Cancer therapeutics in the clinical setting. Need for alternate therapeutics. Target multiple cancer pathways. Circumvent the genetic mutations

Transcription

1 Transcriptional factor Snail and MMP-9 signaling axis controls tumor neovascularization, growth and metastasis in mouse model of human ovarian carcinoma Samar Abdulkhalek 1, Olivia Geen 1, Lacey Brodhagen 1, Fiona Haxho 1, Farah Alghamdi 1, Stephanie Allison 2, Duncan Simmons 1, Leah O Shea 1, Ronald J Neufeld 2, and Myron R Szewczuk 1*. Depts of 1 Biomedical and Molecular Sciences, and 2 Chemical Engineering, Queen s University, Kingston, ON K7L 3N6, Canada. *speaker

2 Cancer therapeutics in the clinical setting Need for alternate therapeutics Target multiple cancer pathways Circumvent the genetic mutations

3 OP: Oseltamivir phosphate Published: TLR-4, -7 & -9 EGFR Trk insulin Identified a new anticancer therapeutic approach: Oseltamivir phosphate (OP) monotherapy or in combination with chemotherapeutics against human ovarian cancer regulates tumour neovascularization, growth and invasiveness.

4 Neu1 sialidase activity is associated with epidermal growth factor (EGF) stimulation of ovarian carcinoma cells Mean fluorescence Control EGF 30ng/ml OP 164µ g/ml + EGF Anti-Neu1 100µ g/ml + EGF MMP9i 50 µ g/ml + EGF Mean fluorescence Control EGF 33ng/ml OP 333µ g/m L + EG F Control EGF 33ng/ml OP 333µ g/ml + EGF

5 Oseltamivir phosphate (OP) decreases the cell viability of, cis, -shrna Slug and -shrna Snail cells cis Stable shrna Slug KD Stable shrna Snail KD LD 50 = 7 μm at 48 hr LD 50 = 4 μm at 48 hr LD 50 > 488 μm at 48hr LD 50 > 488 μm at 48 hr C ell V iab ility (% of Control)+ SEM Cell Viability (% of Control)+ SEM Cell Viability (% of Control) ± SEM Cell Viability (% of Control) ± S.E. OP LD 50 of 7μM for OP LD 50 of 4μM for cisplatin-resistant cis OP LD 50 of 488μM for shrna Slug and Snail KD

6 Cell viability of cells treated with different dosages of OP in combination with 1μM of cisplatin, 5-FU, gemcitabine and paclitaxel Cisplatin 5-FU Gemcitabine Paclitaxel C ell Viability (% of C ontrol) ± S.E. Cell Viability (% of Control) ± S.E. C ell Viability (% of C ontrol) ± S.E. C ell Viability (% of C ontrol) ± S.E. untreated control C isplatin 1 µ M OP 300 µ g/ml + Cis 1µ M OP 600 µ g/m L + C is 1µ M OP 800 µ g/ml + Cis 1µ M untreated control 5-FU 1 µ M OP 300µ g/ml+ 5-FU 1µ M OP 600µ g/ml + 5-FU 1µ M OP 800µ g/m L + 5-FU 1µ M untreated control Gem 1µ M OP 600 µ g/ml + Gem 1µ M OP 300 µ g/ml + Gem 1µ M OP 800 µ g/m L + G em 1µ M untreated control Paclitaxel 1µ M OP 300µ g/ml + Pac 1µ M OP 600µ g/ml + Pac 1µ M OP 800µ g/m L + P ac 1 µ M Beneficial effect using combination therapy of OP and standard clinical chemotherapeutics on amplifying chemo-drug sensitivity. Hypersensitivity reactions to oxaliplatin, gemcitabine are noted complications in cancer patients.

7 human ovarian cancer cell line implanted cutaneously in RAGxCγ double mutant mice A Untreated Control 30mg/kg OP 50mg/kg OP B Tumor volume (mm 3 ) Tumor volume (mm 3 ) Tumor volume (mm 3 ) Control TV

8 H&E staining of microtome 5µm sections of paraffin-embedded necropsy lungs taken from tumor-bearing RAGxCy double mutant mice Lungs Untreated OP 50mg/kg OP 30mg/kg B Number m etastatic clusters per lung untreated O P 30 mg/kg OP 50 mg/kg

9 OVARIAN CANCER human ovarian cancer cell line in RAGxCy double mutant mice Ovarian Ovarian SLUG KD Ovarian SNAIL KD SLUG: represses E-cadherin expression and induces epithelial to mesenchymal transitions SNAIL: repressor of E-cadherin gene expression in epithelial tumour cells

10 H&E staining of microtome 5µm sections of paraffin-embedded necropsy lungs taken from tumor-bearing RAGxCy double mutant mice Necropsy Lungs Untreated Slug shrna Snail shrna B Number m etastatic clusters per lung Slug KD SNAIL KD

11 Necropsy tumors Untreated Slug Snail Untreated A4 mouse SLUG KD B2 mouse SNAIL KD C2 mouse D E-cadherin Untreated N-cadherin A Tumor B Tumor H&E E C Bkg E-cad N-cad relative total staining density VE-cad SLUG KD SNAIL KD

12 Snail-MMP-9 signaling axis potentiates Neu1 sialidase and MMP-9 cross-talk in regulating RTK receptors, tumor neovascularization, growth and invasiveness Abdulkhalek et al 2014 Clinical and Translational Medicine 3:28 (open access) MMP9 Snail Signaling cascade Slug Tumor vasculature

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14 A B Tumor volume (mm 3 ) A4 B4 C4 Confidential unpublished data

15 OVARIAN CANCER human ovarian cancer cell line was established from tumour tissue from an untreated patient Control EGF 30ng/ml Tamiflu 400 μm + EGF Anti-Neu1 100 μg/ml + EGF MMP9i 50 μg/ml + EGF Phase Sialidase activity Snail shrna Sialidase activity Confidential unpublished data

16 A B C C ell V iability (% of C ontrol) + S.E. LD 50 = 50 μm C ell V iability (% of C ontrol) + S.E. cis LD 50 = 8 μm C ell Viability (% of C ontrol) ± S E M (n=6) untreated control C isplatin 1µ M Tam iflu 0.73m M + C isplatin 1 µ M Tamiflu 1.46mM + Cisplatin 1 µ M Tamiflu 1.95mM + Cisplatin 1 µ M Tamiflu 0.73mM Tam iflu 1.46m M Confidential unpublished data

17 A Tumor volume (mm 3 ) B Confidential unpublished data

18 A Untreated 50mg/kg OP 30mg/kg OP Number m etastatic clusters per liver untreated Tamiflu 30 mg/kg Tamiflu 50 mg/kg B Number m etastatic clusters per lung untreated Tamiflu 30 m g/kg Tamiflu 50 mg/kg Confidential unpublished data

19 A Number m etastatic clusters per lung Number m etastatic clusters per liver B Slug KD SNAIL KD Slug KD SNAIL KD Confidential unpublished data

20 Untreated A4 mouse SLUG KD B2 mouse SNAIL KD C2 mouse A Tumor D B C Tumor H&E Bkg E-cad relative total staining density N-cad VE-cad SLUG KD SNAIL KD

21 Novelty Abdulkhalek et al 2013 Research and Reports in Biochemistry 3: SNAIL: a zinc finger transcriptional repressor SLUG KD SNAIL KD Snail genes act primarily as survival factors and inducers of cell movement? Signaling cascade Tumor vasculature secreted cytokines

22 Conclusions Tamiflu provides an unexpected therapeutic benefit in cancer, by interfering with growth factor receptor-induced survival signals and disabling cancer cell survival in acquire chemoresistance. Tamiflu alone or in combination with chemotherapeutics against pancreatic and breast impedes the growth and metastatic spread, tumor neovascularization and chemoresistance in heterotopic xenograft of tumors growing in RAGxCγ double mutant mice.

23 Priority #1: Clinical trials Priority #2: To study the broad range efficacy of Tamiflu therapy in combination with standard chemotherapeutics. Priority #3: To test this novel therapy on surgically obtained patient primary tumors in culture