Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model

Terrick Andey, Srujan Marepally, Apurva Patel, Tanise Jackson, Shubhashish Sarkar, Malaney O'Connell, Rakesh C. Reddy, Srikumar Chellappan, Pomila Singh, Mandip Singh

Research output: Contribution to journalArticle

29 Citations (Scopus)

Abstract

The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199 nm) with a slight positive charge (21.82 mV); CLG-shAnx2 was of similar size (217 nm) with decreased charge (12.11 mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2 h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4 h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

Original languageEnglish (US)
Pages (from-to)67-78
Number of pages12
JournalJournal of Controlled Release
Volume184
Issue number1
DOIs
StatePublished - Jun 28 2014

Fingerprint

Annexin A2
Annexins
Side-Population Cells
Neoplastic Stem Cells
RNA Interference
Small Interfering RNA
Lung Neoplasms
Neoplasm Metastasis
Lipids
Ligands
Growth
Tumor Burden
Neoplasms
Phenotype
Catenins
Lung
docetaxel
gemcitabine
Fluorescence Microscopy
Camptothecin

Keywords

  • Annexin A2 shRNA
  • Gene delivery
  • Liposome delivery
  • Lung cancer stem cell
  • RNA interference
  • Tumor resistance

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Medicine(all)

Cite this

Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model. / Andey, Terrick; Marepally, Srujan; Patel, Apurva; Jackson, Tanise; Sarkar, Shubhashish; O'Connell, Malaney; Reddy, Rakesh C.; Chellappan, Srikumar; Singh, Pomila; Singh, Mandip.

In: Journal of Controlled Release, Vol. 184, No. 1, 28.06.2014, p. 67-78.

Research output: Contribution to journalArticle

Andey, Terrick ; Marepally, Srujan ; Patel, Apurva ; Jackson, Tanise ; Sarkar, Shubhashish ; O'Connell, Malaney ; Reddy, Rakesh C. ; Chellappan, Srikumar ; Singh, Pomila ; Singh, Mandip. / Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model. In: Journal of Controlled Release. 2014 ; Vol. 184, No. 1. pp. 67-78.
@article{7c27eccc7b6a4518aa4a03a8b2910a51,
title = "Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model",
abstract = "The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199 nm) with a slight positive charge (21.82 mV); CLG-shAnx2 was of similar size (217 nm) with decreased charge (12.11 mV), and entrapment and loading efficiencies of 97{\%} and 6.13{\%} respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2 h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92{\%} decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4 h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19{\%} of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.",
keywords = "Annexin A2 shRNA, Gene delivery, Liposome delivery, Lung cancer stem cell, RNA interference, Tumor resistance",
author = "Terrick Andey and Srujan Marepally and Apurva Patel and Tanise Jackson and Shubhashish Sarkar and Malaney O'Connell and Reddy, {Rakesh C.} and Srikumar Chellappan and Pomila Singh and Mandip Singh",
year = "2014",
month = "6",
day = "28",
doi = "10.1016/j.jconrel.2014.03.049",
language = "English (US)",
volume = "184",
pages = "67--78",
journal = "Journal of Controlled Release",
issn = "0168-3659",
publisher = "Elsevier",
number = "1",

}

TY - JOUR

T1 - Cationic lipid guided short-hairpin RNA interference of annexin A2 attenuates tumor growth and metastasis in a mouse lung cancer stem cell model

AU - Andey, Terrick

AU - Marepally, Srujan

AU - Patel, Apurva

AU - Jackson, Tanise

AU - Sarkar, Shubhashish

AU - O'Connell, Malaney

AU - Reddy, Rakesh C.

AU - Chellappan, Srikumar

AU - Singh, Pomila

AU - Singh, Mandip

PY - 2014/6/28

Y1 - 2014/6/28

N2 - The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199 nm) with a slight positive charge (21.82 mV); CLG-shAnx2 was of similar size (217 nm) with decreased charge (12.11 mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2 h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4 h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

AB - The role of side populations (SP) or cancer stem-like cells (CSC) in promoting the resistance phenotype presents a viable anticancer target. Human-derived H1650 SP cells over-express annexin A2 (AnxA2) and SOX2, and are resistant to conventional cytotoxic chemotherapeutics. AnxA2 and SOX2 bind to proto-oncogenes, c-Myc and c-Src, and AnxA2 forms a functional heterotetramer with S100A10 to promote tumor motility. However, the combined role of AnxA2, S100A10 and SOX2 in promoting the resistant phenotype of SP cells has not been investigated. In the current studies, we examined for the first time a possible role of AnxA2 in regulating SA100A10 and SOX2 in promoting a resistant phenotype of lung tumors derived from H1650 SP cells. The resistance of H1650 SP cells to chemotherapy compared to H1650 MP cells was investigated by cell viability studies. A short hairpin RNA targeting AnxA2 (shAnxA2) was formulated in a liposomal (cationic ligand-guided, CLG) carrier and characterized for size, charge and entrapment and loading efficiencies; CLG carrier uptake by H1650 SP cells was demonstrated by fluorescence microscopy, and knockdown of AnxA2 confirmed by qRT-PCR and Western blot. Targeting of xenograft and orthotopic lung tumors was demonstrated with fluorescent (DiR) CLG carriers in mice. The therapeutic efficacy of CLG-AnxA2, compared to that of placebo, was investigated after 2 weeks of treatment in terms of tumor weights and tumor burden in vivo. Compared to mixed population cells, H1650 SP cells showed exponential resistance to docetaxel (15-fold), cisplatin (13-fold), 5-fluorouracil (31-fold), camptothecin (7-fold), and gemcitabine (16-fold). CLG carriers were nanoparticulate (199 nm) with a slight positive charge (21.82 mV); CLG-shAnx2 was of similar size (217 nm) with decreased charge (12.11 mV), and entrapment and loading efficiencies of 97% and 6.13% respectively. Fluorescence microscopy showed high uptake of CLG-shAnxA2 in H1650 SP cells after 2 h resulting in a 6-fold reduction in AnxA2 mRNA expression and 92% decreased protein expression. Fluorescence imaging confirmed targeting of tumors and lungs by DiR-CLG carriers with sustained localization up to 4 h in mice. CLG-shAnxA2 treatment of mice significantly reduced the weights of lung tumors derived from H1650 SP cells and tumor burden was reduced to only 19% of controls. The loss in tumor weights in response to CLG-shAnxA2 was associated with a significant loss in the relative levels of AnxA2, SOX2, total β-catenin and S100A10, both at the RNA and protein levels. These results suggest the intriguing possibility that AnxA2 may directly or indirectly regulate relative levels of β-catenin, S100A10 and SOX2, and that the combination of these factors may contribute to the resistant phenotype of H1650 SP cells. Thus down-regulating AnxA2 using RNAi methods may provide a useful method for targeting cancer stem cells and help advance therapeutic efficacy against lung cancers.

KW - Annexin A2 shRNA

KW - Gene delivery

KW - Liposome delivery

KW - Lung cancer stem cell

KW - RNA interference

KW - Tumor resistance

UR - http://www.scopus.com/inward/record.url?scp=84899802268&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84899802268&partnerID=8YFLogxK

U2 - 10.1016/j.jconrel.2014.03.049

DO - 10.1016/j.jconrel.2014.03.049

M3 - Article

C2 - 24727000

AN - SCOPUS:84899802268

VL - 184

SP - 67

EP - 78

JO - Journal of Controlled Release

JF - Journal of Controlled Release

SN - 0168-3659

IS - 1

ER -