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Incidence of breast cancer among women is highest in North America and Europe
Progression of breast cancer includes proliferation, invasion, migration, angiogenesis, and metastasis of primary tumor
Chemokines and their functional receptors such as CXCR4 and CXCR7 that are embedded on the tumor cell surface have been implicated in promoting the progression of breast cancer
For more than a decade, studies have shown that CXCR4 is involved in inducing directional metastasis of the CXCR4-rich tumor cells by mobilizing them to organs where SDF-1 (Stromal-cell Derived Factor-1), a ligand of CXCR4, is abundant
Targeting CXCR4 with its antagonist or siRNA has been shown to effectively decrease the cancer progression
SDF-1 was thought to be the only ligand of CXCR4 until its another receptor, CXCR7, was recently discovered
The role of CXCR7 has been a subject of debate, as some studies suggest that CXCR7 promotes CXCR4 activity, while other studies suggest that CXCR7 interferes with CXCR4 activity
Both CXCR4 and CXCR7 are over-expressed in many cases of breast cancer, and they may play a synergistic but different role in the cancer progression
We hypothesized that both CXCR4 and CXCR7 promote the progression of breast cancer, and our goal is to synergistically inhibit the progression of the cancer by targeting both receptors
Figure 1. After binding of SDF-1, CXCR4, first studied in context of T cell motility, induces cell motility by activating heterotrimeric G protein.
Figure 2. A, pcDNA3.1+ vector diagram. B, p-BABE-puro vector diagram
Cloning (Figure 2):
CXCR4 sequence and CXCR7 sequence were separately ligated to pcDNA3.1+ plasmid vectors (5’ restriction enzyme: EcoRI and 3’ restriction enzyme: XhoI). Transfection of CXCR4-ligated vector and/or CXCR7-ligated vector to MDA-MB-435 cells was completed to produce MDA-MB-435 sub-clone cell lines expressing CXCR4 and/or CXCR7. p-BABE-puro vector was also co-transfected for clone selection.
Cell Culture:
MDA-MB-231, MDA-MB-435, MDA-MB-435-CXCR4,
MDA-MB-435-CXCR7, and MDA-MB-435-CXCR4/CXCR7 cell lines were cultured in RPMI 1640 medium with 10% FBS and 1% P/S (Incubation condition: 37°C, 5% CO2).
Immunofluorescence:
Each cell line was treated with CXCR4 pAb and CXCR7 mAb. After 1 hour of incubation in room temperature, biotinylated secondary antibodies were used for detection.
RT-PCR and Western Blot:
Trizol was used for RNA isolation and protein isolation. Two-step RT-PCR procedure was used. Gel electrophoresis analysis and SDS-PAGE analysis were compared to each other.
Cell Proliferation Assay:
Cells were seeded in clear 96-well plate. After 24 hours of incubation, a group of cells were treated with 200 ng/mL of SDF-1β. Also, another group of cells were treated with 100 nM of TN 14003, a polypeptide against CXCR4, and/or 200 ng/mL of CXCR7 mAb. After 48 hours of incubation, AQ96 solution was used for detecting absorbance of each well at 490 nm.
Matrigel Invasion Assay:
Matrigel was put into inserts of 24-transwell plate. After 2-hour incubation in room temperature, cells treated with TN 14003 and/or CXCR7 mAb were seeded in the inserts (same concentrations as above). 200-ng/mL SDF-1β was added to each well to induce invasion. After 22 hours, the number of invaded cells were
Figure 3. Specificity of the expression of CXCR4 and/or CXCR7 in five prepared cell lines. A, immunofluorescence staining of CXCR4 and/or CXCR7. B, RT-PCR and Western blot results show different expression levels of CXCR4 and/or CXCR7 from MDA-MB-231, MDA-MB435, and MDA-MB-435 sub-clone cell lines.
Figure 4. Neither CXCR4 nor CXCR7 directly induced cell proliferation in MDA-MB-231 and MDA-MB-435 cell lines. A, SDF-1 did not induce cell proliferation in either cell line. B, targeting CXCR4 and/or CXCR7 by TN 14003 and/or CXCR7 mAb was not cytotoxic to either cell line
Figure 5. Matrigel invasion assay results of five prepared cell lines (cell concentration = 1 x 105/mL). Blocking CXCR4 was effective in MDA-MB-231 cells (A) and MDA-MB-435C4 cells (B). C, invasion assay of SDF-1 positive and negative controls in the five cell lines shows that cells expressing high levels of CXCR7 invade Matrigel efficiently with or without SDF-1, in which the cells’ invasiveness depends on the CXCR4 expression level in a dose-dependent manner.
We hypothesized that both CXCR4 and CXCR7 We hypothesized that both CXCR4 and CXCR7 promote progression of breast cancer. However, little is known about how the two receptors interact with each other. Interestingly, our results indicate that CXCR7 induces CXCR4-expressing cells to be invasive efficiently with or without binding of CXCR4 to SDF-1. This phenomenon complies with a recent study by Levoye et al. suggesting that CXCR7 hetero-dimerizes with CXCR4 to regulate the interaction of CXCR4 and Gαi protein. Signaling through Gαi protein is an essential step for a cell to attain its invasive characteristics, and therefore a molecular mechanism of how CXCR7 induces a change in Gαi signaling, originally regulated through binding of CXCR4 to SDF-1, is an important investigation.
In our cell proliferation assay results, the data show that SDF-1 does not directly trigger increased cell division rate, which may be possible through a specific signal pathway that promotes cellular mitosis. However, many studies suggested that SDF-1 promotes the proliferation of cells expressing CXCR4. This discrepancy may be explainable through that SDF-1 rich environment allows cells to have access to more nutrition and that cells may proliferate better due to their environmental advantages.
In summary, our hypothesis is supported in that CXCR7 assists CXCR4 in abating SDF-1 dependency in cell invasion and migration. Our results specifies one way in which CXCR7 behaves differently from CXCR4
Future Plans
Proliferation Assay with Modifications
Repeating Matrigel Invasion Assay
Wound-healing Migration Assay
Angiogenesis Assay
Soft-agar Colony Formation Assay
Utilizing CXCR7 siRNA or CXCR7 miRNA
In vivo Experiment
This material is based upon work supported by the Howard Hughes Medical Institute under Grant No. 52005873
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