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Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity.

Sriram R, Lo V, Pryce B, Antonova L, Mears AJ, Daneshmand M, McKay B, Conway SJ, Muller WJ, Sabourin LA - Breast Cancer Res. (2015)

Bottom Line: Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer.Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy.This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada. roshankumar2733@yahoo.com.

ABSTRACT

Introduction: Periostin (Postn) is a secreted cell adhesion protein that activates signaling pathways to promote cancer cell survival, angiogenesis, invasion, and metastasis. Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer.

Methods: Using transgenic mice expressing the Neu oncogene in the mammary epithelium crossed into Postn-deficient animals, we have assessed the effect of Postn gene deletion on Neu-driven mammary tumorigenesis.

Results: Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy. Furthermore, we find that loss of Postn in the mammary epithelium does not alter breast tumor initiation or growth in mouse mammary tumor virus (MMTV)-Neu expressing mice but results in an apocrine-like tumor phenotype. Surprisingly, we find that tumors derived from Postn- animals express low levels of Notch protein and Hey1 mRNA but increased expression of androgen receptor (AR) and AR target genes. We show that tumor cells derived from wild-type animals do not proliferate when transplanted in a Postn- environment but that this growth defect is rescued by the overexpression of active Notch or the AR target gene prolactin-induced protein (PIP/GCDFP-15).

Conclusions: Together our data suggest that loss of Postn in an ErbB2/Neu/HER2 overexpression model results in apocrine-like tumors that activate an AR-dependent pathway. This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.

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Active Notch expression can bypass the Postn requirement for heterotopic growth of Met-1 cells. (A) Western blot analysis of tumor lysates showing a marked reduction of active Notch levels in Postn- tumors. This is accompanied by a 2-fold reduction in the levels of Hey1 gene expression as measured by Q-PCR (B; *P <0.01). The data shown are an average of 4 independent tumors performed in triplicate. (C) Western blot analysis of Postn- or NICD-expressing Met-1 cells. Cells expressing Postn showed a marked increase in endogenous NICD levels when compared to vector control (pLPCX). The increase in endogenous NICD was blocked by the addition of a γ-secretase inhibitor (GSI). Overexpression of exogenous NICD was only detectable with a total Notch antibody as the anti-NICD epitope has been deleted in the construct. Similarly, expression of the NLS mutant can only be detected by Q-PCR as it lacks both epitopes recognized by the commercial anti-NICD and anti-Notch antibodies. (D) Quantitation of Hey1 mRNA levels showed a 4- and 70-fold increase in Met-1 cultures overexpressing Postn or NICD, respectively (*P <0.01). No increase was observed in ΔNLS-expressing cells. (E) Tumor volume measurements at 28 days post-injection of Met-1 cells overexpressing NICD or ΔNLS in wildtype or Postn- mice. Cells over-expressing NICD could bypass the Postn requirement for subcutaneous growth in FVB/N females. Little or no growth was observed for ΔNLS-expressing cells (*P <0.01). (F) Expression of dnMAML1 in Met-1 cells impairs their growth in wildtype mice when compared to a GFP-expressing control (*P <0.01).
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Fig4: Active Notch expression can bypass the Postn requirement for heterotopic growth of Met-1 cells. (A) Western blot analysis of tumor lysates showing a marked reduction of active Notch levels in Postn- tumors. This is accompanied by a 2-fold reduction in the levels of Hey1 gene expression as measured by Q-PCR (B; *P <0.01). The data shown are an average of 4 independent tumors performed in triplicate. (C) Western blot analysis of Postn- or NICD-expressing Met-1 cells. Cells expressing Postn showed a marked increase in endogenous NICD levels when compared to vector control (pLPCX). The increase in endogenous NICD was blocked by the addition of a γ-secretase inhibitor (GSI). Overexpression of exogenous NICD was only detectable with a total Notch antibody as the anti-NICD epitope has been deleted in the construct. Similarly, expression of the NLS mutant can only be detected by Q-PCR as it lacks both epitopes recognized by the commercial anti-NICD and anti-Notch antibodies. (D) Quantitation of Hey1 mRNA levels showed a 4- and 70-fold increase in Met-1 cultures overexpressing Postn or NICD, respectively (*P <0.01). No increase was observed in ΔNLS-expressing cells. (E) Tumor volume measurements at 28 days post-injection of Met-1 cells overexpressing NICD or ΔNLS in wildtype or Postn- mice. Cells over-expressing NICD could bypass the Postn requirement for subcutaneous growth in FVB/N females. Little or no growth was observed for ΔNLS-expressing cells (*P <0.01). (F) Expression of dnMAML1 in Met-1 cells impairs their growth in wildtype mice when compared to a GFP-expressing control (*P <0.01).

Mentions: Western blot analysis revealed a 3-fold decrease in the levels of the transcriptionally active Notch intracellular domain fragment (NICD; reviewed in [52]) (Figure 4A). This was accompanied by an approximately 50% downregulation in Hey1 mRNA levels, a NICD target gene (Figure 4B). However, Hes1 levels remained unchanged (not shown), supporting the notion that Notch-mediated activation of Hes1 transcription is context and cell type-dependent [56]. Together our results suggest that the loss of Postn is accompanied by reduced Notch1 activity and a failure to support tumor growth at secondary sites.Figure 4


Loss of periostin/OSF-2 in ErbB2/Neu-driven tumors results in androgen receptor-positive molecular apocrine-like tumors with reduced Notch1 activity.

Sriram R, Lo V, Pryce B, Antonova L, Mears AJ, Daneshmand M, McKay B, Conway SJ, Muller WJ, Sabourin LA - Breast Cancer Res. (2015)

Active Notch expression can bypass the Postn requirement for heterotopic growth of Met-1 cells. (A) Western blot analysis of tumor lysates showing a marked reduction of active Notch levels in Postn- tumors. This is accompanied by a 2-fold reduction in the levels of Hey1 gene expression as measured by Q-PCR (B; *P <0.01). The data shown are an average of 4 independent tumors performed in triplicate. (C) Western blot analysis of Postn- or NICD-expressing Met-1 cells. Cells expressing Postn showed a marked increase in endogenous NICD levels when compared to vector control (pLPCX). The increase in endogenous NICD was blocked by the addition of a γ-secretase inhibitor (GSI). Overexpression of exogenous NICD was only detectable with a total Notch antibody as the anti-NICD epitope has been deleted in the construct. Similarly, expression of the NLS mutant can only be detected by Q-PCR as it lacks both epitopes recognized by the commercial anti-NICD and anti-Notch antibodies. (D) Quantitation of Hey1 mRNA levels showed a 4- and 70-fold increase in Met-1 cultures overexpressing Postn or NICD, respectively (*P <0.01). No increase was observed in ΔNLS-expressing cells. (E) Tumor volume measurements at 28 days post-injection of Met-1 cells overexpressing NICD or ΔNLS in wildtype or Postn- mice. Cells over-expressing NICD could bypass the Postn requirement for subcutaneous growth in FVB/N females. Little or no growth was observed for ΔNLS-expressing cells (*P <0.01). (F) Expression of dnMAML1 in Met-1 cells impairs their growth in wildtype mice when compared to a GFP-expressing control (*P <0.01).
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Fig4: Active Notch expression can bypass the Postn requirement for heterotopic growth of Met-1 cells. (A) Western blot analysis of tumor lysates showing a marked reduction of active Notch levels in Postn- tumors. This is accompanied by a 2-fold reduction in the levels of Hey1 gene expression as measured by Q-PCR (B; *P <0.01). The data shown are an average of 4 independent tumors performed in triplicate. (C) Western blot analysis of Postn- or NICD-expressing Met-1 cells. Cells expressing Postn showed a marked increase in endogenous NICD levels when compared to vector control (pLPCX). The increase in endogenous NICD was blocked by the addition of a γ-secretase inhibitor (GSI). Overexpression of exogenous NICD was only detectable with a total Notch antibody as the anti-NICD epitope has been deleted in the construct. Similarly, expression of the NLS mutant can only be detected by Q-PCR as it lacks both epitopes recognized by the commercial anti-NICD and anti-Notch antibodies. (D) Quantitation of Hey1 mRNA levels showed a 4- and 70-fold increase in Met-1 cultures overexpressing Postn or NICD, respectively (*P <0.01). No increase was observed in ΔNLS-expressing cells. (E) Tumor volume measurements at 28 days post-injection of Met-1 cells overexpressing NICD or ΔNLS in wildtype or Postn- mice. Cells over-expressing NICD could bypass the Postn requirement for subcutaneous growth in FVB/N females. Little or no growth was observed for ΔNLS-expressing cells (*P <0.01). (F) Expression of dnMAML1 in Met-1 cells impairs their growth in wildtype mice when compared to a GFP-expressing control (*P <0.01).
Mentions: Western blot analysis revealed a 3-fold decrease in the levels of the transcriptionally active Notch intracellular domain fragment (NICD; reviewed in [52]) (Figure 4A). This was accompanied by an approximately 50% downregulation in Hey1 mRNA levels, a NICD target gene (Figure 4B). However, Hes1 levels remained unchanged (not shown), supporting the notion that Notch-mediated activation of Hes1 transcription is context and cell type-dependent [56]. Together our results suggest that the loss of Postn is accompanied by reduced Notch1 activity and a failure to support tumor growth at secondary sites.Figure 4

Bottom Line: Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer.Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy.This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Road, Ottawa, ON, K1H 8M5, Canada. roshankumar2733@yahoo.com.

ABSTRACT

Introduction: Periostin (Postn) is a secreted cell adhesion protein that activates signaling pathways to promote cancer cell survival, angiogenesis, invasion, and metastasis. Interestingly, Postn is frequently overexpressed in numerous human cancers, including breast, lung, colon, pancreatic, and ovarian cancer.

Methods: Using transgenic mice expressing the Neu oncogene in the mammary epithelium crossed into Postn-deficient animals, we have assessed the effect of Postn gene deletion on Neu-driven mammary tumorigenesis.

Results: Although Postn is exclusively expressed in the stromal fibroblasts of the mammary gland, Postn deletion does not affect mammary gland outgrowth during development or pregnancy. Furthermore, we find that loss of Postn in the mammary epithelium does not alter breast tumor initiation or growth in mouse mammary tumor virus (MMTV)-Neu expressing mice but results in an apocrine-like tumor phenotype. Surprisingly, we find that tumors derived from Postn- animals express low levels of Notch protein and Hey1 mRNA but increased expression of androgen receptor (AR) and AR target genes. We show that tumor cells derived from wild-type animals do not proliferate when transplanted in a Postn- environment but that this growth defect is rescued by the overexpression of active Notch or the AR target gene prolactin-induced protein (PIP/GCDFP-15).

Conclusions: Together our data suggest that loss of Postn in an ErbB2/Neu/HER2 overexpression model results in apocrine-like tumors that activate an AR-dependent pathway. This may have important implications for the treatment of breast cancers involving the therapeutic targeting of periostin or Notch signaling.

Show MeSH
Related in: MedlinePlus