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119. Screening of Optimized Inhibitors Against Proinflammatory Cytokines in Breast Cancer Metastasis

Darren J. Lighter, Clyde Pruett, Cody Wolf, Joseph Tuccinardi, Riley Olsen, Dr. Matthew D. King, Dr. Ken Tawara, Dr. Don L. Warner, Dr. Cheryl L Jorcyk

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Abstract

Approximately 1 in 8 women in the United States will develop breast cancer in their lifetime. While localized breast cancer has a 5-year survival rate of 99%, metastatic breast cancer has a limited 5-year survival rate of 27%. Prior research from our lab and others have identified a proinflammatory cytokine that induces invasion and detachment of various breast cancer tumor cells. We hypothesize that small molecule inhibitors (SMI), can bind to proinflammatory cytokines and reduce their potential to activate the signaling pathway that promotes metastasis of breast cancer tumor cells. First, an in silico model was used to identify potential parental inhibitors based on prospective binding affinity. Parental molecules were then synthesized through a multi-step organic synthesis, and optimized analogs were presented for potentially increased binding affinity and drug-like properties. Continuing forwards, various in vitro tests were performed, including enzyme linked immunosorbent assay (ELISA) and western blots, which allow us to compare the effectiveness of each SMI to reduce the signaling cascade. Inhibition of these cytokines may lead to novel therapeutics that can be used to prevent breast cancer metastasis.

Introduction

  • Oncostatin M (OSM) is a proinflammatory cytokine associated with decreased patient survival and increased breast cancer metastasis.
  • OSM binds to the receptor complex, consisting of OSM receptor and the gp130 receptor. This induces phosphorylation of STAT3, AKT, JNK and ERK.
  • Computational modeling was used to analyze the induced fit of ~1.65 million compounds to an inhibitory binding pocket in OSM the 26 best compounds were tested in vitro through the use of enzyme-linked immunosorbent assay (ELISA) and WB analysis.

Figure 1. Role of OSM in metastasis of breast cancer Model displaying the role of OSM in metastasis

Figure 1, cross section drawing
OSM can be produced and secreted by the primary tumor cells (A) or by white blood cells such as neutrophils and macrophages (B). OSM binds to its receptor and induces the invasion and migration of breast cancer cells through the extracellular matrix. Intravasation into the blood stream occurs, increasing circulating tumor cell (CTC) numbers (C). (Figure created by Ken T.)

Hypothesis

We hypothesize that small molecule inhibitors (SMIs) will bind to OSM and inhibit it’s signaling cascade, significantly reducing a tumor’s metastatic potential.

Methods

  • Human breast cancer cell lines used: T47D (in vitro), MDA-MB-231 (in vivo)
  • Enzyme-Linked Immunosorbent Assays (ELISAs) and western blots are used to test OSM signaling.
  • Fluorescence quenching used to determine binding affinity.

Figure 2. OSM increases lung and spine metastases, as well as CTC counts

Figure 2, graph
Ex vivo BLI of tumor metastases and CTC counts. A) Mice with MDA-MB-231 luc2 cells injected with OSM showing increased metastasis to lung and spine. B) OSM increased CTC count in mice with tumors. (Figure created by Ken T.)

Figure 3. OSM-SMI-10 analogs inhibits pSTAT3 expression in T47D human breast cancer cell lines

Figure 3, chart
Signaling pathway activation analysis in human breast cancer cell lines with SMI-10 analogs. A) ELISA measuring pSTAT3 in T47D cells after treatment with OSM (10 ng/mL) and SMI-10 analogs (10 µM) for 30 minutes (n=2).

Figure 4. OSM-SMI-10 analogs inhibit pSTAT3 expression in T47D human breast cancer cells

Figure 4, gel stain
Protein expression in human breast cancer cell lines. Western Blot analysis of pSTAT3, pAKT, and pERK expression in T47D cells after 30 minute treatment with OSM and SMI-10 analogs (10 µM). (Figure created by Cody W.)

Figure 5. SMI-10-B binds to OSM with high affinity

Figure 5, graph
Fluorescence Quenching binding assay of OSM with SMI 10-B. Fluorometric data measuring dissociation constant (Kd) used to determine the binding affinity for SMI-10-B to OSM. (figure created by Joey T.)

Figure 6. OSM-SMI-27 analogs inhibit pSTAT3 expression in T47D human breast cancer cells

Figure 6, graph
Signaling pathway activation analysis in T47D human breast cancer cells with SMI-27 analogs. ELISA measuring pSTAT3 expression after treatment with OSM (10 ng/mL) and SMI-27 analogs (10 µM) for 30 minutes (n=3). (Figure created by Cody W.)

Conclusions

  • SMI-10B, and SMI-27-B3, and –B5 are the most effective analogs of parental SMI-10 and SMI-27.
  • Halogen groups at the Aniline increases binding affinity for SMI-26

Future Goals

  • Further optimization of SMIs to achieve stronger binding and specificity.
  • Examination of effectiveness of SMIs in various cell lines.
  • In vivo studies on the most potent candidates.

Acknowledgements

The project described was supported by NIH grants #P20GM103408, and #P20GM109095, the METAvivor Quinn Davis Northwest Arkansas METSquerade Fund, the Smylie Family Cancer Fund, and the Boise State University Biomolecular Research Center. Thank you to Nichole Corbett and Thaaer Muhammed for assistance in synthesis and testing of SMIs.

Additional Information

For questions or comments about this research, contact Darren Lighter at darrenlighter@u.boisestate.edu.

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