT4 , FUT6 , FUT7 , FUT8 mediate cancer cell migration and thereby metastasis, suggesting that altered fucosylation may play an important role in disease progression. There are a significant alterations in FUT3 and FUT5 mRNA expressions in oral cancer patients. Increased fucosylation has been established as a crucial character in invasive and metastatic properties of head and neck cancer stem cells. In breast cancer, high FUT8 protein expression was found to be correlated with lymphatic metastasis and stage status. High expression of FUT8 was also found to be associated with poor survival which can be a significant and independent unfavourable prognostic factor in patients with potentially curatively resected non-small cell Lung Cancer. Further, over expression of FUT8 was found to be associated with aggressive prostate cancer which can serve as a promising target to differentiate between aggressive and non-aggressive prostate tumors. Moreover, altered levels of FUT8 were also significantly linked to the malignant behavior of proliferation and invasion in human hepatocarcinoma cell lines. In addition, FUT4 was found to be associated with the proliferation and metastasis of breast cancer and which can serve as novel biomarker in the diagnosis and prognosis of breast cancer. FUT3 mRNA over expression was found to be responsible for increased SLe^x biosynthesis leading to metastasis in colon carcinoma cell line whereas increased FUT7 levels were observed to be a significant indicator of poor prognosis. The present invention reveal the importance of monitoring fucosylation changes during various stages of cancer progression which can be helpful for early detection and management of cancer patients.

Therefore, alterations in sialylation and fucosylation and its association with tumour burden, invasion and metastasis in a variety of cancers has strengthen our concept that glycosylation is a new hallmark of cancer progression.

Table 1: Clinical Significance of sialylation and fucosylation changes in various cancers

Modification of proteins plays a major role in cell signalling, immune recognition and cell-cell interactions. Aberrant glycosylation has been identified in almost every type of cancer due to significant modification/ alterations in sialylation and fucosyltion by altered expression of various enzymes involved in it. In nut shell, distinctive alterations in tumor-associated glycosylation may provide us a unique feature of cancer cells and therefore grant novel diagnostic and even therapeutic targets. This suggests that altered glycosylation has an important translational value in clinical setting. More interestingly, the development and progression of cancer results in the fundamental changes to the glycome; so, changes in glycosylation can be believed as a brand new hallmark of malignant transformation and a hallmark of translational value in cancer. The drawings and the forgoing description give examples of embodiments. Those skilled in the art will appreciate that one or more of the described elements may well be combined into a single actional element. Alternatively, certain elements may be split into multiple actional elements. Elements from one embodiment may be added to another embodiment. For example, orders of processes described herein may be changed and are not limited to the manner described herein. Also, those acts that are not dependent on other acts may be performed in parallel with the other acts. The scope of embodiments is by no means limited by these specific examples. Numerous variations, whether explicitly given in the specification or not, such as differences in structure, dimension, and use of material, are possible. The scope of embodiments is at least as broad as given by the following claims.

Benefits, other advantages, and solutions to problems have been described above with regard to specific embodiments. However, the benefits, advantages, solutions to problems, and any component(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential feature or component of any or all the claims.

Claims

We Claim:

1. A process for detecting genesis of cancer and monitoring disease status in a patient, said process comprising the steps of:

a. detecting a level of sialylation within a biological sample, wherein said biological sample includes at least one of Blood, Saliva, Tissue from said patient;

b. comparing said level of sialylation with a level of sialylation in a control sample;

c. detecting a level of fucosylation within said biological sample; and d. comparing said level of fucosylation with a level of fucosylation in said control sample;

e. identifying alterations in said level of sialylation and fucosylation detected within said biological sample from said level of sialylation and fucosylation in said control sample respectively for ascertaining aberrant glycosylation, wherein said identifying alterations includes identifying alterations in glycan biosynthesis pathways including altered expression of glycosyltransferases and glycosidases, altered expression of glycoconjugate acceptor together with availability and abundance of the sugar nucleotide donars, altered sugar nucleotide transporter activity and improper function of the golgi structure;

f. determining type of cancer based on identified alterations; and g. determining one more drugs for targeting sialylation and fucosylation for remodelling glycome in cancerous cell and inhibiting determined type of cancer progression.

2. The process as claimed in claim 1 further comprising at least one of:

a. determining an increase in level of glycosylation accompanied by alterations in glycosyltranferases, glycosidases, glycans and mucins (MUC) involved in loss of E- cadherin;

b. coorelating glycosylation with tumor initiation, progression and metastasis.

3. The process as claimed in claim 1, wherein sialylation is governed by sialyltransferases and sialidases.

4. The process as claimed in claim 3, wherein sialyltransferases are selected from ST3Gal (a2, 3-ST), ST6Gal (a2, 6- ST), ST6GalNAc and ST8Sia (a2, 8-ST).

5. The process as claimed in claim 1 further comprising: a. determining alterations in different neuraminidases (NEU) subtypes NEU1, NEU2 and NEU3 and NEU4, wherein said NEU1, NEU2 and NEU3 are localized in the lysosomes, cytosol and plasma membranes, respectively and NEU4 is found in lysosomes or in mitochondria and endoplastic reticulumare; b. correlating said alterations in different neuraminidases (NEU) subtypes with one more cancer cell lines.

6. The process as claimed in claim 1 further comprising determining alterations in enzyme activities of a-2, 3 and a-2, 6 STs in serum, saliva and tissue of patients with OPC and oral cancer patients.

7. The process as claimed in claim 1, wherein fucosylation is catalyzed by fucosyltransferase enzymes (FTs) selected from FUT1 to 11, protein o-fucosyltransferase 1 (POFUT1) and protein o-fucosyltransferase 2 (POFUT2).

8. The process as claimed in claim 7 further comprising: selecting altered expression of various FTs from FUT3, FUT4, FUT6, FUT7, FUT8 to mediate cancer cell migration and thereby metastasis; determining and decrease in FUT3 and FUT5 mRNA expressions in oral cancer patients.

9. The process as claimed in claim 1, wherein FUT4 is associated with the proliferation and metastasis of breast cancer, FUT3 mRNA over expression is responsible for increased SLe^x biosynthesis leading to metastasis in colon carcinoma cell line, and increased FUT7 levels is an indicator of poor prognosis.

10. The process as claimed in claim 1 further comprising determining alterations in composition and decrease in quantity of cell surface fucosylation-associated molecules by fucosidases (FUCA) expression; and limiting invasiveness of cancer cells in early-stage breast tumors, wherein lower FUCA protein levels exhibit increased cell surface fucosylation, which enhances the malignant; an over expression of a-L-fucosidase 1 (FUCA1) exhibits suppressed growth of cancer cells and induced cell death by protein defucosylation.