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Characterization of the Six1 homeobox gene in normal mammary gland morphogenesis.

Coletta RD, McCoy EL, Burns V, Kawakami K, McManaman JL, Wysolmerski JJ, Ford HL - BMC Dev. Biol. (2010)

Bottom Line: Surprisingly, inappropriate expression of Six1 in the adult mammary gland, when levels are normally low to absent, did not inhibit normal mammary differentiation during pregnancy or lactation.Six1 is not critical for normal mammary gland development, since neither loss nor inappropriate overexpression of Six1 adversely affects normal mammary gland development or function.However, as both Six2 and Six4 levels are increased in Six1-/- mammary glands, we postulate that these Six family members are functionally redundant in the gland, as is true of many homeobox gene families.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Colorado Denver, Anschutz Medical Campus, 12800 E, 19th Ave, Aurora, CO 80045, USA.

ABSTRACT

Background: The Six1 homeobox gene is highly expressed in the embryonic mammary gland, continues to be expressed in early postnatal mammary development, but is lost when the mammary gland differentiates during pregnancy. However, Six1 is re-expressed in breast cancers, suggesting that its re-instatement in the adult mammary gland may contribute to breast tumorigenesis via initiating a developmental process out of context. Indeed, recent studies demonstrate that Six1 overexpression in the adult mouse mammary gland is sufficient for initiating invasive carcinomas, and that its overexpression in xenograft models of mammary cancer leads to metastasis. These data demonstrate that Six1 is causally involved in both breast tumorigenesis and metastasis, thus raising the possibility that it may be a viable therapeutic target. However, because Six1 is highly expressed in the developing mammary gland, and because it has been implicated in the expansion of mammary stem cells, targeting Six1 as an anti-cancer therapy may have unwanted side effects in the breast.

Results: We sought to determine the role of Six1 in mammary development using two independent mouse models. To study the effect of Six1 loss in early mammary development when Six1 is normally expressed, Six1-/- embryonic mammary glands were transplanted into Rag1-/- mice. In addition, to determine whether Six1 downregulation is required during later stages of development to allow for proper differentiation, we overexpressed Six1 during adulthood using an inducible, mammary-specific transgenic mouse model. Morphogenesis of the mammary gland occurred normally in animals transplanted with Six1-/- embryonic mammary glands, likely through the redundant functions of other Six family members such as Six2 and Six4, whose expression was increased in response to Six1 loss. Surprisingly, inappropriate expression of Six1 in the adult mammary gland, when levels are normally low to absent, did not inhibit normal mammary differentiation during pregnancy or lactation.

Conclusions: Six1 is not critical for normal mammary gland development, since neither loss nor inappropriate overexpression of Six1 adversely affects normal mammary gland development or function. However, as both Six2 and Six4 levels are increased in Six1-/- mammary glands, we postulate that these Six family members are functionally redundant in the gland, as is true of many homeobox gene families. This data, in conjunction with recent findings that Six1 is capable of promoting breast cancer initiation and progression, suggest that Six1 may serve as a reasonable chemotherapeutic target in a clinical setting, particularly for those women diagnosed with breast cancer in their childbearing years.

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Mammary glands overexpressing Six1 regress normally. H&E sections taken from TOSix and MTB animals after 6 months of dox treatment (beginning at time of first mating) and after three rounds of pregnancy. Tissue was excised four weeks after weaning to ensure complete involution.
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Figure 6: Mammary glands overexpressing Six1 regress normally. H&E sections taken from TOSix and MTB animals after 6 months of dox treatment (beginning at time of first mating) and after three rounds of pregnancy. Tissue was excised four weeks after weaning to ensure complete involution.

Mentions: Since Six1 is known to play a pro-proliferative and anti-apoptotic role in numerous tissues [6-9], we hypothesized that persistent Six1 expression may prevent proper remodelling of the gland following pregnancy, thus contributing to the hyperplasia that eventually arises in these animals [30]. To test this hypothesis, TOSix females from both the 4922 and 6239 lines, as well as MTB controls began dox treatment at 12 weeks of age and were concurrently mated. This experiment was performed on the +dox TOSix group, and not the -dox TOSix group, as both groups were previously shown to equally exhibit hyperplasia after prolonged Six1 expression [30]. After the third pregnancy and 3 weeks of subsequent nursing, pups were weaned, and mothers were allowed to rest for four weeks to ensure complete involution before sacrifice. H&E stained sections reveal that the epithelium in mammary glands expressing Six1, taken from both the 4922 (n = 4) and 6239 (n = 2) transgenic lines, properly regresses, exhibiting similar histology to the glands taken from control MTB animals (n = 7) (Fig. 6). Whole mount analysis also revealed that TOSix mammary glands were fully regressed, similar to MTB mammary glands (data not shown).


Characterization of the Six1 homeobox gene in normal mammary gland morphogenesis.

Coletta RD, McCoy EL, Burns V, Kawakami K, McManaman JL, Wysolmerski JJ, Ford HL - BMC Dev. Biol. (2010)

Mammary glands overexpressing Six1 regress normally. H&E sections taken from TOSix and MTB animals after 6 months of dox treatment (beginning at time of first mating) and after three rounds of pregnancy. Tissue was excised four weeks after weaning to ensure complete involution.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2823684&req=5

Figure 6: Mammary glands overexpressing Six1 regress normally. H&E sections taken from TOSix and MTB animals after 6 months of dox treatment (beginning at time of first mating) and after three rounds of pregnancy. Tissue was excised four weeks after weaning to ensure complete involution.
Mentions: Since Six1 is known to play a pro-proliferative and anti-apoptotic role in numerous tissues [6-9], we hypothesized that persistent Six1 expression may prevent proper remodelling of the gland following pregnancy, thus contributing to the hyperplasia that eventually arises in these animals [30]. To test this hypothesis, TOSix females from both the 4922 and 6239 lines, as well as MTB controls began dox treatment at 12 weeks of age and were concurrently mated. This experiment was performed on the +dox TOSix group, and not the -dox TOSix group, as both groups were previously shown to equally exhibit hyperplasia after prolonged Six1 expression [30]. After the third pregnancy and 3 weeks of subsequent nursing, pups were weaned, and mothers were allowed to rest for four weeks to ensure complete involution before sacrifice. H&E stained sections reveal that the epithelium in mammary glands expressing Six1, taken from both the 4922 (n = 4) and 6239 (n = 2) transgenic lines, properly regresses, exhibiting similar histology to the glands taken from control MTB animals (n = 7) (Fig. 6). Whole mount analysis also revealed that TOSix mammary glands were fully regressed, similar to MTB mammary glands (data not shown).

Bottom Line: Surprisingly, inappropriate expression of Six1 in the adult mammary gland, when levels are normally low to absent, did not inhibit normal mammary differentiation during pregnancy or lactation.Six1 is not critical for normal mammary gland development, since neither loss nor inappropriate overexpression of Six1 adversely affects normal mammary gland development or function.However, as both Six2 and Six4 levels are increased in Six1-/- mammary glands, we postulate that these Six family members are functionally redundant in the gland, as is true of many homeobox gene families.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Obstetrics and Gynecology, University of Colorado Denver, Anschutz Medical Campus, 12800 E, 19th Ave, Aurora, CO 80045, USA.

ABSTRACT

Background: The Six1 homeobox gene is highly expressed in the embryonic mammary gland, continues to be expressed in early postnatal mammary development, but is lost when the mammary gland differentiates during pregnancy. However, Six1 is re-expressed in breast cancers, suggesting that its re-instatement in the adult mammary gland may contribute to breast tumorigenesis via initiating a developmental process out of context. Indeed, recent studies demonstrate that Six1 overexpression in the adult mouse mammary gland is sufficient for initiating invasive carcinomas, and that its overexpression in xenograft models of mammary cancer leads to metastasis. These data demonstrate that Six1 is causally involved in both breast tumorigenesis and metastasis, thus raising the possibility that it may be a viable therapeutic target. However, because Six1 is highly expressed in the developing mammary gland, and because it has been implicated in the expansion of mammary stem cells, targeting Six1 as an anti-cancer therapy may have unwanted side effects in the breast.

Results: We sought to determine the role of Six1 in mammary development using two independent mouse models. To study the effect of Six1 loss in early mammary development when Six1 is normally expressed, Six1-/- embryonic mammary glands were transplanted into Rag1-/- mice. In addition, to determine whether Six1 downregulation is required during later stages of development to allow for proper differentiation, we overexpressed Six1 during adulthood using an inducible, mammary-specific transgenic mouse model. Morphogenesis of the mammary gland occurred normally in animals transplanted with Six1-/- embryonic mammary glands, likely through the redundant functions of other Six family members such as Six2 and Six4, whose expression was increased in response to Six1 loss. Surprisingly, inappropriate expression of Six1 in the adult mammary gland, when levels are normally low to absent, did not inhibit normal mammary differentiation during pregnancy or lactation.

Conclusions: Six1 is not critical for normal mammary gland development, since neither loss nor inappropriate overexpression of Six1 adversely affects normal mammary gland development or function. However, as both Six2 and Six4 levels are increased in Six1-/- mammary glands, we postulate that these Six family members are functionally redundant in the gland, as is true of many homeobox gene families. This data, in conjunction with recent findings that Six1 is capable of promoting breast cancer initiation and progression, suggest that Six1 may serve as a reasonable chemotherapeutic target in a clinical setting, particularly for those women diagnosed with breast cancer in their childbearing years.

Show MeSH
Related in: MedlinePlus