Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics

Author

Connor P. O'Hara, Department of Medicinal Chemistry, School of PharmacyFollow

DOI

https://doi.org/10.25772/QB4B-0024

Author ORCID Identifier

https://orcid.org/0000-0003-1121-6248

Defense Date

2019

Document Type

Thesis

Degree Name

Master of Science

Department

Pharmaceutical Sciences

First Advisor

Dr. Umesh R. Desai

Second Advisor

Dr. Bhaumik B. Patel

Abstract

Connor O’Hara July 29, 2019

Inhibition of Cancer Stem Cells by Glycosaminoglycan Mimetics

In the United States cancer is the second leading cause of death, with colorectal cancer (CRC) being the third deadliest cancer and expected to cause over 51,000 fatalities in 2019 alone.¹ The current standard of care for CRC depends largely on the staging, location, and presence of metastasis.² As the tumor grows and invades nearby lymph tissue and blood vessels, CRC has the opportunity to invade not only nearby tissue but also metastasize into the liver and lung (most commonly).³ The 5-year survival rate for metastasized CRC is <15%, and standard of care chemotherapy regimens utilizing combination treatments only marginally improve survival.^3-5 Additionally, patients who have gone into remission from late-stage CRC have a high risk of recurrence despite advances in treatment.^6-7

The Cancer Stem-like Cell (CSC) paradigm has grown over the last 20 years to become a unifying hypothesis to support the growth and relapse of tumors previously regressed from chemotherapy (Figure 1).⁸ The paradigm emphasizes the heterogeneity of a tumor and its microenvironment, proposing that a small subset of cells in the tumor are the source of tumorigenesis with features akin to normal stem cells.⁹ The CSCs normally in a quiescent state survive this chemotherapy and “seed” tumor redevelopment.¹⁰ First observed in acute myeloid lymphoma models, CSCs have since been identified in various other cancers (to include CRC) by their cell surface antigens and unique properties characterizing them from normal cancer cells.^11-12 These include tumor initiation, limitless self-renewal capacity to generate clonal daughter cells, as well as phenotypically diverse, mature, and highly differentiated progeny.^13-14

Previously our lab has identified a novel molecule called G2.2 (Figure 2) from a unique library of sulfated compounds showing selective and potent inhibition of colorectal CSCs in-vitro.¹⁵ G2.2 is a mimetic of glycosaminoglycans (GAGs) and belongs to a class of molecules called non-saccharide GAG mimetics (NSGMs). Using a novel dual-screening platform, comparisons were made on the potency of G2.2 in bulk monolayer cells, primary 3D tumor spheroids of the same cell line, and subsequent generations of tumor spheroids. This work has shown in-vitro the fold-enhancement of CSCs when culturing as 3D tumor spheroids. Spheroid culture serves as a more accurate model for the physiological conditions of a tumor, as well as the functional importance of upregulating CSCs. Evaluation of G2.2 and other NSGMs was performed in only a few cell lines, developing a need to better understand the ability of G2.2 to inhibit spheroids from a more diverse panel of cancer cells to better understand G2.2’s mechanism.

The last few decades have seen the advancement in fundamental biological and biochemical knowledge of tumor cell biology and genetics.¹⁶ CRC, in particular, has served as a useful preclinical model in recapitulating patient tumor heterogeneity in-vitro.¹⁷ Recent work has characterized the molecular phenotypes of CRC cell lines in a multi-omics analysis, stratifying them into 4 clinically robust and relevant consensus molecular subtypes (CMS).^18-19 Our work was directed to screen a panel of cells from each of the molecular subtypes and characterize the action of G2.2 and 2^nd generation lipid-modified analogs, synthesized to improve the pharmacokinetic properties of the parent compound. Four NSGMs, namely G2.2, G2C, G5C, and G8C (Figure 2) were studied for their ability to inhibit the growth of primary spheroids across a phenotypically diverse panel.

Compound

HT-29 IC₅₀ (μM)

Panel Average IC₅₀ (μM)

G2.2

28 ± 1

185 ± 55

G2C

5 ± 2

16 ± 15

G5C

8 ± 2

63 ± 19

G8C

0.7 ± 0.2

6 ± 3

Primary spheroid inhibition assays were performed comparing the potency of new NSGMs to G2.2. Fifteen cell lines were evaluated in a panel of colorectal adenocarcinoma cell lines with several cell lines representing each CMS. Primary spheroid inhibition assays revealed 3 distinct response with regard to G2.2’s ability to inhibit spheroid growth. Cells from CMS 3 and 4, which display poor clinical prognosis, metabolic dysregulation, and enhanced activation of CSC pathways, showed the most sensitivity to G2.2 (mean IC₅₀ = 89 ± 55 μM). Mesenchymal CMS 4 cell lines were over 3-fold more sensitive to treatment with G2.2 when compared to CMS 1 cell lines. Resistant cell lines were composed entirely of CMS 1 and 2 (mean IC₅₀ = 267 ± 105 μM). In contrast, all lipid-modified analogs showed greater potency than the parent NSGM in almost every CRC cell line. Of the three analogs, G8C showed the greatest potency with a mean IC₅₀ of less than 15 μM. Of the CRC spheroids studied, HT-29 (CMS 3) was most sensitive to G8C (IC₅₀ = 0.73 μM).

To evaluate the selectivity of NSGMs for CSC spheroid inhibition, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) cytotoxicity assays were performed on monolayer cell culture, and the fold-selectivity of NSGM for spheroids was analyzed. Data shows that NSGMs preferentially target CSC-rich spheroids compared with monolayer cellular growth, with G2.2 having over 7-fold selectivity for spheroid conditions. This fold selectivity was enhanced in CMS 3/4, supporting the idea that G2.2 targets a mesenchymal and stem-like phenotype. To further validate this selectivity, limiting dilution assays were performed across the panel to determine the tumor-initiating capacity of each cell line. Cell lines which showed a sensitive response to G2.2 were over 2-fold more likely to develop into spheroids, validating the previous hypothesis. Further characterization was performed analyzing the changes G2.2 induced on CSC markers, as well as the basal expression of a unique pair of cancer cells. Western blots showed a reduction in self-renewal marker across all CMS after treatment with G2.2, and that cell lines sensitive to G2.2-treatment overexpress mesenchymal and stem-like markers. G2.2-resistant cell lines show an epithelial phenotype, lacking this expression.

The positive results observed in these studies enhance the understanding of G2.2 and analogs, and further evaluation with additional cell lines of various tissues would improve the knowledge thus far gained. However, all experiments described take valuable time to perform and analyze. Thus, there became a need to develop a high-throughput screening (HTS) platform for our assays that standardized analysis and enhanced productivity. Initial development of the method for this assay are underway, and recent evidence from these evaluations of breast cancer spheroids suggests that G2.2 and analogs may be tissue-specific compounds for the treatment of cancer. Future work entails refining the application of this method for evaluation of the NCI-60 (National Cancer Institute) tumor cell panel.

Overall, these results make several suggestions concerning the NSGMs evaluated against the panel. First, G2.2 selectively targets CSCs with limited toxicity to monolayer cells of the same cell line. Further, G2.2 has the greatest potency with CMS 3/4, whose mesenchymal phenotypes are associated with poor clinical prognosis and enrichment of CSCs. Supporting evidence include that sensitive cell lines are highly tumorigenic and show enhanced expression of mesenchymal/CSC markers compared to resistant cell lines. Lipid-modification of G2.2 enhances in-vitro potency against spheroid growth, with nM potency reached in the most sensitive cell lines. Evidence in the development of a HTS platform also suggests these NSGMs show tissue specificity to cancers of the intestine. Further work characterizing the mechanism of NSGMs in a broader multi-tissue panel will enhance our understanding of the compounds as a potential therapy to dramatically improve patient survival through specific targeting of tumorigenesis.

References

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Rights

© Connor P. O'Hara

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

8-1-2019