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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Int J Cancer. Author manuscript; available in PMC Aug 15, 2011.
Published in final edited form as:
PMCID: PMC3150590
NIHMSID: NIHMS185384

A novel role of gap junction connexin46 protein to protect breast tumors from hypoxia

Abstract

Connexin proteins are the principle structural components of the gap junctions. Colocalization and tissue-specific expression of diverse connexin molecules are reported to occur in a variety of organs. Impairment of gap junctional intercellular communication, caused by mutations, gain of function, or loss of function of connexins, is involved in a number of diseases including the development of cancer. Here we show that human breast cancer cells, MCF-7, and breast tumor tissues express a novel gap junction protein, connexin 46 (Cx46) and it plays a critical role in hypoxia. Previous studies have shown that connexin46 is predominantly expressed in lens and our studies find that Cx46 protects human lens epithelial cells (HLEC) from hypoxia induced death. Interestingly, we find that Cx46 is upregulated in MCF-7 breast cancer cells and human breast cancer tumors. Downregulation of Cx46 by siRNA promotes 40% MCF-7 cell death at 24 hour under hypoxic conditions. Furthermore, direct injection of anti-Cx46 siRNA into xenograft tumors prevents tumor growth in nude mice. This finding will provide an exciting new direction for drug development for breast cancer treatment and suggests that both normal hypoxic tissue (lens) and adaptive hypoxic tissue (breast tumor) utilize the same protein, Cx46, as a protective strategy from hypoxia.

Keywords: Coonexin46, hypoxia, Breast Cancer, Cell Viability, MCF-7 cells

Introduction

Gap junctions are intercellular membrane channels that maintain direct intercellular communication through the exchange of ions, small molecules and cellular metabolites between neighboring cells. Gap junction channels are formed through the hexameric oligomerization of transmembrane proteins, called connexins1. Over the years, several studies have shown that the gap junctions are involved in the regulation of important cellular processes including cellular homeostasis, cell growth and differentiation, morphogenesis and cell death2.

For more than forty years the loss of gap junction communication has been shown to be an important step in the development of carcinoma3. There is also increasing evidence that loss of connexin protein expression or dysfunction of gap junction intercellular communication (GJIC) is associated with breast cancer malignant progression46. The connexin, well documented for tumor-suppressive functions in breast carcinoma, is connexin 43 (Cx43)711. This connexin is required for differentiation and proper development of normal mammary gland. Cx43 is not expressed in normal breast stem cells but is expressed in the normal breast epithelial cells derived from these breast stem cells1214. Studies have shown that Cx43 was downregulated at the mRNA and protein level in human breast tumors and several human mammary tumor cell lines7. Re-expression of Cx43 reverses the malignancy of human mammary carcinoma cells MDA-MB-4359. Similarly, overexpression of Cx43 in MDA-MB-231 cells inhibits the tumor growth in mice10. Cx43 is also considered to be involved in the inhibition of angiogenesis in breast cancer. For instance, downregulation of Cx43 by siRNA in breast cancer Hs578t cells results in the reduced expression of angiogenesis inhibitor thrombospondin-1 (TSP-1) and aggressive cell phenotype11.

Another important feature in breast carcinoma is adaptation to hypoxia that favors tumor growth and survival. Several studies have shown that the inside oxygen levels, in a solid breast tumor, can go down as low as 1%15, 16. Hypoxia which arises due to lack of formation of new blood vessels in a developing tumor mass induces hypoxia-inducible factor-1α (HIF-1α)17. HIF-1α is a key transcription factor that activates gene expression of pro-angiogenic factor vascular endothelial growth factor (VEGF)18. In solid tumors, HIF-1α also induces expression of other growth factor such as transforming growth factor –β and platelet-derived growth factor to promote cell proliferation1921. Hypoxia also regulates the gene expression of several other signaling pathways including apoptosis and glycolysis to enhance tumor progression and metastasis2225.

There is another tissue that is naturally hypoxic, vertebrate lens. The oxygen concentration in the cortical region of lens is below 5% and around (or below) 1% in the nucleus2629. Lens requires hypoxia to prevent opacification and maintain optimum transparency3032. Lens tissue endogenously express three connexin isoforms Cx43 (α1), Cx50 (α8) and Cx46 (α3)3336. Connexin46 (Cx46, Mw ~ 46kD) is preferentially expressed in lens fiber cells, inside the even more hypoxic center of the lens37. Cx46 is believed to be essential for maintaining proper Ca2+ homeostasis in fiber cells to prevent cataract formation. Cx46 knockout mice show a marked increase in nuclear cataract 38 with increased Ca2+.

In this study, we investigated a novel role of Cx46 protein to protect cells from hypoxia-induced death. We found Cx46 protein is highly expressed in MCF-7 breast cancer cells and human breast tumors. We hypothesized that the presence of Cx46 protein aids breast tumors growth, and also lens under hypoxia. Downregulation of Cx46 in hypoxia-resistant human lens epithelial cells (HLEC) and MCF-7 breast cancer cells confer susceptibility to hypoxia. In contrast, overexpression of Cx46 in hypoxia-sensitive neuronal N2A cells endows resistance against hypoxia. Further studies demonstrated that knockdown of Cx46 in vivo inhibited human breast tumor xenograft growth in nude mice. Thus Cx46, unlike other connexins, may play a significant role in the growth and development of breast tumors.

Materials and Methods

Cell Culture and transfection

Rabbit lens epithelial NN1003A cells and human lens epithelial cells (HLEC) and murine neuronal N2A cells were cultured as described previously39. MCF-7 cells were grown as recommended by American Type Culture Collection (ATCC).

To generate a stable line of neuronal N2A cells expressing Cx46 or Cx43, the cDNA of rat Cx46 or rat Cx43 was cloned in pEGFP-N3 vector (BD Biosciences, MD) and transfection was carried out using Lipofectamine 2000 (Invitrogen, CA). The selection of transfected cells was done by growing cells in the presence of G418 antibiotic (500ug/ml) for six weeks.

Antibodies

Rabbit polyclonal anti-Cx46 was purchased from USBiological, MA. Anti-Cx50, anti-Cx26 and mouse anti-α-tubulin was purchased from Zymed-Invitrogen, CA. Anti-β-actin was purchased from Sigma, MO.

Whole cell lysate preparation and Western Blot

NN1003A, HLEC, MCF-7 or MEC cell lysates were prepared as previously described39. Western blot was performed as previously described39.

Reverse Transcriptase (RT)-PCR

Total RNA was purified from MEC, MCF-7 and HLEC cells using RNeasy Mini Kit (Qiagen, CA) and RT-PCR was performed using one step RT-PCR kit (Qiagen, CA) according to the instruction manual with Cx46 specific primers and β-actin primers as control. The primers for Cx46 cDNA were 5’-CTG GCC CTG CTG GCC TTG-3’ and 5’-CCA CCA CCT GCT GAT GAC-3’. The primers for β-actin were 5’-GAA ATC GTG CGT GAC ATT AAG-3’ and 5’-CTA GAA GCA TTT GCG GTG GAC GAT-3’

Immunohistochemical study

Immunohistochemsitry was performed on paraffin-embedded human breast tumor tissue (Histologic type: Infiltrating ductal carcinoma; Histologic grade: Nottingham grade 3; Tumor type: PT2, PN0, PMO with undetected lymphovascular invasion) by following standard protocol for 3, 3 diaminobenzidine (DAB) and using rabbit anti-Cx46 antibody (USBiological, MA).

Hypoxia

Hypoxia conditions were considered as 1% O2, 5% CO2 at 37°C and 100% relative humidity. Hypoxia conditions were created by a Proox C21 hypoxic chamber (BioSpherix, NY) using nitrogen and CO2 as displacement gases. Normoxia conditions were considered as 21% O2, 5% CO2 at 37°C and 100% relative humidity. For hypoxia studies, 6 ×105 rabbit NN1003A cells were pre-incubated with DMEM low glucose media (supplemented with 10% FBS) at 21% O2, 5% CO2 (normoxic conditions) for 12 hr. Following this incubation, the media was replaced with DMEM low glucose complete media that was pre-equilibrated to 1% O2. Then the cells were incubated under hypoxic conditions (1% O2, 5% CO2) in Proox C21 hypoxic chamber (BioSpherix, NY) and harvested after 1–7 days. Whole cell lysates were run on 8% SDS-PAGE followed by western blot to check the expression levels of different proteins.

siRNA transfection

Anti-Cx46 siRNA (Target sequence: CGC ATG GAA GAG AAG AAG AAA) and negative non-silencing control siRNA were purchased from Qiagen, CA. HLEC or MCF-7 cells were cultured in their respective medium under normoxic conditions (5% CO2, 21% O2) on 60 mm dishes. 2 × 106 HLEC or MCF-7 cells were transfected with 512 ng of Cx46 siRNA (Final conc.10nM) or 512 ng negative control siRNA and 20 uL of HiPerFect transfectant reagent (Qiagen, CA). After transfection, cells were incubated under normoxic conditions (21% O2, 5% CO2) and harvested at different time intervals (24 hr and 48 hr for HLEC and 12 hr, 24 hr and 48 hr, for MCF-7 cells). Whole cell lysates were analyzed by western blot.

Cell viability assay

Cell viability assay was performed using CellTiter-Blue® Cell Viability Assay kit (Promega, WI). The kit applies the fluorometric detection of resorufin converted from resazurin40 by viable cells. The amount of fluorescence measured is directly proportional to the number of viable cells. For this assay, 3 × 104 HLEC or MCF-7 cells, in 100 uL of DMEM low glucose media or MEM media respectively, were seeded into each well in 96 well micro-titer plates and cultured for 14 hr at 37°C under normoxic conditions (21% O2, 5% CO2). The cells were then transfected with 10ng (per well, final conc.10nM) of Cx46 siRNA or 10 ng negative non-silencing siRNA along with 0.75ul (per well) of HiPerFect transfectant reagent and incubated for 24 hr under normoxic conditions (21% O2, 5% CO2). Cells with no siRNA treatment were considered as controls. Following this incubation, the media for HLEC or MCF-7 cells were changed with 100uL (per well) of their respective media that was equilibrated to 1% O2. Then the cells were incubated under hypoxic conditions (21% O2, 5% CO2) in Proox C21 hypoxic chamber (BioSpherix, CA) for different time intervals depending on cell type (6 hr, 12 hr, 18 hr and 36 hr for HLEC and 12 hr and 24 hr for MCF-7 cells). Another set of 96-well plates of HLEC or MCF-7 cells with similar siRNA treatment containing 100uL media but equilibrated to 21% O2 were incubated under normoxic conditions (21% O2, 5% CO2) for the same interval of time. For the estimation of number of viable cells, 20 uL of Cell Titer-Blue reagent was added in each well for MCF-7 or HLEC cells incubated under hypoxic or normoxic conditions and following incubation, for 4 hr for HLEC or 3 hr for MCF-7 cells, at 37°C, fluorescence was recorded at 560(5)Ex/ 590(5)Em.

To determine the cell viability of wild type N2A cells and N2A cells overexpressing Cx46-GFP or Cx43 GFP under hypoxic or normoxic conditions, 3 × 104 cells (per well of 96 well micro-titer plate) were cultured in 100uL DMEM low glucose media for 14 h at 37°C under normoxic conditions (21% O2, 5% CO2). Then the media was replaced with 100ul (per well) DMEM low glucose media equilibrated to 1% O2 or 21% O2. The cells were then incubated under hypoxic or normoxic conditions and cell viability was assessed every 4 hr for up to 24 hr. To measure the number of viable cells 20 uL of Cell Titer-Blue reagent was added in each well, incubated for 4 hr at 37°C and fluorescence was recorded at 560(5)Ex/ 590(5)Em.

Human breast tumor and normal tissues

The human breast tissue lysates were purchased from Protein Biotechnologies, CA. The characterizations of human breast tumors tissues are as follows; Tumor breast tissue 1: Infiltrating Ductal Carcinoma, grade 2, stage IIA. T2N0M0, source: female, 42 years; Tumor breast tissue 2: Invasive Ductal Carcinoma, grade 2, stage IIA. T2N0M0, source: female, 42 years. The Human adult normal tissue lysate was purchased from Novus Biologicals, CO. 20 ug of total protein of each lysate was loaded and run in 8% agarose gel. The blot was probed with rabbit polyclonal anti-Cx46 antibody (USBiological, MA) and developed as described previously39.

Xenograft tumors of MCF-7 cells in Nu/Nu Mice

Nu/Nu mice (strain NuFoxn1) were purchased from Charles River Laboratory. Mice were implanted with 17 β-estradiol (1.7 mg/pellet) one week before the injection of 1 × 107 MCF-7 breast cancer cells subcutaneously into the inguinal region of mammary fat pad. Cell viability of MCF-7 cells was performed prior to the injection. Mice were observed for any change in behavior, appearance or weight. Two weeks after MCF-7 cell injection, tumors were injected with 7.5 ug of anti-Cx46 siRNA or negative non-silencing siRNA or no siRNA (control) every 48 h for minimum of 10 days to maximum of 18 days. Depending on the size of each tumor the siRNAs were injected directly at two or three different locations. The tumor size was measured in two dimensions by a caliper every other day before injection. The tumor volume was estimated by the formula: tumor volume = a (b2)/2, where a and b is the tumor length and width respectively41 in mm. The tumor size measured prior to first siRNA injection is considered day 0 measurement. After 10 days of first injection, two anti-Cx46 siRNA treated, one negative non-silencing siRNA treated and one control (no siRNA) tumors were dissected out. The size of two anti-Cx46 siRNA treated tumors became almost undetectable at day 12. Three anti-Cx46 siRNA treated tumors were dissected out at day 16. The rest of the anti-Cx46 siRNA treated tumors were isolated from euthanized mice at day 18. Tumors were dissected out, homogenized and lysed in RIPA buffer. The lysates were sonicated for 10 sec for three times. Whole tumor tissue lysates were quantitated by Bio-Rad Protein Assay and analyzed by western blot.

Statistical analysis

The level of significance (see * in figure legends) was considered at P < 0.01 or P <0.001 using paired-t test analyses. All data are presented as mean ± s.e.m. of at least three independent experiments.

Results

Cx46 protects human lens epithelial cells (HLEC) form hypoxia-induced death

To determine the role of Cx46 in breast tumors, we first established the role of Cx46 in hypoxia in lens cells. We found that the Cx46 protein level was elevated during 4–7 days of hypoxia in rabbit NN1003A lens epithelial cells (Fig. 1a). However, normoxia treatment (21% O2, 5% CO2) had no effect on Cx46 protein level (Fig. S1a). Since Cx46 protein was responsive to hypoxia, we speculated whether lens epithelial cells are hypoxia resistant due to Cx46 protein. We downregulated the Cx46 protein in human lens epithelial cells (HLEC) using siRNA (Fig. 1b) and investigated the effect of Cx46 downregulation on cell viability under hypoxic conditions (1% O2, 5% CO2). About 85% selective knockdown of Cx46 protein expression was achieved in HLEC after 24 hr and 48 hr of anti-Cx46 siRNA transfection (Fig. 1c). HLEC with Cx46 knocked-down showed significant reduction in cell viability under hypoxic conditions (Fig. 1d). Anti-Cx46 siRNA treated HLEC viability was reduced to 23% at 6 hr, 38% at 12 hr and 42% at 18 hr compared to untreated (control) or non-silencing siRNA treated cells (Fig. 1d) under hypoxia. Interestingly, Cx46 downregulation had no effect on HLEC viability under normoxic conditions (Fig. S1b) which suggested that Cx46 provides protections to lens cells only against hypoxia-induced death.

Figure 1
Cx46 downregulated Human lens epithelial cells (HLEC) are susceptible to hypoxia induced death. (a) The upregulation of Cx46 in response to 1% O2 in rabbit lens epithelial NN 1003A cells. Cells were subjected to hypoxia or normoxia and harvested at 1–7 ...

Overexpression of Cx46 increases the cell viability of hypoxia-susceptible neuronal N2A cells in hypoxic conditions

To further establish the role of connexin46, Cx46 and Cx43 were stably overexpressed as a GFP tagged fusion protein in hypoxia-sensitive murine neuronal N2A cells and cell viability was assessed under hypoxic conditions. N2A cells do not express endogenous Cx46 or Cx43 proteins (Fig. S2a). N2A cells transfected with Cx43-GFP or Cx46-GFP expressed fusion proteins of predicted molecular weight of ~71kDa and 73kDa, respectively (Fig. 2a) as determined by western blot using anti-GFP antibody. These N2A cells overexpressing Cx46-GFP or Cx43-GFP were incubated under hypoxic conditions (1% O2, 5% CO2) or at normoxic conditions (21% O2, 5% CO2) and cell viability was assessed by fluorometric resazurin reduction method34. Wild type N2A cells were hypoxia sensitive as they began to die after 4 hr under hypoxic conditions and cell viability was reduced by 40% at 12 h and almost by 90% at 24 hr (Fig. 2b). The cell viability of N2A cells overexpressing Cx43-GFP showed the same pattern as wild type N2A cells with cell viability reducing significantly during 4–24 hr time period at 1% O2 (Fig. 2b). But interestingly, N2A cells overexpressing Cx46-GFP remained viable to a considerable extent even after 12 h at 1% O2. The cell viability of these cells was only reduced by 3% at 12 h and 51% at 24 h at 1% O2. No reduction in cell viability was observed for wild type, Cx46-GFP or Cx43-GFP tagged stable cell lines under normoxic conditions (Fig. S2b). Taken together, the results suggested that Cx46 can confer protection to a hypoxia sensitive cell while another connexin, Cx43, cannot.

Figure 2
Overexpression of Cx46 in hypoxia sensitive neuronal N2A cells confers survival in hypoxia (1% O2). (a) Western blot analyses of Cx46-GFP and Cx43-GFP proteins overexpressed in neuronal N2A cells. The blot was probed with antibody against GFP. Stable ...

Cx46 expression in breast cancer cells and breast tumor tissues

Tissues, other than lens, reported to express Cx46 protein are lung adenoma and lung alveolar42, 43, schwann cells44 and bone osteoblastic cells45. Here we showed, for the first time, that human breast cancer cells and human breast tumor tissue express Cx46 protein. Expression of Cx46 protein was detected in MCF-7 human breast cancer cells as determined by western blot analyses, however, in normal human mammary epithelial (HMEC) the expression was not seen (Fig. 3a). The expression of Cx46 was further confirmed by RT-PCR which showed the presence of Cx46 mRNA in MCF-7 cells but not in HMEC cells (Fig. 3b). Next, we investigated the expression of Cx46 protein in human breast tumor tissue. Indeed, Cx46 protein was present in pre-metastatic breast tumor as determined by immunohistochemistry (Fig. 3c). In addition, immunoblot analyses showed that the Cx46 protein was upregulated in tumor tissues as compared to normal breast tissue (Fig. 3c).

Figure 3
Cx46 is expressed in human breast cancer cells and human breast tumors. Breast cancer cell MCF-7 express Cx46 protein as determined by western blot (a) and RT-PCR study (b). HMEC: Human mammary epithelial cells, (c) Immunohistochemistry showing the expression ...

Downregulation of Cx46 in MCF-7 cells increases hypoxia-induced cell death

The presence of Cx46 in MCF-7 cells and absence in HMEC cells led us to hypothesize that breast cancer cells and breast tumors also use Cx46, as an adaptation to hypoxia, to survive and grow. To confirm this, Cx46 protein was downregulated in MCF-7 cells using anti-Cx46 siRNA (Fig. 3a) to more than 60% and 80% (Fig. 3c) after 24 hr and 48 hr of transfection respectively. Downregulation of Cx46 remarkably reduced the MCF-7 cell viability to approximately 34% at 12 hr and 40% at 24 hr under hypoxic conditions (1% O2, 5% CO2) as compared to control (no siRNA) or negative non-silencing siRNA treated cells (Fig. 3d). As seen in the case for HLEC cells, the downregulation of Cx46 had no effect on MCF-7 cell viability under normoxia (Fig. S3). These data clearly demonstrated that human breast cancer cells MCF-7 also utilize Cx46 to survive against death caused by hypoxia.

Downregulation of Cx46 shows reduced growth of MCF-7 tumor xenografts in nude mice

To further investigate the function of Cx46 as a hypoxia survival factor we carried forward our work to an in vivo human tumor-bearing xenograft nude mouse system. We asked whether Cx46 plays a similar and significant hypoxia-protective role in the growth of human breast tumors in vivo. Estrogen stimulated immunodeficient Nu/Nu mice were injected with 1 × 107 MCF-7 cells into the inguinal region of mammary fat pad to develop tumors of human origin. Two weeks after MCF-7 cells injection, newly formed tumors were directly injected with 7.5 ug of anti-Cx46 siRNA (n=10 tumors) or negative non-silencing siRNA (n=7 tumors) or no siRNA (control, n=7 tumors) every 48 h for minimum of 10 to maximum of 18 days. Tumor size was measured every alternate day prior to siRNA injection and hence the tumor size measured prior to first injection was considered as day 0 (measurement). We found that after 10 days of siRNA injection (total of five siRNA injections with injection every 48 h), all the tumors with no siRNA (n= 7 tumors) or non-silencing (n=6 tumors) siRNA treatment increased in size with time, with average increase about 426% and 482%, respectively when compared to day 0 (prior to first injection)(Fig. 5a). No notable reduction in size of control (no siRNA) or non-silencing siRNA injected tumors was observed even after 18 days (Table S1, S3). However, anti-Cx46 siRNA treatment inhibited the tumor growth significantly with average increase of only about 30% (compared to day 0) after 10 days of injection (Fig. 5a–c). Any noteworthy increase in size of anti-Cx46 siRNA treated tumors was not noticed from day 10 to day 18 (Table S2).

Figure 5
siRNA mediated downregulation of Cx46 in mouse tumor xenografts inhibits the growth of breast cancer tumors. (a) Mice were implanted with 17β-estradiol (1.7 mg/pellet) followed by the injection of 1 × 107 MCF-7 cells subcutaneously into ...

To confirm that the inhibition of anti-Cx46 siRNA treated tumor growth is due to Cx46 protein downregulation, we analyzed the tumors by western blot. Effective knockdown of Cx46 protein was only seen in tumors injected with anti-Cx46 siRNA (Fig. 5d). Out of ten anti-Cx46 siRNA injected tumors, six tumors (tumors 16 and 17 at day 10, tumor 10 at day 16, tumors 11, 14 and 15 at day 18) presented reduced level of Cx46 protein (Fig. 5d, S4), two tumors (tumors 8 and 13 at day 16) showed no reduction in Cx46 protein expression (Fig. S4) and two tumors (tumors 9 and 12 see Table S2) became too small to be analyzed by western blot. We also measured Cx26 protein level in all tumors to investigate any nonspecific effect of the siRNA but no change of Cx26 protein was observed. This suggested that decreased tumor growth in xenograft mice resulted due to selective knockdown of Cx46 by anti-Cx46 siRNA.

Discussion

In the present study we demonstrated that Cx46 maintains cells to survive under hypoxia. We observed an upregulation of Cx46 protein in HLEC under hypoxic conditions at 4–7 days but no change in protein expression was found under normoxia treatment (Fig. 1a). HLEC are naturally hypoxic cells as they survive for weeks at 1% O2. But when Cx46 was downregulated (by siRNA) the HLEC viability reduced significantly even after 12 hr under hypoxia (Fig. 1d). These indicate that lens epithelial cells survive under hypoxia by upregulating Cx46 protein.

If HLEC are able to survive in hypoxia because of Cx46 protein, then overexpression of Cx46 should confer resistance to a hypoxia susceptible cell that lacks endogenous Cx46. Indeed, we observed that overexpression of Cx46 in hypoxia susceptible murine neuronal N2A cells significantly increased the cell viability under hypoxic conditions (Fig. 2b). However no such effect on N2A cell survival at 1% O2 was observed when another connexin, Cx43, was overexpressed. It suggests that Cx46 can also help cells other than in lens to survive in hypoxic condition.

To date, the gap junction connexins, Cx26, Cx32 and low level of Cx43 have been detected in breast cancer MCF-7 cells. Here, for the first time, we show that human breast cancer cells and human breast tumors also express Cx46 (Fig. 3). Both Cx46 mRNA and protein were detected in breast cancer MCF-7 cells but not in normal human mammary epithelial (HMEC) cells (Fig. 3a–b). Moreover, higher expression of Cx46 protein was observed in human breast tumor tissues than in normal breast tissues raising the possibility that Cx46 may be used as a marker in breast oncogenesis.

Our results clearly demonstrated that Cx46 functions to protect MCF-7 cells against hypoxia-induced death. MCF-7 cells with downregulated Cx46 protein showed significant decrease in cell viability at 1% O2 (Fig. 3d). However downregulation of Cx46 had no effect on cell viability under normoxia treatment (Fig. S3). Like in HLEC, Cx46 may be involved only in hypoxia-signaling pathways in breast cancer cells. The function of Cx46 in human breast tumor formation was investigated, in vivo, in human tumor xenograft-bearing nude mice. The tumor of human origin in nude mice was generated by injecting MCF-7 cells at inguinal region of mammary fat pad. Since levels of Cx46 protein was reduced maximally after 48 hours of anti-Cx46 siRNA transfection in MCF-7 cells in vitro, we injected anti-Cx46 siRNA directly into the MCF-7 xenograft tumors every 48 hours. Consistent with the result of decreased cell viability of MCF-7 cells in vitro under hypoxia, dramatic inhibition of tumor growth was also observed in MCF-7 xenograft tumors (Fig. 5), in vivo, treated with anti-Cx46 siRNA even for 18 days. Thus Cx46 appeared to play a significant role in early breast tumor development.

We achieved significant knockdown of Cx46 protein in vivo by injecting siRNA directly to the xenograft tumors in nude mice. The anti-Cx46 siRNA treated xenograft tumors that were associated with reduced tumor growth (tumors 10, 11, 14, 15, 16 and 17) presented low level of Cx46 protein (Fig 5d). Two anti-Cx46 siRNA treated tumors (tumors 8 and 13) manifested increased tumor growth and showed no significant reduction in Cx46 protein level (Fig. S4). It clearly suggests that downregulation of Cx46 protein accounted for the reduced growth of xenograft tumors.

The breast cancer tumor progression is generally believed to be associated with loss of gap junction intracellular communications (GJIC). The decreased communication of tumor cells with neighboring normal cells due to defective intercellular gap junctions has been shown to be beneficial for tumor progression. Therefore, the other gap junction connexins, Cx43, Cx26 and Cx32, expressed in breast tissue, are generally deemed tumor suppressors. Loss of Cx43 is correlated with breast tumor progression and restoration of Cx43 protein level promoted controlled growth and proliferation of cancer cells 6,9,10,46. In contrast, our studies identify a unique gap junction protein, Cx46, which is upregulated and acts as an oncogene in early breast cancer tumors.

In conclusion, our data provide evidence that the presence of Cx46 and its function as a hypoxia-critical-factor can be targeted for the treatment of breast cancer. This is the first report to utilize a natural hypoxic tissue (lens) to understand the complexity of an adaptive hypoxic tissue such as solid tumor for their survival strategies involving a novel gap junction protein. Our findings also revealed that local administration of siRNA can mediate effective knock down of this target protein with minimal nonspecific effects and prevent tumor formation. These results will have a high impact to breast cancer therapeutics in designing an entirely new line of drugs to control and potentially eliminate early breast cancers while at the early hypoxic stage.

Novelty and Impact of the work

  1. This work is first to report that breast cancer MCF-7 cells and human breast tumor tissues express Cx46 protein. The gap junction connexins are usually deemed tumor suppressors and are, therefore, often suppressed during tumor formation (example, connexin 43). However this manuscript shows that Cx46 is upregulated and aids human breast cancer cells and solid tumor tissues to survive under hypoxia, one of the survival mechanisms of solid tumor. Our findings also reveal that local administration of siRNA can mediate effective knock down of this target protein and prevent tumor formation in vivo.
  2. The findings of this manuscript will provide an exciting new direction for drug development for breast cancer treatment. In addition Cx46 could provide an early marker for hypoxia. Therefore these findings could have broader significance for other hypoxia related diseases.
Figure 4
Downregulation of Cx46 in breast cancer MCF-7 cells confers susceptibly to hypoxia induced death. (a) The siRNA mediated selective downregulation of Cx46 protein in MCF-7 cells. 2 × 106 MCF-7 cells were transfected with 512 ng anti-Cx46 siRNA ...

Supplementary Material

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Acknowledgements

This work was supported in part by funds provided by grants NIH EY13421 to DJT, NIH P20RR017686 to ATN, Terry C. Johnson Cancer Center Innovative Research Grant to DJT and Terry C. Johnson Cancer Center Summer Stipend to DB and NIH K-INBRE grant P20RR016475 to AH and DM.

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