The Role Of Cathepsin Biology Essay

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The biological environment in which cancer arises plays a critical role in inhibiting tumor development; conversely it can also be manipulated by cancerous cells into providing factors that support tumor progression. In recent years, there has been heightened interest in the functional role of the microenvironment and modulators of this tumor niche on tumorigenesis, distant metastasis and drug resistance. The interaction, inhibitory and stimulatory regulation of various tumor bed associated elements, with increased focus on the specific roles of proteolytic enzymes, cancer associated fibroblast (CAF), tumor associated macrophage (TAM), and para-tumor cytokines, have also received attention. Our long-term objective is to understand the molecular crosstalk between tumor and the stromal cells, and to determine the mechanisms by which host tissues are co-opted to facilitate cancer progression.

Cathepsin S (Cat S), a cysteine protease with documented endoproteolytic activity and broad pH profile, is up-regulated in many human and murine cancers and its increased expression is associated with malignant progression and poor patient prognosis. Despite the accentuated interest and increasing volume of work regarding the mechanism of Cat S-induced loss of tissue architectural pattern or destruction, and cancer progression, the specific molecular mechanisms by which they facilitate tumor aggressiveness and mediate stemness are still inadequately defined. In this study, attention is drawn to a cysteine protease, secreted almost exclusively by endotheliocytes and antigen presenting cells, especially macrophages, which has an immunosuppressive effect and debatable anti-apoptotic activities in various cell types, including TAM and transformed cells.

This proposal focuses on the contribution of the Cat S to tumor-host molecular interactions, tumor 'malignantization' and its putative role as a marker of cancer stemness. Our hypothesis is that Cat S is crucial for distinct steps in acquiring each distinct 'hallmark capability' of cancer, and phenotypic characteristics of cancer stem cell in colorectal carcinoma (CRC). We will investigate by what signalling pathway Cat S modulates tumor progression. We will test the hypothesis that Cat S not only promotes tumor invasion by a molecular mechanism requiring the extracellular cleavage of E-cadherin, and facilitate angiogenesis through the degradation and release of vascular basement membrane proteins, but also that Cat S is a marker of stemness in CRC. We will also determine if the cellular sources and substrates of Cat S in the neoplastic microenvironment are necessary for its pro-tumorigenic functions and clonigenicity. To achieve these aims, we will analyze Cat S with a combination of our lab-designed novel cell co-culture system, biochemical and pharmacological experiments.

These studies will provide vital insights into the biology of Cat S in cancer, the role of the microenvironment in tumor progression, and enable the development and application of therapeutic cancer strategies based on studies in this pre-clinical co-culture system model.

Keywords:

Cathepsin, Cathepsin S, Proteases, Cysteine proteinases, Proteolytic enzymes, Cancer stem cells, Tumor initiating cells, Stemness, Tumor associated macrophages, Microenvironment, Macrophage, Colon, CRC, Malignant, Tumor, Invasion

Project Generic Title:

A Prospective Study of Proteolytic Factors as Surrogate Markers in Human Malignant Diseases

Project Title (1):

The Role of Cathepsin S in Malignant Colon Carcinoma Progression, Stemness and its Regulation Mechanism

Problem Definition:

In recent years, globally and more particularly in resource-limited communities, malignancies are becoming a common diagnosis with great economic, emotional and physical consequence. This is probably not because it is a novel health condition invading the world but rather due to increased awareness, better diagnostic attempts and improved patronage of institutionalized orthodox medical facilities. However, it is unfortunate that many turn to orthodox medicine at advanced or end stage, owing largely to inaccessibility to available facilities and/or high cost of orthodox medical aid in these resource-limited communities.

The World Health Organization 2008 mortality factsheet shows that about 70% of all cancer related deaths occurred in low- and middle-income countries, and projects that cancer deaths worldwide would continue to rise to over 13.1 million in 2030. (1) Thus, this underscores the magnitude of the cancer burden, and the need for more pragmatic approach to unravelling an innovative, inexpensive, readily available and accessible cancer screening tool in these poor communities.

Project Background:

Colorectal cancer (CRC), a largely preventable disease, presents an enormous socio-economic and clinical challenge, being the second (2nd) most prevalent malignant morbidity amongst females, third (3rd), amongst males and the fourth (4th) major cause of mortality worldwide. Most recent published figures from WHO showed that global cancer mortality was 7.6 million with CRC accounting for about 8.5% of this mortality - a worrisome 647 121 deaths (2), and an annual incidence of over one million newly diagnosed cases with almost no preferential gender discrimination.

Colorectal cancer, like many other human malignancies, is not mono-etiological in pathogenesis; it is rather a product of a combination of various genetic, epigenetic, somatic, environmental and endogenous factors. CRC is an epithelial-cell-derived gastrointestinal malignancy beginning with benign adenomatous precursors (polyps) and is best appreciated in light of the conventional adenoma-carcinoma sequence. Characterized by torpidly progressive dysplastic epithelial overgrowth, impaired differentiation reflected as an expansion of nascent cells with corresponding depletion of mature cells, and increasing invasive cell loci, CRC is a multi-seriate pathology fuelled by aberrations that serially enhance malignant traits.

It is pertinent at this juncture to state that the author does not consider CRC to be a simplistic monoclonal or homogenous assembly of mutant cells. Drawing from enormous body of evidence, we view colorectal carcinoma, like most other types of human malignancies, to be a composite tissue system characterized by deregulated entropic growth, modulated by a small intra-tumor congregation of mutant malignant cells with an inherent capacity for self-renewal, and maintenance of post-differentiation phenotypic heterogeneity, as well as, an acquired aggressive and hyper-proliferative propensity. The author addresses this later.

The transition from normal epithelium to benign adenomatous polyps and subsequently to the malignant stage takes one to three decades, accompanied by a plethora of epigenetic and genetic alterations and is often accelerated in the confines of a permissive tumor bed. By tumor bed, we refer to the neoplastic microenvironment made up of the transformed cells and their surrounding supporting milieu. The support structure is a cast of normal non-cancerous matrix stroma cells (epithelial cells, fibroblasts, adipocytes), vascular elements (endothelial cells, pericytes), immune and inflammatory components (T-lymphocytes, B-lymphocytes, natural killer and dendritic cells, mast cells, monocytes / macrophages), as well as the numerous associated chemokines and cytokines.

Communication between the colonic tumor cells and their microenvironment helps drive the process of tumor growth and evolution. Tumor progression is dependent on what happens around the tumor cells, as much as what takes place within them, thus, tissue architecture with a malignant phenotype is necessary for colonic tumor cell survival and maintenance. Enhanced angiogenic activity and increased invasiveness - two (2) key hallmarks of tumor progression, are actually microenvironment-dependent. Tumor masses produce substances capable of attracting a constant influx of pro - and anti-inflammatory cells. These cells once in the tumor bed or microenvironment as it is conventionally called, are polarized towards an alternative stimuli and begin to enhance cellular proliferation, stroma remodelling and tumor progression. (3, 4, 5) Though controversial, many studies show that CRC predisposition is positively regulated by the infiltration and colonization of the tumor bed by immune cells, especially, macrophages. (3)

The immune-inflammatory component of the tumor bed, particularly the so called tumor associated macrophages (TAMs), plays no small role in the amplification of tumor-susceptibility. Despite general knowledge that the immune system plays a vital role in the identification and subsequent elimination of transformed cells, a function termed 'cancer immunosurveillance', it is however, not all culprits that are prosecuted, as some tumor cells often escape the immunosurveillance machinery. Having evaded recognition and elimination by the activated Natural killers (NKs) and other T lymphocytes, these tumor cells now have their growth facilitated by the once 'hostile' immune system. How? Via the TAM mediated neovascularisation pathway. (6, 7)

As first line of defence in most neoplastic terrain, macrophages, due to their characteristic capacity for rapid infiltration and colonization of lesion sites, in addition to their ability to produce cytokines which attract and activate NKs and dendritic cells (DCs), are considered very important in tumor pathogenesis and evolution; (8) howbeit, the 'law enforcement agent' often time may turn out as an 'accomplice'.

Contrary to the generally accepted anti-pathogenic or pathogenocidal role of macrophages, increasing evidence reveal that TAMs under the varying modulation of several enzymes, cytokines and chemokines, possess a M1 < M2 (M1/M2 < 1) polarization ratio and as such display an immunosuppressive, pro-angiogenesis and pro-neoplastic propensity.

This paradoxical role of TAM in tumor evolution is increasingly being supported by evidence from various works. Alberto Mantovani, finely showed in his work on TAM's role in progression of neoplasm, how a friendly macrophage (M1 phenotype) effecting tumor rejection could also act as foe (M2 phenotype) promoting tumor proliferation, angiogenesis and metastasis (9), hypothesizing that this pluriphenotypic and multifunctional nature of TAM relies on the state of its microenvironment, since their activation and function is modulated by local stimuli. (10)

Macrophage recruitment and migration to tumor mass is mediated by chemokine (C-C motif) ligand 2 (CCL2) also known as monocyte chemotactic protein-1 (MCP-1) and chemokine (C-C motif) ligand 5 (CCL5) known also as RANTES (regulated and normal T cell expressed and secreted). The quantity of macrophage recruited is independent of CCL2, yet, macrophage-associated angiogenesis is highly dependent on it. Aside CCL2 and RANTES, other chemokines such as CCL3, CCL4, CCL8, CXCL8, CCL22 and CCL18 have been implicated in tumor mass colonization by TAM, however, the precise extent of their involvement needs more elucidation. (11 - 16)

The role of cytokines on TAM activity cannot be overstated. Colony stimulating factor 1 (CSF-1), which is produced by monocytes, macrophages and a number of other cells, stimulates the recruitment, proliferation, differentiation and survival of monocytes/macrophages in the tumor mass. Elevated level of CSF 1 activates and upregulates the tumor bed macrophage content; these in turn positively modulate angiogenesis via VEGF mediation and thus enhances tumor proliferation. Vasculo-endothelial growth factor (VEGF) have been shown to trigger circulating myeloid cells homing in adult neovascularisation sites and they sustain the survival of recruited paravascular cells via CXCL12 stroma cell derived factor-1 expression.

Of great relevance in the cancer biology is the role of proteolytic enzymes, and more specifically, the cysteine peptidases. (17 - 23) Based on available evidences from substantial body of literature, this group of proteases are facilitators of several steps in cancer progression. Several cysteine proteases have been implicated in tumor growth, tumor cell invasion, metastasis, and more recently, in tumor-related angiogenesis, thus, of no less significance in tumor initiation, progression, and putative stemness acquisition, is the role played by the well-known cysteine protease with an evolving structural, functional and regulatory characterisation, Cathepsin S (Cat S). (17, 18)

Cat S, a potent elastin and collagen degrading enzyme, with a restricted or tissue-specific expression pattern and approximately 30 kDa molecular mass, (17, 24) belong to the cysteine proteinases family (CA1) of the Papain super family, comprising of other proteases such as cathepsins B, L, C and H.(25) In addition to its notable spatial association with antigen processing, and peptide presentation in immunocytes, especially the professional antigen-presenting cells (APCs) and its role in bio-reparative processes such as wound healing and bone remodelling, (26) the plausible work of Guo-Ping Shi et al., on the characterisation of Cat S in 1994, and the 1995 Vivek Reddy et al. demonstration of the pericellular mobilization of Cat S by human monocyte-derived macrophages (hMDM), an immunocyte, and a non-redundant element of the tumor niche, aroused interest in their role in tumor evolution, resulting in mounting evidence implicating Cat S in tumor initiation and progression via its mitogenic, anti-apoptotic, pro-invasion, pro-epithelial-mesenchymal transformation, potent angiogenic and pro-osteogenesis activities.(17, 25, 26, 27)

Predominantly expressed in certain cellular components of the tumour microenvironment, Cat S is upregulated in several preclinical models of various malignancies, with a positive correlation between their degree of expression, and/or, secretion, and increased tumor cell aggressiveness cum 'malignantization'. (28)

In addition to its restricted tissue distribution, by exhibiting a significant portion of its proteolytic activity at neutral pH, Cat S differs from other cysteine cathepsin isoforms in its propensity for secretion and extracellular bioactivity. (29) Cat S is an effective mediator of the irreversible posttranslational modification of protein via the catalysis of the amide bond hydrolysis. The maintenance of its proteolytic and associated attributes even after long incubation at neutral pH, projects the preferential suitability of Cat S as potent effector of tissue dissolution and extracellular matrix (ECM) loss of architectural pattern in the non-acidic extracellular niche. (30) This ECM loss of structural integrity or remodelling, and by inference, epithelial mesenchymal transition (EMT) is characteristic of the histopathologically defined CRC adenoma-carcinoma tumor progression sequence and metastasis. Metastasis is the process by which tumor cells acquire an aggressive phenotype. It is a multiseriate process involving the loss of cell-cell and cell-host bond, detachment of malignant cells and their invasion of the para-tumor tissues. Increasing evidence reveals that tumor metastasis and not the primary tumor is the principal cause of the soaring mortality among CRC patients. In fact, depending on the tumor stage, an estimated 20% to 70% and 10% to 20% of CRC-related deaths are secondary to intrahepatic and intrapulmonary metastasis respectively. (31) The high incidence of these metastatic cases and tumor recurrence contribute to CRC ranking as one of the most frequent human cancer with a prognosis that remains poor.

In several cancer types, Cat S is translocated from its usual intracellular lysosomal habitat into the extracellular milieu, where it is believed to promote ECM remodelling and eventually, tumor invasion and progression in the tumor niche. (32) Thus, implicated as a culprit in matrix degradation, and by extension in the EMT and metastatic process, we will evaluate Cat S activity and expression in CRC against variations in epithelial and mesenchymal markers. Upregulation of mesenchymal markers such as N-cadherin, fibronectin, laminin, beta-tubulin, and vimentin, in contrast with, downregulation of epithelial markers such as cytokeratin, E-cadherin and beta-catenin, are defining attributes of EMT. E-cadherin has been shown to be a substrate of Cat S. The loss of E-cadherin, a key component of the adherens junction, as well as a mediator of cell-cell adhesion and epithelial organization, facilitates the adenoma - carcinoma sequence and thus enhances tumor aggressiveness. (33, 34)

Beyond its anti-apoptotic activity, (34) the endogenous expression of Cat S by endothelial cells and tumour associated macrophages is speculated to rapidly increased in response to nascent and sustained inflammatory stimuli. In certain cancer types, such as CRC, where chronic inflammation precedes and enhances tumor formation, and many others, where inflammatory signal transduction is triggered by intrinsic tumor cell and tumor host/ microenvironment interaction, it is speculated that the upregulation of Cat S expression in the tumor microenvironment may be indicative of Mantovani's tumor-promoting 'smouldering inflammation', which enhances not only macrophage survival, but the survival, proliferation, angiogenesis, increased invasiveness and migratory capability of malignant cells, while subverting adaptive immunity and altering tumor response to hormones and chemotherapy.(35)

In recent years, growing evidence show that local and distant dissemination of malignant cells, as well as cancer recurrence, is linked to the activities of a phenotypic small subpopulation of tumor cells believed to possess tumor-growth initiating abilities; (reviewed in 36) and our background knowledge of the implication of Cat S in epithelial disorganization plus enhanced tumor cell aggressiveness; We hypothesize that Cat S may not be an innocent bystander in the cancer stemness arena. We speculate that Cat S is an active and non-redundant player in the initiation and/or maintenance of this phenotypic subset of tumor cells, functionally referred to as cancer stem cells (CSCs), or more preferably, tumor initiating cells (TICs). This subset of malignant cells is attributed with the capability to produce tumor cells (tumorigenicity) which not only maintain a differentiated phenotype, but also reproduce a complete phenotypic and morphologic heterogeneity of the parental mutants (clonogenicity). (37) This study will thus include an evaluation of the possible involvement of Cat S in the maintenance of these cancer stem-like 'side-population' (SP) cells in CRC cell lines, and their speculative role in resistance to conventional chemotherapy and radiotherapy. Validation of our hypothesis on the involvement of Cat S in acquisition and/or maintenance of tumor stemness would indicate the potential clinical usability of Cat S, not only as an effective prognostic tool, but also as a potent arsenal in overcoming chemoresistance secondary to the activities of the so-called cancer stem cells.

Despite increased volume of work relating to cysteine cathepsins and cancer biology, yet, the molecular mechanism underlying Cat S secretion and over-expression has not been fully elucidated, nor has the intrinsic relationship between the cysteine cathepsin expression and tumor progression, as well as stemness.

We project that by exploring, characterizing and cataloguing the distinct role of Cat S in cancer evolution and stemness, we would be adding to the body of knowledge by which the long-term goal of establishing a cancer screening tool that could be molecularly targeted, as an effective oncoprophylaxis and anticancer therapeutic strategy.

This study seeks to gain insight into the role of Cat S in the initiation and progression of malignancies, especially colorectal cancer, investigate the association of Cat S and tumor stemness, and attempt to proffer an efficient therapeutic strategy. The objective of this proposal is to clarify the molecular mechanism underlying Cat S- tumor promoting role. At this stage, this project will focuses on the use of human colorectal cancer cell lines.

Illustrations & Charts:

(38)

(38)

(39)

In this project, the specific objectives are as follows:

Research Objectives & Hypotheses

Objectives:

To investigate the endogenous expression of Cat S in colorectal carcinoma spheroid cells

To evaluate the correlation between Cat S expression and tumor stage/progression

To investigate the effect of Cat S inhibition on tumor initiating cell activities

To validate the potentials of Cat S as therapeutic target in chemoresistant CRC

Hypothesis:

1.

2.

3.

4.

Experimental Design:

A paired panel (parental and spheroid) of selected colorectal cancer cell lines with varying degree of aggressiveness will be generated in compliance with our lab standardized protocol. Validation of the spheroid cells as CRC stem cells will be done using anti-EpCAM and anti-CD 44 antibodies. (40) Using the western blot/ immunoblotting assay, endogenous expression status of Cat S in the cells lines will be investigated and a comparative analysis of expression levels in corresponding pair done. Quantitative PCR (q-PCR) will be employed to evaluate correlation between Cat S messenger and protein level. Our sister lab- generated cell co-culture system model will be used to test the hypotheses and ascertain to what extent M2 and/or M1 macrophages and the apoptosis inhibitor of macrophages (AIM) negatively modulate Cat S expression. The role of Cat S in tumor progression would be subsequently validated using the matrigel invasion, and wound healing assays. HUVEC angiogenesis assay would also be done to examine angiogenic effects of Cat S activation and silencing. In vivo activities would be evaluated using highly malignant colon cell lines and CRC stem cell lines xenograft studies.

Materials and Methods

Selection and culture of CRC cell lines

DLD-1, SW480, SW620, HCT-116, and HT 29 colorectal cell lines will be chosen for this study

SP analysis and purification using flow-cytometry

Culture of SP cells into tumor spheroid cells

Colony formation of SP and non-SP cells

Wound healing assay to evaluate if and how EMT in wound healing occurs under gain and loss of Cat S functions

Matrigel Invasion assay to evaluate the migration ability of the cell lines under gain and loss of Cat S functions

Western Blotting to confirm Cathepsin S expression in the panel of cell lines

Reverse transcriptase (RT)- and Real time (Q)-PCR

Co-culture of Cat S expressing CRC cell lines with THP-1 cell line

Data compilation and statistical analysis

What this Study will add to the field?

At the end of this study, we look to demystify the currently unclear molecular mechanism underlying Cat S activities, its regulatory role in the tumor microenvironment and how it enhances tumor progression.

To the best of our knowledge, this is the first study with intent to evaluate Cat S ability to induce and/or maintain tumor stemness. No previous study has shown the relationship between Cat S expression and so-called cancer stem cell activities.

Validating our hypothesis that Cat S activity promotes cancer cell stemness would be a milestone in our journey towards attenuation of tumor aggressiveness and elimination of CSC-related chemo- and radio-therapy.

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