IGF-1 regulates Cyr61 induced breast cancer cell proliferation and invasion.

BackgroundStudies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation.MethodsSeveral ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation.ResultsCells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells.ConclusionThe findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion

Vitamin D receptor gene polymorphisms and prognosis of breast cancer among African-American and Hispanic women.

BackgroundVitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). Although African-Americans have the lowest levels of serum vitamin D, there is a dearth of information on VDR gene polymorphisms and breast cancer among African-Americans and Hispanics. This study examines whether VDR gene polymorphisms are associated with breast cancer in these cohorts.MethodsBlood was collected from 232 breast cancer patients (Cases) and 349 non-cancer subjects (Controls). Genotyping for four polymorphic variants of VDR (FokI, BsmI, TaqI and ApaI) was performed using the PCR-RFLP method.ResultsAn increased association of the VDR-Fok1 f allele with breast cancer was observed in African-Americans (OR = 1.9, p = 0.07). Furthermore, the FbTA, FbtA and fbtA haplotypes were associated with breast cancer among African-Americans (p<0.05). Latinas were more likely to have the VDR-ApaI alleles (Aa or aa) (p = 0.008). The VDR-ApaI aa genotype was significantly associated with poorly-differentiated breast tumors (p = 0.04) in combined Cases. Kaplan-Meier survival analysis showed decreased 5-year disease-free-survival (DFS) in breast cancer patients who had the VDR-Fok1 FF genotype (p<0.05). The Cox regression with multivariate analysis revealed the independent predictor value of the VDR-FokI polymorphism for DFS. The other three variants of VDR (BsmI, TaqI and ApaI) were not associated with disease outcome.ConclusionsVDR haplotypes are associated with breast cancer in African-Americans, but not in Hispanic/Latinas. The VDR-FokI FF genotype is linked with poor prognosis in African-American women with breast cancer

IGF-1 regulates Cyr61 induced breast cancer cell proliferation and invasion.

BackgroundStudies from our laboratory and others have shown that cysteine-rich 61 (Cyr61) may be involved in tumor proliferation and invasion. In earlier studies, we demonstrated increased insulin-like growth factor-I (IGF-1) is associated with breast tumor formation and poor clinical outcomes. In our current study we have investigated IGF-1 regulation of Cyr61 and whether targeting IGF-1 could inhibit Cyr61 induced tumor growth and proliferation.MethodsSeveral ATCC derived normal and breast cancer cell lines were used in this study: MDA-MB231, BT474, MCF-7, and SKBR3. We also tested cells stably transfected in our laboratory with active Akt1 (pAkt; SKBR3/AA and MCF-7/AA) and dominant negative Akt1 (SKBR3/DN and MCF-7/DN). In addition, we used MCF-7 cells transfected with full length Cyr61 (CYA). Monolayer cultures treated with IGF-1 were analyzed for Cyr61 expression by RT-PCR and immunohistochemical staining. Migration assays and MTT based proliferation assays were used to determine invasive characteristics in response to IGF-1/Cyr61 activation.ResultsCells with activated Akt have increased levels of Cyr61. Conversely, cells with inactive Akt have decreased levels of Cyr61. IGF-1 treatment increased Cyr61 expression significantly and cells with high level of Cyr61 demonstrate increased invasiveness and proliferation. Cyr61 overexpression and activation led to decrease in E-cadherin and decrease in FOXO1. Inhibition of the PI3K and MAPK pathways resulted in significant decrease in invasiveness and proliferation, most notably in the PI3K pathway inhibited cells.ConclusionThe findings of this study show that IGF-1 upregulates Cyr61 primarily through activation of the Akt-PI3K pathway. IGF-1 induced MAPK plays a partial role. Increase in Cyr61 leads to increase in breast cancer cell growth and invasion. Hence, targeting Cyr61 and associated pathways may offer an opportunity to inhibit IGF-1 mediated Cyr61 induced breast cancer growth and invasion

Inverse Relationship between IGF-1 induced Cyr61 upregulation and decreased E-cadherin and FOXO1 expression is confirmed in two cell lines.

<p>The mRNA expression of Cyr61, E-cadherin, and FOXO1 were assessed by RT-Q-PCR. All mRNA levels were adjusted for the housekeeping gene, 18S. (<b>A</b>) Expression of Cyr61, E-cadherin, and FOXO1 in response to IGF-1 induction (100 ng/mL) following serum starvation in MCF-7 WT cells; and (<b>B</b>) MDAMB231 cells. P-values were assessed relative to the untreated condition within each cell line, and P<0.05 considered statistically significant.</p

Proposed Schematic of IGF-1 mediated signaling in relation to Cyr61 expression and activity.

<p>Proposed Schematic of IGF-1 mediated signaling in relation to Cyr61 expression and activity.</p

Cyr61 Levels in Various Breast Cancer Cell Lines – Baseline and in Response to IGF-1.

<p>(<b>A</b>) Baseline mRNA expression of Cyr61 in Wildtype MCF-7, SKBR3, BT474, and MDAMB231 as determined by RT-Q-PCR. (<b>B–D</b>) Cyr61 expression after IGF-1 treatment at baseline, 20 minutes, 4 hours, and 24 hours in (<b>B</b>) MCF-7 WT, (<b>C</b>) MCF-7 DN, and (<b>D</b>) MCF-7 AA cells. Cyr61 is increased by treatment of IGF-1, especially at 20 minutes. (<b>E</b>) Expression of Cyr61 mRNA in MCF-7 WT cells compared to MCF-7 AA (transfectant with constitutively active Akt), and MCF-7 DN (transfectant with dominant negative constitutively inactive Akt). (<b>F</b>) The results obtained in the MCF-7 cell line were confirmed by a second breast cancer cell line, SKBR3. All mRNA levels were adjusted for the housekeeping gene, 18S. P-values were calculated relative to MCF-7 WT in (A,E), to 0 Minutes in (B–D), and SKBR3 WT in (F). P<0.05 is statistically significant.</p

Effects of IGF-1 and PD98059 Induction on Proliferation, Invasion, and Cyr61 Expression in MCF-7 WT and MCF-7 CYA.

<p>(<b>A</b>) The mRNA expression of Cyr61 was assessed by RT-Q-PCR. All mRNA levels were adjusted for the housekeeping gene, 18S. (<b>B</b>) Proliferation assay summary data after 48 hour IGF-1 (100 ng/mL) and PD98059 (30 µM) treatment in MCF-7 WT and MCF-7 CYA. Each condition was plated into 6 identical wells in a 96 well plate. All treatments were standardized relative to the untreated condition for each cell line. (<b>C</b>) Results from Invasion assay after 40 hour IGF-1 (100 ng/mL) and PD98059 (30 µM) treatment in MCF-7 WT and MCF-7 CYA. P-values were assessed relative to the untreated condition in each assay, with P<0.05 considered statistically significant.</p

Confirmation of Cyr61 levels and Invasiveness in MCF-7 WT and MCF-7 CYA.

<p>(<b>A</b>) The mRNA expression of Cyr61 was assessed by RT-Q-PCR and (<b>B</b>) by immunofluorescent staining (FITC - green – Cyr61; DAPI – blue – nuclei). (<b>C</b>) Quantification of invasiveness from invasion chamber. (<b>D</b>) Red arrows demonstrate positive examples from invasion chamber. CYA cells are 9 times more invasive than the MCF-7 WT cells. All mRNA levels were adjusted for the housekeeping gene, 18S. P-values were calculated relative to MCF-7 and P<0.05 is statistically significant.</p

Effects of IGF-1 and LY294002 Induction on Proliferation, Invasion, and Cyr61 Expression in MCF-7 WT and CYA.

<p>(<b>A</b>) The mRNA expression of Cyr61 was assessed by RT-Q-PCR. All mRNA levels were adjusted for the housekeeping gene, 18S. (<b>B</b>) Results from proliferation assay after 48 hour IGF-1 (100 ng/mL) and LY294002 (50 µM) treatment in MCF-7 WT and MCF-7 CYA. Each condition was plated into 6 identical wells in a 96 well plate. All treatments were standardized relative to the untreated condition for each cell line. (<b>C</b>) Results from Invasion assay after 40 hour IGF-1 (100 ng/mL) and LY294002 (50 µM) treatment in MCF-7 WT and MCF-7 CYA. P-values were assessed relative to the untreated condition in each assay, with P<0.05 considered statistically significant.</p

Vitamin D receptor gene polymorphisms and prognosis of breast cancer among African-American and Hispanic women.

Vitamin D plays a role in cancer development and acts through the vitamin D receptor (VDR). Although African-Americans have the lowest levels of serum vitamin D, there is a dearth of information on VDR gene polymorphisms and breast cancer among African-Americans and Hispanics. This study examines whether VDR gene polymorphisms are associated with breast cancer in these cohorts.Blood was collected from 232 breast cancer patients (Cases) and 349 non-cancer subjects (Controls). Genotyping for four polymorphic variants of VDR (FokI, BsmI, TaqI and ApaI) was performed using the PCR-RFLP method.An increased association of the VDR-Fok1 f allele with breast cancer was observed in African-Americans (OR = 1.9, p = 0.07). Furthermore, the FbTA, FbtA and fbtA haplotypes were associated with breast cancer among African-Americans (p<0.05). Latinas were more likely to have the VDR-ApaI alleles (Aa or aa) (p = 0.008). The VDR-ApaI aa genotype was significantly associated with poorly-differentiated breast tumors (p = 0.04) in combined Cases. Kaplan-Meier survival analysis showed decreased 5-year disease-free-survival (DFS) in breast cancer patients who had the VDR-Fok1 FF genotype (p<0.05). The Cox regression with multivariate analysis revealed the independent predictor value of the VDR-FokI polymorphism for DFS. The other three variants of VDR (BsmI, TaqI and ApaI) were not associated with disease outcome.VDR haplotypes are associated with breast cancer in African-Americans, but not in Hispanic/Latinas. The VDR-FokI FF genotype is linked with poor prognosis in African-American women with breast cancer