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TGF-β2: A Novel Target of CD44-Promoted Breast Cancer Invasion.

Ouhtit A, Madani S, Gupta I, Shanmuganathan S, Abdraboh ME, Al-Riyami H, Al-Farsi YM, Raj MH - J Cancer (2013)

Bottom Line: Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tetracycline at 18, 24, and 48 h post-HA stimulation compared to the parental cells.More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation, and subsequently inhibited BC cell invasion in vitro.In addition to identifying TGF-β2 as a target for HA/CD44 signaling, this data suggests that ATF/CREB might be a potential transcription factor linking HA/CD44 activation to TGF-β2 transcription and additional experiments are required for a better understanding of the molecular mechanisms underpinning the novel function of the CD44/ TGF-β2 signaling pathway in breast cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Genetics.

ABSTRACT
We have developed a tetracycline (tet)-off regulated expression of CD44s gene in the breast cancer (BC) cell line MCF-7 (B5 clone) and identified TGF-β2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44-downstream transcriptional target by microarray analysis. To further validate this finding, the same RNA samples, used for microarray analysis and their corresponding protein lysates, collected from the BC cell line MCF-7-B5, were examined for CD44 expression in the presence of HA. Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tetracycline at 18, 24, and 48 h post-HA stimulation compared to the parental cells. Furthermore, the TGF-β2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tetracycline. More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation, and subsequently inhibited BC cell invasion in vitro. In addition to identifying TGF-β2 as a target for HA/CD44 signaling, this data suggests that ATF/CREB might be a potential transcription factor linking HA/CD44 activation to TGF-β2 transcription and additional experiments are required for a better understanding of the molecular mechanisms underpinning the novel function of the CD44/ TGF-β2 signaling pathway in breast cancer metastasis.

No MeSH data available.


Related in: MedlinePlus

Time-course western-blot validation of the tetracycline (tet) regulated CD44 expression in MCF7F-B5 breast cancer cell line. Protein lysates were collected at different time points (18, 24 and 48hrs) following withdrawal of tet, in the presence of hyaluronan (HA). The level of expression of CD44s showed a time-dependent increase and was maintained up to 48 hrs.
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Figure 1: Time-course western-blot validation of the tetracycline (tet) regulated CD44 expression in MCF7F-B5 breast cancer cell line. Protein lysates were collected at different time points (18, 24 and 48hrs) following withdrawal of tet, in the presence of hyaluronan (HA). The level of expression of CD44s showed a time-dependent increase and was maintained up to 48 hrs.

Mentions: We have previously generated a tet-off-regulated CD44s expression system in the parental MCF-7 cell line, expressing minimal amounts of CD44s to obtain an inducible MCF7-B5 BC cell line (10). To validate that this system functions properly in vitro, upregulation of both CD44s mRNA and protein was examined in the MCF7-B5 clone throughout a 48-h time course. Total RNA and protein lysates were collected from MCF7F-B5 cells at 18, 24, and 48 h in the presence and absence of dox and examined for CD44s expression by western blot. In the absence of dox, the level of expression of CD44s showed a time-dependent increase and was maintained up to 48 h. A single immunoreactive band was detected at a molecular mass equivalent to 85 kDa, which is consistent with the presence of CD44s (Fig. 1). However in the presence of dox, the MCF7F-B5 cells cultured showed barely detectable CD44s expression, indicating that the tet-regulated CD44s suppression system was properly functional in the MCF7F-B5 cell line.


TGF-β2: A Novel Target of CD44-Promoted Breast Cancer Invasion.

Ouhtit A, Madani S, Gupta I, Shanmuganathan S, Abdraboh ME, Al-Riyami H, Al-Farsi YM, Raj MH - J Cancer (2013)

Time-course western-blot validation of the tetracycline (tet) regulated CD44 expression in MCF7F-B5 breast cancer cell line. Protein lysates were collected at different time points (18, 24 and 48hrs) following withdrawal of tet, in the presence of hyaluronan (HA). The level of expression of CD44s showed a time-dependent increase and was maintained up to 48 hrs.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3753531&req=5

Figure 1: Time-course western-blot validation of the tetracycline (tet) regulated CD44 expression in MCF7F-B5 breast cancer cell line. Protein lysates were collected at different time points (18, 24 and 48hrs) following withdrawal of tet, in the presence of hyaluronan (HA). The level of expression of CD44s showed a time-dependent increase and was maintained up to 48 hrs.
Mentions: We have previously generated a tet-off-regulated CD44s expression system in the parental MCF-7 cell line, expressing minimal amounts of CD44s to obtain an inducible MCF7-B5 BC cell line (10). To validate that this system functions properly in vitro, upregulation of both CD44s mRNA and protein was examined in the MCF7-B5 clone throughout a 48-h time course. Total RNA and protein lysates were collected from MCF7F-B5 cells at 18, 24, and 48 h in the presence and absence of dox and examined for CD44s expression by western blot. In the absence of dox, the level of expression of CD44s showed a time-dependent increase and was maintained up to 48 h. A single immunoreactive band was detected at a molecular mass equivalent to 85 kDa, which is consistent with the presence of CD44s (Fig. 1). However in the presence of dox, the MCF7F-B5 cells cultured showed barely detectable CD44s expression, indicating that the tet-regulated CD44s suppression system was properly functional in the MCF7F-B5 cell line.

Bottom Line: Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tetracycline at 18, 24, and 48 h post-HA stimulation compared to the parental cells.More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation, and subsequently inhibited BC cell invasion in vitro.In addition to identifying TGF-β2 as a target for HA/CD44 signaling, this data suggests that ATF/CREB might be a potential transcription factor linking HA/CD44 activation to TGF-β2 transcription and additional experiments are required for a better understanding of the molecular mechanisms underpinning the novel function of the CD44/ TGF-β2 signaling pathway in breast cancer metastasis.

View Article: PubMed Central - PubMed

Affiliation: 1. Department of Genetics.

ABSTRACT
We have developed a tetracycline (tet)-off regulated expression of CD44s gene in the breast cancer (BC) cell line MCF-7 (B5 clone) and identified TGF-β2 (Transforming Growth Factor beta-2; 3 fold induction) as a potential CD44-downstream transcriptional target by microarray analysis. To further validate this finding, the same RNA samples, used for microarray analysis and their corresponding protein lysates, collected from the BC cell line MCF-7-B5, were examined for CD44 expression in the presence of HA. Our results showed that TGF-β2 mRNA levels were significantly elevated following the removal of tetracycline at 18, 24, and 48 h post-HA stimulation compared to the parental cells. Furthermore, the TGF-β2 precursor protein increased in a time-dependent pattern upon HA-stimulation and in the absence of tetracycline. More interestingly, inhibition of CD44 gene by RNAi method decreased TGF-β2 expression upon HA-stimulation, and subsequently inhibited BC cell invasion in vitro. In addition to identifying TGF-β2 as a target for HA/CD44 signaling, this data suggests that ATF/CREB might be a potential transcription factor linking HA/CD44 activation to TGF-β2 transcription and additional experiments are required for a better understanding of the molecular mechanisms underpinning the novel function of the CD44/ TGF-β2 signaling pathway in breast cancer metastasis.

No MeSH data available.


Related in: MedlinePlus