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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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Pregnancy occurs normally in TOSix animals. (A) Real-time quantitative PCR (qRT-PCR), using transgene specific primers and probe, reveals that HASix1 is not expressed in the MTB control dox treated animals, but is expressed at low levels in the uninduced (-dox) mammary glands, and at high levels in the induced (+dox) mammary glands. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox or sucrose vehicle-control at time of mating. Error bars denote mean ± sem. (B) H&E sections taken from MTB and TOSix mice. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox at time of mating. (C) H&E sections taken from TOSix mice sacrificed at P18 after beginning treatment with sucrose-vehicle control at time of mating.
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Figure 4: Pregnancy occurs normally in TOSix animals. (A) Real-time quantitative PCR (qRT-PCR), using transgene specific primers and probe, reveals that HASix1 is not expressed in the MTB control dox treated animals, but is expressed at low levels in the uninduced (-dox) mammary glands, and at high levels in the induced (+dox) mammary glands. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox or sucrose vehicle-control at time of mating. Error bars denote mean ± sem. (B) H&E sections taken from MTB and TOSix mice. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox at time of mating. (C) H&E sections taken from TOSix mice sacrificed at P18 after beginning treatment with sucrose-vehicle control at time of mating.

Mentions: qRT-PCR data has shown that the Six1 transgene is expressed in aged, uninduced TOSix animals at low levels [30], demonstrating that the expression of Six1 is not completely dependent on dox induction in aged animals. Interestingly, tumor formation occurs at a higher frequency in the animals that express low levels of Six1, indicating that Six1 may act in a dose-dependent manner to induce mammary tumors [30]. Therefore, we sought to determine if Six1 is expressed in younger, uninduced TOSix animals, the focus of our mammary developmental studies, and whether Six1 was tightly controlled by dox in these animals. Twelve week old TOSix and MTB females were mated to wild-type males, and concurrently began continuous treatment with water containing 2 mg/ml dox or with sucrose as a vehicle control. At P18, females were sacrificed and their mammary glands excised for analysis. qRT-PCR data demonstrates that the Six1 transgene is expressed at low levels in the uninduced, sucrose-treated animals (-dox), but at much higher levels in the induced animals (+dox) (Fig. 4A). Thus, as was observed in the aged population, low levels of Six1 transgene expression can be observed in the mammary glands in the absence of dox induction. Therefore, we included animals from both cohorts in a number of the following studies to determine if the dose of Six1 has any affect on normal mammary development.


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)

Pregnancy occurs normally in TOSix animals. (A) Real-time quantitative PCR (qRT-PCR), using transgene specific primers and probe, reveals that HASix1 is not expressed in the MTB control dox treated animals, but is expressed at low levels in the uninduced (-dox) mammary glands, and at high levels in the induced (+dox) mammary glands. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox or sucrose vehicle-control at time of mating. Error bars denote mean ± sem. (B) H&E sections taken from MTB and TOSix mice. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox at time of mating. (C) H&E sections taken from TOSix mice sacrificed at P18 after beginning treatment with sucrose-vehicle control at time of mating.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: Pregnancy occurs normally in TOSix animals. (A) Real-time quantitative PCR (qRT-PCR), using transgene specific primers and probe, reveals that HASix1 is not expressed in the MTB control dox treated animals, but is expressed at low levels in the uninduced (-dox) mammary glands, and at high levels in the induced (+dox) mammary glands. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox or sucrose vehicle-control at time of mating. Error bars denote mean ± sem. (B) H&E sections taken from MTB and TOSix mice. Animals were sacrificed at pregnancy day 18 (P18) after beginning treatment with dox at time of mating. (C) H&E sections taken from TOSix mice sacrificed at P18 after beginning treatment with sucrose-vehicle control at time of mating.
Mentions: qRT-PCR data has shown that the Six1 transgene is expressed in aged, uninduced TOSix animals at low levels [30], demonstrating that the expression of Six1 is not completely dependent on dox induction in aged animals. Interestingly, tumor formation occurs at a higher frequency in the animals that express low levels of Six1, indicating that Six1 may act in a dose-dependent manner to induce mammary tumors [30]. Therefore, we sought to determine if Six1 is expressed in younger, uninduced TOSix animals, the focus of our mammary developmental studies, and whether Six1 was tightly controlled by dox in these animals. Twelve week old TOSix and MTB females were mated to wild-type males, and concurrently began continuous treatment with water containing 2 mg/ml dox or with sucrose as a vehicle control. At P18, females were sacrificed and their mammary glands excised for analysis. qRT-PCR data demonstrates that the Six1 transgene is expressed at low levels in the uninduced, sucrose-treated animals (-dox), but at much higher levels in the induced animals (+dox) (Fig. 4A). Thus, as was observed in the aged population, low levels of Six1 transgene expression can be observed in the mammary glands in the absence of dox induction. Therefore, we included animals from both cohorts in a number of the following studies to determine if the dose of Six1 has any affect on normal mammary development.

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