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Cancer Biology & Therapy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcbt20
Down-regulating HIF-1α by lentivirus-mediated shRNA for therapy of triple negative breast cancer ab
c
b
b
Shuang Li , Qingzhu Wei , Qin Li , Bin Zhang & Qiang Xiao a
b
Graduate School of Southern Medical University, Guangzhou 510515, China
b
Department of Plastic Surgery, General Hospital of Guangzhou Military Command, 510010, Guangzhou, China c
Department of Pathology, The Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China Accepted author version posted online: 28 Apr 2015.
Click for updates To cite this article: Shuang Li, Qingzhu Wei, Qin Li, Bin Zhang & Qiang Xiao (2015): Down-regulating HIF-1α by lentivirusmediated shRNA for therapy of triple negative breast cancer, Cancer Biology & Therapy To link to this article: http://dx.doi.org/10.1080/15384047.2015.1040958
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Down-regulating HIF-1α by lentivirus-mediated shRNA for therapy of triple negative breast cancer Shuang Li,1,2 Qingzhu Wei,3 Qin Li,2* Bin Zhang,2 Qiang Xiao2 1
Graduate School of Southern Medical University, Guangzhou 510515, China
2
Department of Plastic Surgery, General Hospital of Guangzhou Military Command, 510010,
Guangzhou, China 3
Department of Pathology, The Third Affiliated Hospital of Southern Medical University,
Downloaded by [] at 11:38 15 May 2015
Guangzhou 510630, China *Corresponding author: Qin Li, Department of Plastic Surgery, General Hospital of Guangzhou Military Command, 111 Liuhua Street, Guangzhou 510010, China; Phone/fax number: +86-020-88653260/+86-020-88224856 Email: [email protected] Running title: Targeting HIF-1α for TNBC treatment Abstract Hypoxia is associated with poor response to treatment in various cancers. Hypoxia inducible factor 1 (HIF-1) is a major transcription factor that mediates adaptation of cancer cells to a hypoxic environment and regulates many genes that are involved in key cellular functions, including cell immortalization, stem cell maintenance, autocrine growth/survival, angiogenesis, invasion/metastasis, and resistance to chemotherapy. HIF-1α has been considered as an attractive therapeutic target for cancer treatment, but there is limited success in this research field.
In the
present study, we designed a recombinant lentivirus containing HIF-1α siRNA, developed stably transfected cell lines, and tested the anticancer effects of the siRNA on cancer cells in vitro and in vivo. Our results indicated that the stable downregulation of HIF-1α reversed chemoresistance, inhibited proliferation, migration and invasion of cancer cells, and slowed down the tumor
1
growth in breast cancer xenograft models. In conclusion, the recombinant lentivirus containing HIF-1α siRNA provides a new avenue for developing novel therapy for triple negative breast cancer. Key words: HIF-1α, triple negative breast cancer, stably transfected cell lines，recombinant
Downloaded by [] at 11:38 15 May 2015
lentivirus, siRNA therapy, gene therapy, apoptosis
2
Introduction Breast cancer is one of the most common invasive cancers in women and is responsible for at least 13.7% of cancer-related deaths in women.1,2 Although recent advances in detection and treatment of breast cancer have been made, overall survival rate of patients with advanced breast cancer remains low.3,4 Currently available treatments for late-stage breast cancer rarely result in long-term benefits. Breast cancer is particularly difficult to treat when it metastasizes and/or becomes resistant to anti-estrogen therapies.5 In particular, patients with triple negative breast cancer (TNBC) have the poorest prognosis, with the five-year survival rate being lower than 30%.6,7 Therefore, there is an urgent need for developing novel approaches to overcoming the
Downloaded by [] at 11:38 15 May 2015
limitations of current breast cancer therapies. Hypoxia is a characteristic feature of most solid tumors and related to poor response to treatment. In response to hypoxia, cancer cells undergo a variety of adoptive changes, including activation of signaling involve in cancer cells survival, proliferation, apoptosis, invasion, chemoresistance, and angiogenesis.8,
9
HIF-1 is one of the major transcription factors that
mediate this adaptive process of cancer cells to the hypoxic environment. HIF-1 is a heterodimer composed of the rate limiting factor HIF1α and the constitutively expressed HIF-1β;10 the latter is also called the aryl hydrocarbon receptor nuclear translocator and heterodimerises with several other factors, such as the Ahr transcription factor.11 HIF-1 controls the expression of almost 70 genes involved in cell proliferation, senescence, apoptosis, differentiation, angiogenesis, glucose metabolism and other cellular functions.12 For instance, VEGF is a typical downstream gene of HIF-1. It binds to its receptor VEGFR2 and is a major regulator of angiogenesis, which induces migration and proliferation of vascular vessel endothelial cells, promoting the increase in vasopermeability and angiogenesis and ameliorates wound blood supply.13 HIF-1α is induced by hypoxia, and also by oncogenes, such as HER-2/neu, v-src, and ras.14 In addition to oxygen-independent mechanisms, the PI3K, AKT and MAPK pathways mediate primarily non-hypoxic HIF regulation under normoxic environment. Consequently, HIFs and other pathways (PI3K/AKT and mTOR/p70S6K1) that lead to normoxic HIF activation are considered
3
potential therapeutic targets.15 Despite mounting evidence implicates HIF-1α plays a major role in carcinogenesis and cancer development and progression, it has been regarded as “undruggable”. 16,17 A number of gene-therapy approaches have been developed for various cancers.18-20 HIF-1α is viewed as a viable prospective target for novel pharmacologic approaches to the clinical management of solid tumors as it is the major regulator of the hypoxic adaptive response.21,22 Multiple cellular pathways resulting in HIF activation could be successfully inhibited by use of different drugs (e.g., topotecan and inhibitors of heat shock protein 90 and mTOR).23 In the present study, we designed a recombinant lentivirus containing HIF-1α siRNA and obtained
Downloaded by [] at 11:38 15 May 2015
stably lentivirus-transfected cell lines and then tested its therapeutic effects on cancer cells under normoxic conditions. We found that downregulation of HIF-1α reversed chemoresistance and inhibited capability of proliferation, migration and invasion of breast cancer cells in vitro. Furthermore, the recombinant lentivirus significantly inhibited the growth of breast cancer xenografts in nude mice. The results suggest that the recombinant lentivirus targeting HIF-1α could be a new approach to breast cancer treatment. Results IF-1α is stably knockdown via lentivirus carrying a HIF-1α shRNA We observed the expression of GFP which was contained in lentivirus under an inverted fluorescence microscope. There were no significant changes in cell morphology between different groups (Figure 1A). We then demonstrated that the expression of HIF-1α decreased dramatically at both the mRNA and protein levels (Figures 1B). The inhibition rate reached 95% at protein level (Figures 1C). Downregulation of HIF-1α in MDA-MB-231 inhibits cell growth, migration and invasion capacity in vitro We next investigated the effects of downregulated HIF-1α expression by lentivirus on the cell proliferation, migration, and invasion of MDA-MB-231 cells. As shown in Figure 1D, inhibition 4
of proliferation was observed in cells treated with HIF-1α shRNA. We next compared migratory ability and invasive potential among the cells treated with HIF-1α shRNA or NC shRNA and untreated MDA-MB-231. The number of cells crossing through the Transwell with Matrigel was significantly reduced in the HIF-1α shRNA-treated group (Figure 2A). The number of cells crossing through in NC shRNA treated and untreated MDA-MB-231 cells was 1.63-fold and 1.76-fold higher than that of the cells treated with HIF-1α shRNA, respectively (Figure 2B). In addition, compared with the NC shRNA treated cells, the HIF-1α shRNA treated cells showed 1.77- and 1.75-fold decreases in the number of cells migrating across the wound at 24 and 48 h, respectively (Fig. 2C) and the same figures were , 2.05- and 1.84-fold decreases, compared with
Downloaded by [] at 11:38 15 May 2015
that of MDA-MB-231, respectively (Fig. 2D). Downregulation of HIF-1α promotes apoptosis and induces cell cycle arrest in G2/M phase Both early and late apoptotic cell counts were increased in HIF-1α shRNA treated cells; the percentages of apoptotic cells were 5.87±0.91% in untreated MDA-MB-231 cells, 6.5±1.23% in NC shRNA treated cells, and 10.93±1.12% in HIF-1α shRNA treated cells, respectively (Figure 3). The number of cells in G2/M phase in HIF-1α shRNA cells were significantly increased, compared with the NC shRNA-treated cells (p=0.002) and untreated MDA-MB-231 (p
Cancer Biology & Therapy Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kcbt20
Down-regulating HIF-1α by lentivirus-mediated shRNA for therapy of triple negative breast cancer ab
c
b
b
Shuang Li , Qingzhu Wei , Qin Li , Bin Zhang & Qiang Xiao a
b
Graduate School of Southern Medical University, Guangzhou 510515, China
b
Department of Plastic Surgery, General Hospital of Guangzhou Military Command, 510010, Guangzhou, China c
Department of Pathology, The Third Affiliated Hospital of Southern Medical University, Guangzhou 510630, China Accepted author version posted online: 28 Apr 2015.
Click for updates To cite this article: Shuang Li, Qingzhu Wei, Qin Li, Bin Zhang & Qiang Xiao (2015): Down-regulating HIF-1α by lentivirusmediated shRNA for therapy of triple negative breast cancer, Cancer Biology & Therapy To link to this article: http://dx.doi.org/10.1080/15384047.2015.1040958
Disclaimer: This is a version of an unedited manuscript that has been accepted for publication. As a service to authors and researchers we are providing this version of the accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proof will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to this version also.
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Down-regulating HIF-1α by lentivirus-mediated shRNA for therapy of triple negative breast cancer Shuang Li,1,2 Qingzhu Wei,3 Qin Li,2* Bin Zhang,2 Qiang Xiao2 1
Graduate School of Southern Medical University, Guangzhou 510515, China
2
Department of Plastic Surgery, General Hospital of Guangzhou Military Command, 510010,
Guangzhou, China 3
Department of Pathology, The Third Affiliated Hospital of Southern Medical University,
Downloaded by [] at 11:38 15 May 2015
Guangzhou 510630, China *Corresponding author: Qin Li, Department of Plastic Surgery, General Hospital of Guangzhou Military Command, 111 Liuhua Street, Guangzhou 510010, China; Phone/fax number: +86-020-88653260/+86-020-88224856 Email: [email protected] Running title: Targeting HIF-1α for TNBC treatment Abstract Hypoxia is associated with poor response to treatment in various cancers. Hypoxia inducible factor 1 (HIF-1) is a major transcription factor that mediates adaptation of cancer cells to a hypoxic environment and regulates many genes that are involved in key cellular functions, including cell immortalization, stem cell maintenance, autocrine growth/survival, angiogenesis, invasion/metastasis, and resistance to chemotherapy. HIF-1α has been considered as an attractive therapeutic target for cancer treatment, but there is limited success in this research field.
In the
present study, we designed a recombinant lentivirus containing HIF-1α siRNA, developed stably transfected cell lines, and tested the anticancer effects of the siRNA on cancer cells in vitro and in vivo. Our results indicated that the stable downregulation of HIF-1α reversed chemoresistance, inhibited proliferation, migration and invasion of cancer cells, and slowed down the tumor
1
growth in breast cancer xenograft models. In conclusion, the recombinant lentivirus containing HIF-1α siRNA provides a new avenue for developing novel therapy for triple negative breast cancer. Key words: HIF-1α, triple negative breast cancer, stably transfected cell lines，recombinant
Downloaded by [] at 11:38 15 May 2015
lentivirus, siRNA therapy, gene therapy, apoptosis
2
Introduction Breast cancer is one of the most common invasive cancers in women and is responsible for at least 13.7% of cancer-related deaths in women.1,2 Although recent advances in detection and treatment of breast cancer have been made, overall survival rate of patients with advanced breast cancer remains low.3,4 Currently available treatments for late-stage breast cancer rarely result in long-term benefits. Breast cancer is particularly difficult to treat when it metastasizes and/or becomes resistant to anti-estrogen therapies.5 In particular, patients with triple negative breast cancer (TNBC) have the poorest prognosis, with the five-year survival rate being lower than 30%.6,7 Therefore, there is an urgent need for developing novel approaches to overcoming the
Downloaded by [] at 11:38 15 May 2015
limitations of current breast cancer therapies. Hypoxia is a characteristic feature of most solid tumors and related to poor response to treatment. In response to hypoxia, cancer cells undergo a variety of adoptive changes, including activation of signaling involve in cancer cells survival, proliferation, apoptosis, invasion, chemoresistance, and angiogenesis.8,
9
HIF-1 is one of the major transcription factors that
mediate this adaptive process of cancer cells to the hypoxic environment. HIF-1 is a heterodimer composed of the rate limiting factor HIF1α and the constitutively expressed HIF-1β;10 the latter is also called the aryl hydrocarbon receptor nuclear translocator and heterodimerises with several other factors, such as the Ahr transcription factor.11 HIF-1 controls the expression of almost 70 genes involved in cell proliferation, senescence, apoptosis, differentiation, angiogenesis, glucose metabolism and other cellular functions.12 For instance, VEGF is a typical downstream gene of HIF-1. It binds to its receptor VEGFR2 and is a major regulator of angiogenesis, which induces migration and proliferation of vascular vessel endothelial cells, promoting the increase in vasopermeability and angiogenesis and ameliorates wound blood supply.13 HIF-1α is induced by hypoxia, and also by oncogenes, such as HER-2/neu, v-src, and ras.14 In addition to oxygen-independent mechanisms, the PI3K, AKT and MAPK pathways mediate primarily non-hypoxic HIF regulation under normoxic environment. Consequently, HIFs and other pathways (PI3K/AKT and mTOR/p70S6K1) that lead to normoxic HIF activation are considered
3
potential therapeutic targets.15 Despite mounting evidence implicates HIF-1α plays a major role in carcinogenesis and cancer development and progression, it has been regarded as “undruggable”. 16,17 A number of gene-therapy approaches have been developed for various cancers.18-20 HIF-1α is viewed as a viable prospective target for novel pharmacologic approaches to the clinical management of solid tumors as it is the major regulator of the hypoxic adaptive response.21,22 Multiple cellular pathways resulting in HIF activation could be successfully inhibited by use of different drugs (e.g., topotecan and inhibitors of heat shock protein 90 and mTOR).23 In the present study, we designed a recombinant lentivirus containing HIF-1α siRNA and obtained
Downloaded by [] at 11:38 15 May 2015
stably lentivirus-transfected cell lines and then tested its therapeutic effects on cancer cells under normoxic conditions. We found that downregulation of HIF-1α reversed chemoresistance and inhibited capability of proliferation, migration and invasion of breast cancer cells in vitro. Furthermore, the recombinant lentivirus significantly inhibited the growth of breast cancer xenografts in nude mice. The results suggest that the recombinant lentivirus targeting HIF-1α could be a new approach to breast cancer treatment. Results IF-1α is stably knockdown via lentivirus carrying a HIF-1α shRNA We observed the expression of GFP which was contained in lentivirus under an inverted fluorescence microscope. There were no significant changes in cell morphology between different groups (Figure 1A). We then demonstrated that the expression of HIF-1α decreased dramatically at both the mRNA and protein levels (Figures 1B). The inhibition rate reached 95% at protein level (Figures 1C). Downregulation of HIF-1α in MDA-MB-231 inhibits cell growth, migration and invasion capacity in vitro We next investigated the effects of downregulated HIF-1α expression by lentivirus on the cell proliferation, migration, and invasion of MDA-MB-231 cells. As shown in Figure 1D, inhibition 4
of proliferation was observed in cells treated with HIF-1α shRNA. We next compared migratory ability and invasive potential among the cells treated with HIF-1α shRNA or NC shRNA and untreated MDA-MB-231. The number of cells crossing through the Transwell with Matrigel was significantly reduced in the HIF-1α shRNA-treated group (Figure 2A). The number of cells crossing through in NC shRNA treated and untreated MDA-MB-231 cells was 1.63-fold and 1.76-fold higher than that of the cells treated with HIF-1α shRNA, respectively (Figure 2B). In addition, compared with the NC shRNA treated cells, the HIF-1α shRNA treated cells showed 1.77- and 1.75-fold decreases in the number of cells migrating across the wound at 24 and 48 h, respectively (Fig. 2C) and the same figures were , 2.05- and 1.84-fold decreases, compared with
Downloaded by [] at 11:38 15 May 2015
that of MDA-MB-231, respectively (Fig. 2D). Downregulation of HIF-1α promotes apoptosis and induces cell cycle arrest in G2/M phase Both early and late apoptotic cell counts were increased in HIF-1α shRNA treated cells; the percentages of apoptotic cells were 5.87±0.91% in untreated MDA-MB-231 cells, 6.5±1.23% in NC shRNA treated cells, and 10.93±1.12% in HIF-1α shRNA treated cells, respectively (Figure 3). The number of cells in G2/M phase in HIF-1α shRNA cells were significantly increased, compared with the NC shRNA-treated cells (p=0.002) and untreated MDA-MB-231 (p
