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Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression.

Centritto F, Paroni G, Bolis M, Garattini SK, Kurosaki M, Barzago MM, Zanetti A, Fisher JN, Scott MF, Pattini L, Lupi M, Ubezio P, Piccotti F, Zambelli A, Rizzo P, Gianni' M, Fratelli M, Terao M, Garattini E - EMBO Mol Med (2015)

Bottom Line: Luminal and ER(+) (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity.Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity.All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.

No MeSH data available.


Related in: MedlinePlus

RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7 cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A–D groups and T1–T3 tertiles).Left: The upper graph indicates the averages expression levels of the RARα protein in T1–T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER− cell lines (middle graph) and the indicated classes of HER2+ and HER2− cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.
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fig08: RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7 cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A–D groups and T1–T3 tertiles).Left: The upper graph indicates the averages expression levels of the RARα protein in T1–T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER− cell lines (middle graph) and the indicated classes of HER2+ and HER2− cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.

Mentions: Given the observed relevance of the RARα3 transcript in our models, the basal levels of the corresponding RARα protein were determined in breast cancer cell lines with a specific antibody (Fig8A and B). The average levels of RARα are significantly higher in T1 versus T3 ATRA score groups (Fig8C), and the same trend is observed if the analysis is restricted to Luminal cells. Larger amounts of RARα are also observed in Luminal versus Basal and ER+ versus ER− cell lines. As for possible correlations with the RARα mRNA variants across the cell lines, the highest R2 values were calculated for the RARα protein and the RARα3/RARα4 mRNAs (Fig8D). This indicates that the protein is encoded by either the RARα3 or the RARα4 transcript. Given the low relative expression levels of RARα4, we favor RARα3.


Cellular and molecular determinants of all-trans retinoic acid sensitivity in breast cancer: Luminal phenotype and RARα expression.

Centritto F, Paroni G, Bolis M, Garattini SK, Kurosaki M, Barzago MM, Zanetti A, Fisher JN, Scott MF, Pattini L, Lupi M, Ubezio P, Piccotti F, Zambelli A, Rizzo P, Gianni' M, Fratelli M, Terao M, Garattini E - EMBO Mol Med (2015)

RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7 cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A–D groups and T1–T3 tertiles).Left: The upper graph indicates the averages expression levels of the RARα protein in T1–T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER− cell lines (middle graph) and the indicated classes of HER2+ and HER2− cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4520659&req=5

fig08: RARα protein and ATRA sensitivity in breast cancer cell linesTotal proteins were extracted from logarithmically growing cell lines and subjected to Western blot analysis.The panel illustrates representative Western blots for a number of cell lines. The blots were sequentially developed with RARα-specific and control tubulin antibodies. The positions of the RARα and tubulin bands (right) along with the position of a relevant molecular weight marker (left) are indicated. To normalize the Western blot signals in different gels, the same preparation of RARα-transfected COS-7 cell extracts (COS-7) and MCF7 extracts was loaded in each gel. The blots are representative of at least two independent experiments providing similar results.The quantitative results obtained after densitometric analysis of the RARα bands are plotted against the ATRA scores. Cell lines are grouped according to the ATRA score (A–D groups and T1–T3 tertiles).Left: The upper graph indicates the averages expression levels of the RARα protein in T1–T3 cell lines. The middle and lower graphs indicate the average expression levels after stratification of the cell lines for the Luminal (six cell lines in each of the T1 and T3 groups) and the Basal (six cell lines in each of the T1 and T3 groups) phenotype, respectively. Right: The levels of the RARα protein in Basal and Luminal cell lines (upper graph), in ER+, ER−, and Luminal ER− cell lines (middle graph) and the indicated classes of HER2+ and HER2− cell lines (lower graph) are shown. *Significantly different (P-value < 0.05, Student's t-test). **Significantly different (P-value < 0.01, Student's t-test).The plots show the correlation curves between the levels of the indicated RARα-variant transcripts and the RARα protein.
Mentions: Given the observed relevance of the RARα3 transcript in our models, the basal levels of the corresponding RARα protein were determined in breast cancer cell lines with a specific antibody (Fig8A and B). The average levels of RARα are significantly higher in T1 versus T3 ATRA score groups (Fig8C), and the same trend is observed if the analysis is restricted to Luminal cells. Larger amounts of RARα are also observed in Luminal versus Basal and ER+ versus ER− cell lines. As for possible correlations with the RARα mRNA variants across the cell lines, the highest R2 values were calculated for the RARα protein and the RARα3/RARα4 mRNAs (Fig8D). This indicates that the protein is encoded by either the RARα3 or the RARα4 transcript. Given the low relative expression levels of RARα4, we favor RARα3.

Bottom Line: Luminal and ER(+) (estrogen-receptor-positive) cell lines are generally sensitive to ATRA, while refractoriness/low sensitivity is associated with a Basal phenotype and HER2 positivity.Pathway-oriented analysis of the constitutive gene-expression profiles in the cell lines identifies RARα as the member of the retinoid pathway directly associated with a Luminal phenotype, estrogen positivity and ATRA sensitivity.All this is paralleled by similar effects on ATRA-dependent inhibition of cell motility, indicating that RARα may mediate also ATRA anti-metastatic effects.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Molecular Biology, IRCCS-Istituto di Ricerche Farmacologiche "Mario Negri", Milano, Italy.

No MeSH data available.


Related in: MedlinePlus