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Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Lee JK, Garbe JC, Vrba L, Miyano M, Futscher BW, Stampfer MR, LaBarge MA - Front Cell Dev Biol (2015)

Bottom Line: Cells from all resulting immortal lines exhibited normal karyotypes.Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines.Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA, USA.

ABSTRACT
Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus

Phase diagram summarizing our hypothesis of the impacts of chronological age and the means of bypassing stasis on the intrinsic subtype of immortal HMEC. The color red indicated basal-like subtypes, and green represents luminal-like subtypes.
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Figure 7: Phase diagram summarizing our hypothesis of the impacts of chronological age and the means of bypassing stasis on the intrinsic subtype of immortal HMEC. The color red indicated basal-like subtypes, and green represents luminal-like subtypes.

Mentions: Breast cancers have been categorized by lineage markers into intrinsic subtypes that differ in prognosis and response to treatment. The mechanisms responsible for determining subtype have not been clearly defined; cell of origin, specific oncogenic insults, and cellular microenvironment have been proposed to influence lineage expression in cancer cells (Sims et al., 2007; Prat and Perou, 2011). However, lineage specificity in immortal and malignantly transformed cells is neither exact nor obvious by comparison to the normal lineages in vivo—they are caricatures of normal at the best. Indeed, every cell line had some level of heterogeneity, with varying distributions of cells representing the luminal and myoepithelial lineages. Here we have shown that expression of lineage-related markers in immortalized HMEC is influenced by both chronological age and the method by which the normal HMEC escape an initial tumor-suppressive senescence barrier, stress-associated stasis. A comparison of HMEC from young (<30 years) vs. older (>60 years) women showed that increased age biased toward generation of immortalized lines with greater expression of luminal phenotypes. A comparison of immortalized lines generated by using either p16sh or cyclin D1/CDK2 to bypass stasis showed a bias toward a luminal phenotype when cyclin D1/CDK2 was utilized, independent of age. Advanced age combined with cyclin D1-mediated bypass of stasis generated a line that was qualitatively the most luminal, exhibiting expression of K19, CD227, ERα, and other luminal transcripts. While a younger strain transduced with cyclin D1 also gave rise to a K19 and CD227-expressing luminal line, it did not produce ERα. In contrast, the younger lines that bypassed stasis with p16sh were qualitatively very basal-like (K19−/K14+, CD227− and basal transcripts). These results are based on our currently small number of cell lines, due in part to the length of time required to generate and examine each line. As more immortal cell lines are generated using this methodology the relative importance of age and stasis bypass in the etiology of intrinsic subtypes will become more obvious. Using a phase diagram we have expressed our hypothesis that the relative luminal vs. basal phenotype of immortalized HMEC is influenced by both chronological age and the method of stasis bypass (Figure 7).


Age and the means of bypassing stasis influence the intrinsic subtype of immortalized human mammary epithelial cells.

Lee JK, Garbe JC, Vrba L, Miyano M, Futscher BW, Stampfer MR, LaBarge MA - Front Cell Dev Biol (2015)

Phase diagram summarizing our hypothesis of the impacts of chronological age and the means of bypassing stasis on the intrinsic subtype of immortal HMEC. The color red indicated basal-like subtypes, and green represents luminal-like subtypes.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4356162&req=5

Figure 7: Phase diagram summarizing our hypothesis of the impacts of chronological age and the means of bypassing stasis on the intrinsic subtype of immortal HMEC. The color red indicated basal-like subtypes, and green represents luminal-like subtypes.
Mentions: Breast cancers have been categorized by lineage markers into intrinsic subtypes that differ in prognosis and response to treatment. The mechanisms responsible for determining subtype have not been clearly defined; cell of origin, specific oncogenic insults, and cellular microenvironment have been proposed to influence lineage expression in cancer cells (Sims et al., 2007; Prat and Perou, 2011). However, lineage specificity in immortal and malignantly transformed cells is neither exact nor obvious by comparison to the normal lineages in vivo—they are caricatures of normal at the best. Indeed, every cell line had some level of heterogeneity, with varying distributions of cells representing the luminal and myoepithelial lineages. Here we have shown that expression of lineage-related markers in immortalized HMEC is influenced by both chronological age and the method by which the normal HMEC escape an initial tumor-suppressive senescence barrier, stress-associated stasis. A comparison of HMEC from young (<30 years) vs. older (>60 years) women showed that increased age biased toward generation of immortalized lines with greater expression of luminal phenotypes. A comparison of immortalized lines generated by using either p16sh or cyclin D1/CDK2 to bypass stasis showed a bias toward a luminal phenotype when cyclin D1/CDK2 was utilized, independent of age. Advanced age combined with cyclin D1-mediated bypass of stasis generated a line that was qualitatively the most luminal, exhibiting expression of K19, CD227, ERα, and other luminal transcripts. While a younger strain transduced with cyclin D1 also gave rise to a K19 and CD227-expressing luminal line, it did not produce ERα. In contrast, the younger lines that bypassed stasis with p16sh were qualitatively very basal-like (K19−/K14+, CD227− and basal transcripts). These results are based on our currently small number of cell lines, due in part to the length of time required to generate and examine each line. As more immortal cell lines are generated using this methodology the relative importance of age and stasis bypass in the etiology of intrinsic subtypes will become more obvious. Using a phase diagram we have expressed our hypothesis that the relative luminal vs. basal phenotype of immortalized HMEC is influenced by both chronological age and the method of stasis bypass (Figure 7).

Bottom Line: Cells from all resulting immortal lines exhibited normal karyotypes.Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines.Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression.

View Article: PubMed Central - PubMed

Affiliation: Life Sciences Division, Lawrence Berkeley National Laboratory Berkeley, CA, USA.

ABSTRACT
Based on molecular features, breast cancers are grouped into intrinsic subtypes that have different prognoses and therapeutic response profiles. With increasing age, breast cancer incidence increases, with hormone receptor-positive and other luminal-like subtype tumors comprising a majority of cases. It is not known at what stage of tumor progression subtype specification occurs, nor how the process of aging affects the intrinsic subtype. We examined subtype markers in immortalized human mammary epithelial cell lines established following exposure of primary cultured cell strains to a two-step immortalization protocol that targets the two main barriers to immortality: stasis (stress-associated senescence) and replicative senescence. Cell lines derived from epithelial cells obtained from non-tumorous pre- and post-menopausal breast surgery tissues were compared. Additionally, comparisons were made between lines generated using two different genetic interventions to bypass stasis: transduction of either an shRNA that down-regulated p16(INK4A), or overexpressed constitutive active cyclin D1/CDK2. In all cases, the replicative senescence barrier was bypassed by transduction of c-Myc. Cells from all resulting immortal lines exhibited normal karyotypes. Immunofluorescence, flow cytometry, and gene expression analyses of lineage-specific markers were used to categorize the intrinsic subtypes of the immortalized lines. Bypassing stasis with p16 shRNA in young strains generated cell lines that were invariably basal-like, but the lines examined from older strains exhibited some luminal features such as keratin 19 and estrogen receptor expression. Overexpression of cyclin D1/CDK2 resulted in keratin 19 positive, luminal-like cell lines from both young and old strains, and the lines examined from older strains exhibited estrogen receptor expression. Thus age and the method of bypassing stasis independently influence the subtype of immortalized human mammary epithelial cells.

No MeSH data available.


Related in: MedlinePlus