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Hedgehog overexpression leads to the formation of prostate cancer stem cells with metastatic property irrespective of androgen receptor expression in the mouse model.

Chang HH, Chen BY, Wu CY, Tsao ZJ, Chen YY, Chang CP, Yang CR, Lin DP - J. Biomed. Sci. (2011)

Bottom Line: In the hyperplastic lesions and tumors, the progeny of PCSCs differentiated into cells of basal-intermediate and intermediate-luminal characteristics, whereas rare ChgA+ neuroendocrine differentiation was seen.Furthermore, in the metastatic loci within lymph nodes, kidneys, and lungs, the P63+ PCSCs formed prostate-like glandular structures, characteristic of the primitive structures during early prostate development.Besides, androgen receptor (AR) expression was detected heterogeneously during tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Optometry, Chung Shan Medical University, Taichung 402, Taiwan.

ABSTRACT

Background: Hedgehog signalling has been implicated in prostate tumorigenesis in human subjects and mouse models, but its effects on transforming normal basal/stem cells toward malignant cancer stem cells remain poorly understood.

Methods: We produced pCX-shh-IG mice that overexpress Hedgehog protein persistently in adult prostates, allowing for elucidation of the mechanism during prostate cancer initiation and progression. Various markers were used to characterize and confirm the transformation of normal prostate basal/stem cells into malignant cancer stem cells under the influence of Hedgehog overexpression.

Results: The pCX-shh-IG mice developed prostatic intraepithelial neoplasia (PIN) that led to invasive and metastatic prostate cancers within 90 days. The prostate cancer was initiated through activation of P63+ basal/stem cells along with simultaneous activation of Hedgehog signalling members, suggesting that P63+/Patch1+ and P63+/Smo+ cells may serve as cancer-initiating cells and progress into malignant prostate cancer stem cells (PCSCs). In the hyperplastic lesions and tumors, the progeny of PCSCs differentiated into cells of basal-intermediate and intermediate-luminal characteristics, whereas rare ChgA+ neuroendocrine differentiation was seen. Furthermore, in the metastatic loci within lymph nodes, kidneys, and lungs, the P63+ PCSCs formed prostate-like glandular structures, characteristic of the primitive structures during early prostate development. Besides, androgen receptor (AR) expression was detected heterogeneously during tumor progression. The existence of P63+/AR-, CK14+/AR- and CD44+/AR- progeny indicates direct procurement of AR- malignant cancer trait.

Conclusions: These data support a cancer stem cell scenario in which Hedgehog signalling plays important roles in transforming normal prostate basal/stem cells into PCSCs and in the progression of PCSCs into metastatic tumor cells.

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Differentiation status and AR expression profile of PCSCs under the influence of Hedgehog overexpression. The boxed areas in the pictures are further magnified and shown in the corresponding lower or right pictures. (A) Increased CK14+ cells along with tumorigenic progression in the pCX-shh-IG-injected prostate ((b), (c) and (d)), as compared to the normal prostate ((a)). (B) Western blot analysis indicated up-regulation of CK14, CD44 and CK8 in the pCX-shh-IG-injected prostates as compared to the normal pCX-IG-injected prostates. (C) Characterization of PCSCs by double-immunofluorescence staining showing differentiation toward CK14+ progeny ((a), (b) and (c)), CK14+ cells toward CD44+ progeny ((d), (e) and (f)), and CK14+ cells toward CK8+ progeny ((g), (h) and (i)). CK14+ differentiation involved Hedgehog signalling activation, as indicated by co-localized Patch1 expression ((j), (k) and (l)). The basal-intermediate (CK14+/CD44+) and intermediate-luminal (CK14+/CK8+) populations were increased in PIN and CaP of pCX-shh-IG-injected prostates as compared to those of the pCX-IG-injected vehicle controls (D and E). (F) Some PCSCs were AR- as indicated by arrows in (a), (b), (c), (d), and (e), even though they were P63+, CK14+, CK8+, CD44+, or Patch1+. The relative proportions of AR+ and AR- cells among P63+, CK14+, CK8+, CD44+, and Patch1+ cell populations were shown respectively in (f). All scale bars represent 10 μm in length. CaP: prostate cancer; PIN: prostatic intraepithelial neoplasia; HGPIN: high grade prostatic intraepithelial neoplasia.
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Figure 5: Differentiation status and AR expression profile of PCSCs under the influence of Hedgehog overexpression. The boxed areas in the pictures are further magnified and shown in the corresponding lower or right pictures. (A) Increased CK14+ cells along with tumorigenic progression in the pCX-shh-IG-injected prostate ((b), (c) and (d)), as compared to the normal prostate ((a)). (B) Western blot analysis indicated up-regulation of CK14, CD44 and CK8 in the pCX-shh-IG-injected prostates as compared to the normal pCX-IG-injected prostates. (C) Characterization of PCSCs by double-immunofluorescence staining showing differentiation toward CK14+ progeny ((a), (b) and (c)), CK14+ cells toward CD44+ progeny ((d), (e) and (f)), and CK14+ cells toward CK8+ progeny ((g), (h) and (i)). CK14+ differentiation involved Hedgehog signalling activation, as indicated by co-localized Patch1 expression ((j), (k) and (l)). The basal-intermediate (CK14+/CD44+) and intermediate-luminal (CK14+/CK8+) populations were increased in PIN and CaP of pCX-shh-IG-injected prostates as compared to those of the pCX-IG-injected vehicle controls (D and E). (F) Some PCSCs were AR- as indicated by arrows in (a), (b), (c), (d), and (e), even though they were P63+, CK14+, CK8+, CD44+, or Patch1+. The relative proportions of AR+ and AR- cells among P63+, CK14+, CK8+, CD44+, and Patch1+ cell populations were shown respectively in (f). All scale bars represent 10 μm in length. CaP: prostate cancer; PIN: prostatic intraepithelial neoplasia; HGPIN: high grade prostatic intraepithelial neoplasia.

Mentions: Since our data had indicated that ChgA+ neuroendocrine cells did not constitute the main cellular lineage under the influence of Hh overexpression, the differentiation status of PCSCs was examined with some cell markers, including P63 (primitive basal cells), CK14 (advanced and hyperplastic basal cells), CD44 (intermediate cells), and CK8 (mature luminal cells). In the normal vehicle control prostates, fewer CK14+ basal cells lay flat along the basement membrane (Figure 5A; arrowhead-indicated in (a)), whereas in the PIN and CaP lesions of pCX-shh-IG-injected prostates, CK14+ cells were increased with neoplastic transformation (Figure 5A; arrow-indicated in (b), (c) and (d)). By western blot analysis, we found that CK14, CD44, and CK8 markers were up-regulated in the prostate tumors as compared to the normal prostates (Figure 5B). Double labelling of P63 and CK14 markers showed P63+/CK14(low or -) cells in the normal prostates (Figure 5C; arrowhead-indicated in (a)). In the pCX-shh-IG-injected prostates, as the prostates were induced into PIN and progressed into CaP status, these P63+/CK14(low or -) cells appeared to be differentiated into P63+/CK14+ (Figure 5C; arrowhead-indicated in (b)) and further into P63 (low)/CK14+ (Figure 5C; arrowhead-indicated in (c)) and P63-/CK14+ (Figure 5C; arrow-indicated in (b) and (c)) cells. The loss of P63 expression revealed that the PCSCs were differentiated toward the luminal progeny. Comparably, the CK14+/CD44+ cancer cells in the PIN and CaP lesions (Figure 5C; arrow (2)-indicated in (e) and (f)) were likely to be originated from CK14(low or -)/CD44(low or -) cells in the normal prostates (Figure 5C; arrowhead-indicated in (d)) or from CK14+/CD44(low or -) cells (Figure 5C; arrow (1)-indicated in (e) and (f)) and CK14(low or -)/CD44+ cells (Figure 5C; arrow (3)-indicated in (e) and (f)), as the cells were transformed into PIN and progressed into CaP conditions. Additionally, the CK14+/CK8+ cancer cells in the PIN and CaP lesions (Figure 5C; arrow-indicated in (h) and (i)) might be originated from CK14(low or -)/CK8- cells in the normal prostates (Figure 5C; arrowhead-indicated in (g)), and then differentiated into CK14low/CK8(high or +) progeny (Figure 5C; arrowhead-indicated in (i)). The increase of basal-intermediate (CK14+/CD44+) (Figure 5D) and intermediate-luminal (CK14+/CK8+) (Figure 5E) progeny in the PIN and CaP lesions of pCX-shh-IG-injected prostates were significant when compared to the vehicle controls. The involvement of Hh signalling during PCSCs differentiation in the PIN and CaP lesions was indicated by double labelling of Patch1 and CK14 (Figure 5C; arrow-indicated in (k) and (l)), in contrast to the only few Patch1+ cells detected in the normal vehicle controls (Figure 5C; arrowhead-indicated in (j)).


Hedgehog overexpression leads to the formation of prostate cancer stem cells with metastatic property irrespective of androgen receptor expression in the mouse model.

Chang HH, Chen BY, Wu CY, Tsao ZJ, Chen YY, Chang CP, Yang CR, Lin DP - J. Biomed. Sci. (2011)

Differentiation status and AR expression profile of PCSCs under the influence of Hedgehog overexpression. The boxed areas in the pictures are further magnified and shown in the corresponding lower or right pictures. (A) Increased CK14+ cells along with tumorigenic progression in the pCX-shh-IG-injected prostate ((b), (c) and (d)), as compared to the normal prostate ((a)). (B) Western blot analysis indicated up-regulation of CK14, CD44 and CK8 in the pCX-shh-IG-injected prostates as compared to the normal pCX-IG-injected prostates. (C) Characterization of PCSCs by double-immunofluorescence staining showing differentiation toward CK14+ progeny ((a), (b) and (c)), CK14+ cells toward CD44+ progeny ((d), (e) and (f)), and CK14+ cells toward CK8+ progeny ((g), (h) and (i)). CK14+ differentiation involved Hedgehog signalling activation, as indicated by co-localized Patch1 expression ((j), (k) and (l)). The basal-intermediate (CK14+/CD44+) and intermediate-luminal (CK14+/CK8+) populations were increased in PIN and CaP of pCX-shh-IG-injected prostates as compared to those of the pCX-IG-injected vehicle controls (D and E). (F) Some PCSCs were AR- as indicated by arrows in (a), (b), (c), (d), and (e), even though they were P63+, CK14+, CK8+, CD44+, or Patch1+. The relative proportions of AR+ and AR- cells among P63+, CK14+, CK8+, CD44+, and Patch1+ cell populations were shown respectively in (f). All scale bars represent 10 μm in length. CaP: prostate cancer; PIN: prostatic intraepithelial neoplasia; HGPIN: high grade prostatic intraepithelial neoplasia.
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Related In: Results  -  Collection

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Figure 5: Differentiation status and AR expression profile of PCSCs under the influence of Hedgehog overexpression. The boxed areas in the pictures are further magnified and shown in the corresponding lower or right pictures. (A) Increased CK14+ cells along with tumorigenic progression in the pCX-shh-IG-injected prostate ((b), (c) and (d)), as compared to the normal prostate ((a)). (B) Western blot analysis indicated up-regulation of CK14, CD44 and CK8 in the pCX-shh-IG-injected prostates as compared to the normal pCX-IG-injected prostates. (C) Characterization of PCSCs by double-immunofluorescence staining showing differentiation toward CK14+ progeny ((a), (b) and (c)), CK14+ cells toward CD44+ progeny ((d), (e) and (f)), and CK14+ cells toward CK8+ progeny ((g), (h) and (i)). CK14+ differentiation involved Hedgehog signalling activation, as indicated by co-localized Patch1 expression ((j), (k) and (l)). The basal-intermediate (CK14+/CD44+) and intermediate-luminal (CK14+/CK8+) populations were increased in PIN and CaP of pCX-shh-IG-injected prostates as compared to those of the pCX-IG-injected vehicle controls (D and E). (F) Some PCSCs were AR- as indicated by arrows in (a), (b), (c), (d), and (e), even though they were P63+, CK14+, CK8+, CD44+, or Patch1+. The relative proportions of AR+ and AR- cells among P63+, CK14+, CK8+, CD44+, and Patch1+ cell populations were shown respectively in (f). All scale bars represent 10 μm in length. CaP: prostate cancer; PIN: prostatic intraepithelial neoplasia; HGPIN: high grade prostatic intraepithelial neoplasia.
Mentions: Since our data had indicated that ChgA+ neuroendocrine cells did not constitute the main cellular lineage under the influence of Hh overexpression, the differentiation status of PCSCs was examined with some cell markers, including P63 (primitive basal cells), CK14 (advanced and hyperplastic basal cells), CD44 (intermediate cells), and CK8 (mature luminal cells). In the normal vehicle control prostates, fewer CK14+ basal cells lay flat along the basement membrane (Figure 5A; arrowhead-indicated in (a)), whereas in the PIN and CaP lesions of pCX-shh-IG-injected prostates, CK14+ cells were increased with neoplastic transformation (Figure 5A; arrow-indicated in (b), (c) and (d)). By western blot analysis, we found that CK14, CD44, and CK8 markers were up-regulated in the prostate tumors as compared to the normal prostates (Figure 5B). Double labelling of P63 and CK14 markers showed P63+/CK14(low or -) cells in the normal prostates (Figure 5C; arrowhead-indicated in (a)). In the pCX-shh-IG-injected prostates, as the prostates were induced into PIN and progressed into CaP status, these P63+/CK14(low or -) cells appeared to be differentiated into P63+/CK14+ (Figure 5C; arrowhead-indicated in (b)) and further into P63 (low)/CK14+ (Figure 5C; arrowhead-indicated in (c)) and P63-/CK14+ (Figure 5C; arrow-indicated in (b) and (c)) cells. The loss of P63 expression revealed that the PCSCs were differentiated toward the luminal progeny. Comparably, the CK14+/CD44+ cancer cells in the PIN and CaP lesions (Figure 5C; arrow (2)-indicated in (e) and (f)) were likely to be originated from CK14(low or -)/CD44(low or -) cells in the normal prostates (Figure 5C; arrowhead-indicated in (d)) or from CK14+/CD44(low or -) cells (Figure 5C; arrow (1)-indicated in (e) and (f)) and CK14(low or -)/CD44+ cells (Figure 5C; arrow (3)-indicated in (e) and (f)), as the cells were transformed into PIN and progressed into CaP conditions. Additionally, the CK14+/CK8+ cancer cells in the PIN and CaP lesions (Figure 5C; arrow-indicated in (h) and (i)) might be originated from CK14(low or -)/CK8- cells in the normal prostates (Figure 5C; arrowhead-indicated in (g)), and then differentiated into CK14low/CK8(high or +) progeny (Figure 5C; arrowhead-indicated in (i)). The increase of basal-intermediate (CK14+/CD44+) (Figure 5D) and intermediate-luminal (CK14+/CK8+) (Figure 5E) progeny in the PIN and CaP lesions of pCX-shh-IG-injected prostates were significant when compared to the vehicle controls. The involvement of Hh signalling during PCSCs differentiation in the PIN and CaP lesions was indicated by double labelling of Patch1 and CK14 (Figure 5C; arrow-indicated in (k) and (l)), in contrast to the only few Patch1+ cells detected in the normal vehicle controls (Figure 5C; arrowhead-indicated in (j)).

Bottom Line: In the hyperplastic lesions and tumors, the progeny of PCSCs differentiated into cells of basal-intermediate and intermediate-luminal characteristics, whereas rare ChgA+ neuroendocrine differentiation was seen.Furthermore, in the metastatic loci within lymph nodes, kidneys, and lungs, the P63+ PCSCs formed prostate-like glandular structures, characteristic of the primitive structures during early prostate development.Besides, androgen receptor (AR) expression was detected heterogeneously during tumor progression.

View Article: PubMed Central - HTML - PubMed

Affiliation: School of Optometry, Chung Shan Medical University, Taichung 402, Taiwan.

ABSTRACT

Background: Hedgehog signalling has been implicated in prostate tumorigenesis in human subjects and mouse models, but its effects on transforming normal basal/stem cells toward malignant cancer stem cells remain poorly understood.

Methods: We produced pCX-shh-IG mice that overexpress Hedgehog protein persistently in adult prostates, allowing for elucidation of the mechanism during prostate cancer initiation and progression. Various markers were used to characterize and confirm the transformation of normal prostate basal/stem cells into malignant cancer stem cells under the influence of Hedgehog overexpression.

Results: The pCX-shh-IG mice developed prostatic intraepithelial neoplasia (PIN) that led to invasive and metastatic prostate cancers within 90 days. The prostate cancer was initiated through activation of P63+ basal/stem cells along with simultaneous activation of Hedgehog signalling members, suggesting that P63+/Patch1+ and P63+/Smo+ cells may serve as cancer-initiating cells and progress into malignant prostate cancer stem cells (PCSCs). In the hyperplastic lesions and tumors, the progeny of PCSCs differentiated into cells of basal-intermediate and intermediate-luminal characteristics, whereas rare ChgA+ neuroendocrine differentiation was seen. Furthermore, in the metastatic loci within lymph nodes, kidneys, and lungs, the P63+ PCSCs formed prostate-like glandular structures, characteristic of the primitive structures during early prostate development. Besides, androgen receptor (AR) expression was detected heterogeneously during tumor progression. The existence of P63+/AR-, CK14+/AR- and CD44+/AR- progeny indicates direct procurement of AR- malignant cancer trait.

Conclusions: These data support a cancer stem cell scenario in which Hedgehog signalling plays important roles in transforming normal prostate basal/stem cells into PCSCs and in the progression of PCSCs into metastatic tumor cells.

Show MeSH
Related in: MedlinePlus