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CL-siSOX2 inhibits expression markers of tumor growth
The SOX2 embryonic stem cell marker has gained attention for its wide-ranging transcriptional effects on enabling tumor growth and resistance in different types of cancer; for example, SOX2 promoted tumor growth and acquired resistance to radiation and chemotherapy in oral cancer and transcriptionally modulated tumor growth in gastric cancer.25,26 In lung cancer cells, SOX2 in cooperation with beta-catenin and POU5F1 was associated with increased IGF-R1 signaling and poor prognosis.10 Further, a positive feedback loop between SOX2 and EGFR promoted self-renewal and chemo-resistance in lung cancer.25 Central to the role of SOX2 in promoting the resistance phenotype in cancer is the existence of SOX2-enriched side populations (SPs) of cancer C11 BODIPY 581/591 (cancer stem cells, CSCs) that are reposed with a functional capacity for
Figure 5. Cationic lipoplex loaded with siSOX2 (CL-siSOX2) inhibits expression of markers of tumor resistance in mice xenograft tumors. C.B.17 SCID mice with H1650 SP cells as tumor xenografts received treatment as described. (A) Immunoblotting of tumor lysates shows protein expression of (B) Wnt3a, (C) Wnt5a/b, (D) phospho-β-catenin, (E) Dvl2 and (F) ABCG2. Results were calculated as protein/β-actin ratio and presented as mean percent of the CL-siScr with SD. Statistical analysis: student t test: (treatment vs. siScr: ****P b 0.0001, treatment vs. cisplatin: ##P b 0.01; ####P b 0.0001, and treatment vs. CL-siSOX2:
promoting self-renewal and resistance in cancer.27 Therefore, for treatment of cancer to be effective over the long term, the spectrum of therapies must include targeting of these quiescent, residual CSCs.8,28 We proposed the use of a therapeutic small interfering RNA (siRNA) targeting SOX2 formulated in a cationic lipoplex (CL-siSOX2) for passive targeting and treatment of CSC-derived murine tumors.
Sustained proliferation is elemental to the survival, growth and metastatic dissemination of tumors. Indeed, cancer cells have long been associated with a higher potential for proliferation compared to normal cells, and inhibition of cell proliferation is an empirical measure of the potential clinical efficacy of anticancer therapy. SOX2-enriched H1650 SP cells were associated with higher proliferative and metastatic potential, which is evidenced by increased rates of sphere and spheroid formation, as well as increased migration and resistance to cisplatin compared to H1650 MP cells. As with other types of anticancer therapies (i.e. radiation and chemotherapy), targeted therapies also suffer from the fate of rapid progression towards acquired tumor resistance facilitated via extrinsic factors (e.g. secretion of enzymes and/or growth factors by stromal cells of the tumor microenvironment that deactivate drugs and/or
activate primary and secondary resistance inside cancer cells respectively), and intrinsic factors (e.g. mutation of oncogenic signaling pathways, upregulation of efflux transporters, etc.).29–31 Nanoparticle drug formulation strategies provide opportunities for both active and passive targeted delivery of drugs to tumors while circumventing the effects of deactivating and/or inhibitory intrinsic and extrinsic resistance factors to improve cellular internalization and enhanced therapeutic efficacy, while limiting adverse effects due to improved targeting. Nanoparticles employing specific targeting, versatile drug loading, and imaging capabilities have been used in targeted delivery, imaging and treatment of different types of cancer.32–34 We proposed the use of fluorescent lipoplexes for targeted delivery of siRNA targeting SOX2 for imaging and treatment of lung tumors of stem cell origin.
We successfully, formulated siSOX2 in a cationic lipoplex with high drug loading. The formulation parameters were optimized to ensure nano-sized lipoplexes, which we predicted will facilitate lung tumor targeting will be sufficiently aided by an enhanced targeting of tumor vasculature resulting in increased extravasation into tumor tissue aided by the cationic charge of the lipoplexes; our predication was based on the effect of size
Figure 6. Cationic lipoplex loaded with siSOX2 (CL-siSOX2) inhibits markers of tumor invasion and metastasis in mice xenograft tumors. C.B.17 SCID mice with H1650 SP cells as tumor xenografts received treatment as described. (A) Immunoblotting of tumor lysates shows expression of (B) Slug, (C) E-cadherin and (D) N-cadherin. CL-siSOX2 and CL-siSOX2 + cisplatin downregulated the pro-invasion and metastatic Slug and N-cadherin, and induced the expression of
Figure 7. Cationic lipoplex loaded with siSOX2 (CL-siSOX2) inhibits expression of inflammatory markers in mice xenograft tumors. C.B.17 SCID mice with H1650 SP cells as tumor xenografts received treatment as described. (A) Immunoblotting of tumor lysates shows protein expression of (B) TLR9, (C) TLR1 and