Abstract B26: A novel naturally-derived withalonglide synergizes with cisplatin to block self-renewal, migration, and EMT transition to enhance apoptosis via targeting of translational initiation
Authors: Subramanian Chitra , Pierce Robin , Clague Michaela , Timmermann Barbara N. , Cohen Mark S. 
Published: May 2017

Introduction: Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer in the world with greater than 300,000 fatalities annually. Despite advances in surgery, chemotherapy and radiotherapy, the five-year survival rate of HNSCC has not changed much over the last four decades due to recurrence and metastasis. Current first-line standard treatment for advanced HNSCC patients is chemotherapy with a platinum agent (cisplatin) and radiotherapy, which carry problems with resistance and toxicity, creating a critical need for novel therapeutics that are not toxic, more efficacious or can mitigate cisplatin limitations. Through structure activity relationships we have identified and characterized a novel, naturally derived withalongolide A-triacetate (WGA-TA) from Physalis longifolia as a lead compound for targeting several cancers including HNSCC. WGA-TA selectively targets several key pathways including PI3K/mToR through modulation of HSP90-cdc37 chaperon function and has a high therapeutic index. Therefore, we hypothesize that combining WGA-TA with cisplatin can be synergistic and lower toxicity in combination.

Methods: Validated HNSCC cell lines MDA-1986, UMSCC-22B and UMSCC-12 were treated with serial dilutions of WGA-TA and cisplatin starting from 10 nM to 100 μM and the viability of the cells were determined by MTS assay. The IC50 values for single drug treatment was determined using GraphPad Prism. Based on the IC50 values the single drug treatment cells were treated with varying combinations of cisplatin and WGA-TA that are above their IC50 values and the combination index (CI) was calculated using CompuSyn. CI&lt;1 was considered synergistic. Targeting of translational initiation complex proteins an as well as the EMT transition was measured after treating MDA-1986 and UMSCC 22B cells with the combinations that were synergistic. Induction of apoptosis was measured by western blot analysis for the cleavage of PARP and further validated by Flow cytometry. Self-renewal was assessed by sphere formation assay. Boyden-chamber assay was used to measure invasion and migration.

Results: When HNSCC cells were treated with varying concentrations of either WGA-TA alone or cisplatin alone for 72 h, a dose dependent decrease in viability was observed. The IC50 value for WGA-TA treatment was 1.7μM, 0.611μM and 0.75μM and that for cisplatin treatment was 6.639 μM, 4.141 μM and 5 μM for UMSCC-12, MDA1986 and UMSCC-22B respectively. When cells were treated with varying combinations of cisplatin and WGA-TA for 72 h or 24 h, we observed CI value of &lt;1 when several different concentrations of WGA-TA are combined with 1.25 and 2.5 μM cisplatin for both MDA-1986 and UMSCC-22B indicating that combining cisplatin with WGA-TA has synergistic effect (p&lt;0.01).

To further evaluate whether WGA-TA in combination with cisplatin could target translational initiation complex proteins, we have treated UMSCC-22B and MDA-1986 with either 1.25 μM cisplatin or 2.5 μM cisplatin along with varying concentrations of WGA-TA for 24 h and evaluated modulation of translational complex proteins by western blot analysis. Our results indicated down regulation of p-e1F4B by 50-90%, pe1F4G by 80-90% and p4EBP by 45-70% for combination treatments with no changes in e1F4B, e1F4G, 4-EPB1, e1F4E, pe1F4E, e1F4A and e1F4A1. Consistent with down regulation of translational initiation, PARP cleavage as well as decrease in the levels of vimentin and increase in the expression of e-cadherin was also observed by immuno blot analysis indicating induction of apoptosis and blocking of EMT transition. Finally, we demonstrate that cancer stem cell functions such as self-renewal, migration and invasion was also blocked significantly using the combination therapy.

Conclusion: WGA-TA when combined with cisplatin synergizes to effectly target translational initiation complex proteins. Enhanced apoptosis observed while combining very low concentration of cisplatin with WGA-TA indicates that the combination treatment is a much safer and effective option for targeting HNSCC that mitigates the dose and toxicity associated with standard cisplatin treatment. Further in vivo validation studies are warranted to define this synergistic effect for rapid clinical translation.

Citation Format: Chitra Subramanian, Robin Pierce, Michaela Clague, Barbara N. Timmermann, Mark S. Cohen. A novel naturally-derived withalonglide synergizes with cisplatin to block self-renewal, migration, and EMT transition to enhance apoptosis via targeting of translational initiation. [abstract]. In: Proceedings of the AACR Special Conference on Translational Control of Cancer: A New Frontier in Cancer Biology and Therapy; 2016 Oct 27-30; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2017;77(6 Suppl):Abstract nr B26.