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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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Six1-overexpressing dams lactate normally. (A) Average pup weight from TOSix and MTB litters, treated with dox or sucrose-vehicle as noted. Litters were normalized to 8 pups and weights were taken daily from lactation day 1 (L1) to L21. Error bars denote mean ± sem. n = average number of pups weighed (B) H&E stained sections taken from TOSix and MTB animals at L2. Animals began dox treatment at time of mating.
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Figure 5: Six1-overexpressing dams lactate normally. (A) Average pup weight from TOSix and MTB litters, treated with dox or sucrose-vehicle as noted. Litters were normalized to 8 pups and weights were taken daily from lactation day 1 (L1) to L21. Error bars denote mean ± sem. n = average number of pups weighed (B) H&E stained sections taken from TOSix and MTB animals at L2. Animals began dox treatment at time of mating.

Mentions: To determine if Six1 may affect normal lactation and milk production, pup weights were recorded for litters born to Six1-overexpressing dams. At 12 weeks of age, female animals from both the 4922 and 6239 lines began dox or vehicle-control treatment and MTB animals began dox treatment. All females were concurrently mated to wild-type males and were allowed to undergo three rounds of pregnancy. Litter sizes were normalized to eight pups and weights were taken each day from lactation day 1 (L1) to L21 during one of the three pregnancies, selected at random. No profound differences in pup weights were identified between litters born to TOSix +/- dox and MTB + dox dams, suggesting that TOSix dams lactate at a proficient level to meet the nutritional needs of their pups (Fig. 5A). Interestingly, we did note that a high percentage (approximately 80%) of 6239 TOSix +dox dams lost at least one of their litters during the three rounds of pregnancy as compared to 30-40% in MTB, 4922 line TOSix +/- dox, and 6239 line TOSix -dox animals (data not shown). However, this litter loss was inconsistent and did not appear to follow a clear trend. Some dams lost their litter only once during the three pregnancies, and in dams that lost litters, the loss didn't always occur in the same sequence (i.e. first, second, or third). We thus cannot attribute this finding to increased levels of Six1.


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)

Six1-overexpressing dams lactate normally. (A) Average pup weight from TOSix and MTB litters, treated with dox or sucrose-vehicle as noted. Litters were normalized to 8 pups and weights were taken daily from lactation day 1 (L1) to L21. Error bars denote mean ± sem. n = average number of pups weighed (B) H&E stained sections taken from TOSix and MTB animals at L2. Animals began dox treatment at time of mating.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Six1-overexpressing dams lactate normally. (A) Average pup weight from TOSix and MTB litters, treated with dox or sucrose-vehicle as noted. Litters were normalized to 8 pups and weights were taken daily from lactation day 1 (L1) to L21. Error bars denote mean ± sem. n = average number of pups weighed (B) H&E stained sections taken from TOSix and MTB animals at L2. Animals began dox treatment at time of mating.
Mentions: To determine if Six1 may affect normal lactation and milk production, pup weights were recorded for litters born to Six1-overexpressing dams. At 12 weeks of age, female animals from both the 4922 and 6239 lines began dox or vehicle-control treatment and MTB animals began dox treatment. All females were concurrently mated to wild-type males and were allowed to undergo three rounds of pregnancy. Litter sizes were normalized to eight pups and weights were taken each day from lactation day 1 (L1) to L21 during one of the three pregnancies, selected at random. No profound differences in pup weights were identified between litters born to TOSix +/- dox and MTB + dox dams, suggesting that TOSix dams lactate at a proficient level to meet the nutritional needs of their pups (Fig. 5A). Interestingly, we did note that a high percentage (approximately 80%) of 6239 TOSix +dox dams lost at least one of their litters during the three rounds of pregnancy as compared to 30-40% in MTB, 4922 line TOSix +/- dox, and 6239 line TOSix -dox animals (data not shown). However, this litter loss was inconsistent and did not appear to follow a clear trend. Some dams lost their litter only once during the three pregnancies, and in dams that lost litters, the loss didn't always occur in the same sequence (i.e. first, second, or third). We thus cannot attribute this finding to increased levels of Six1.

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