Adult murine prostate basal and luminal cells are self-sustained lineages that can both serve as targets for prostate cancer initiation

Abstract

The prostate epithelial lineage hierarchy and the cellular origin for prostate cancer remain inadequately defined. Using a lineage-tracing approach, we show that adult rodent prostate basal and luminal cells are independently self-sustained in vivo. Disrupting the tumor suppressor Pten in either lineage led to prostate cancer initiation. However, the cellular composition and onset dynamics of the resulting tumors are distinctive. Prostate luminal cells are more responsive to Pten null-induced mitogenic signaling. In contrast, basal cells are resistant to direct transformation. Instead, loss of Pten activity induces the capability of basal cells to differentiate into transformation-competent luminal cells. Our study suggests that deregulation of epithelial differentiation is a critical step for the initiation of prostate cancers of basal cell origin.

Fig. 1. Lineage tracing shows that prostate basal cells only generate basal cells in vivo

(A) Schematic illustration of the lineage tracing strategy. (B–D) Co-staining of GFP with K5 and K8 (B), K5 and P63 (C), and Synaptophysin (D) in tamoxifen-treated K14-mTmG mice. Arrowheads in B and C indicate GFP-labeled basal cells. Arrowhead in D points to a neuroendocrine cell. (E) Timeline for androgen deprivation and replacement experiments. (F–H) Co-staining of GFP with K5 and K8 (F), K5 and P63 (G), and Synaptophysin (H) in tamoxifen-treated K14-mTmG mice after induced epithelial turnover. Arrowheads in F and G indicate GFP-labeled basal cells. Arrowhead in H points to a neuroendocrine cell. (I) Bar graph shows the percentage of GFP-labeled basal cells in lateral prostate lobes of K14-mTmG mice 5 days after tamoxifen induction (6 weeks) and after 2 cycles of epithelial regression-regeneration. Data represent means with SD. Also see Table S1. (J) GFP-labeled basal cells (arrowhead) incorporated BrdU. See also Fig. S1 and Table S1.

Fig. 2. The prostate luminal cell lineage is self-sustained in vivo

(A–B) Co-staining of GFP with K5 and K8 (A), and Synaptophysin (B) in tamoxifen-treated K8-mTmG mice. Arrowheads and asterisks in A denote basal cells that are not GFP-labeled and GFP-labeled luminal cells, respectively. Arrow in B points to a neuroendocrine cell. (C–D) Co-staining of GFP with K5 and K8 (C), and Synaptophysin (D) in tamoxifen-treated K8-mTmG mice after induced epithelial turnover. Arrowheads indicate that all basal cells are GFP negative. Arrow points to a neuroendocrine cell. (E) Bar graph shows the percentage of GFP-labeled luminal cells 5 days after tamoxifen induction (6 weeks, black bars), after 4-month-ageing (red bars), after the first androgen deprivation (blue bars) and after 2 cycles of androgen deprivation-replacement experiments (green bars) in anterior (AP), ventral (VP), dorsal (DP) and lateral (LP) prostate lobes. Data represent means with SD. See Table S2–4 for more details. (F) Timeline for investigating the origin of newly formed luminal cells by BrdU labeling. (G) Bar graph shows that the frequency of newly formed GFP-positive luminal (GFP+K8+BrdU+/ K8+BrdU+) cells reflects that of GFP-labeled luminal (GFP+K8+/K8+) cells in all four prostate lobes. Data represent means with SD. Also see Table S2–4. (H) A representative image showing BrdU incorporated into luminal cells during prostate regeneration. See also Fig. S2 and Tables S2–4.

Fig. 3. Prostate cancer initiated from basal cell-specific loss-of-function of PTEN

(A): Costaining of basal cell marker P63 with pAKT in K14-Pten mice 1 month post tamoxifen induction. Arrowheads point to pAKT⁺P63⁺ cells. (B) H&E staining of a K14-Pten prostate 3-month post tamoxifen induction reveals the formation of PIN lesions (asterisks). (C) PIN lesions (asterisks), but not adjacent normal glands, express pAKT. (D–E) PIN lesions contain both K5-expressing basal cells and K8-expressing luminal cells. Arrowheads indicate K5-expressing basal cells that encapsulate glands. (F) Quantification of the expansion of luminal cells in PIN lesions (region B) as compared in normal glands (region A). Data represent means with SD. (G–H) Pten is disrupted in some basal cells. Arrowheads in G and G′ denote a P63-expressing basal cell that expresses pAKT. Arrowhead in H points to an anatomically typical basal cell that expresses pAKT. See also Fig. S3.

Fig. 4. Progression of prostate cancer in the K14-Pten model

(A-A′) Representative images of urogenital organs and dissected prostate lobes from K14-Pten mice 8 months after induction with vehicle (A′) or tamoxifen (A). (B–E) Representative images of H&E staining of total prostate (B), anterior (AP, C), dorsolateral (DLP, D), and ventral (VP, E) prostate lobes of a K14-Pten mouse 8 months after tamoxifen treatment. (F–H) Immunostaining of K5 and K8 of a K14-Pten mouse 3 months after tamoxifen treatment (F) and a 4-month old ARR2PB-Pten mouse (G). (H) Quantification of the percentage of K5⁺K8⁺ and K5⁺K8⁻ cells in the two models. (I–J) Immunostaining of K5 and P63 in K14-Pten mice (I). (J) Quantification of the percentage of K5⁺P63⁺ and K5⁺P63⁻ cells. (K) Synaptophysin-expressing neuroendocrine cells (arrow) are rare in K14-Pten prostate tumor. Arrowheads point to P63-expressing basal cells. (L-L″) Prostate basal cells in normal glands in tamoxifen-treated K14-Pten mice express pAKT. Arrowheads point to P63-expressing basal cells that express pAKT. (M–O) Representative images of H&E staining show that only mild focal PIN lesions (dot-circled or arrow-pointed regions) are developed in many K14-Pten mice 6–8 months post tamoxifen treatment. (P–S) Dissociated prostate cells from K14-Pten tumors are capable of regenerating abnormal glandular structures. (P) H&E staining of the outgrown tissues. (Q–S) Immunostaining of K5 (Q, arrow) and K8 (Q, arrowhead), P63 (R, arrowhead), and pAKT (S, arrowhead). Insets in S indicate that both basal (arrowhead) and luminal (arrow) cells express pAKT. See also Fig. S4.

Fig. 5. Prostate cancer derived from luminal cell specific disruption of PTEN

(A–C) H&E staining of anterior (AP), ventral (VP), and dorsolateral (DLP) prostate lobes of K8-Pten mice at 1, 3, and 6 months post tamoxifen induction. Arrows point to focal hyperplasia in AP. (D–F) Immunostaining of K5 and K8 reveals a distinct lineage composition among VP (D), AP (E), and DLP (F). (G–I) P63-expressing basal cells (arrowheads) do not express pAKT in VP (G), AP (H), and DLP (I). (J–L) Only K5-expressing basal cells residing at the basement membrane express P63 (arrowheads). VP (J), AP (K), and DLP (L). (M–R) K8-Pten prostate tumor can repopulate. Representative images of H&E staining of outgrown tissues from dissociated K8-Pten tumor tissues (M–O). Immunostaining of K5 and K8 (P and Q), and pAKT (R) of outgrown tissues. Arrows indicate cancerous foci. See also Fig. S5.

Fig. 6. Castration resistant prostate cancer cells exist in K8-Pten prostate tumors

(A-A′) Representative images of urogenital organs excluding seminal vesicles from K8-Pten mice 2 month after a mock surgery (A) and castration (A′). BL: bladder. UR: Urethra. AP: anterior prostate. VP: ventral prostate. DP: dorsal prostate. LP: lateral prostate. (B–G) H&E staining (B–D) and immunostaining of K5 and K8 (E–G) of VP (B, E), AP (C, F), and DLP (D, G) of K8-Pten mice before castration (B–D), and 10 days (B′–D′), and 2 months (B″–D″) after castration. Insets in 6E–6G show staining of K5 and K8 at bracketed regions in respective images.

Fig. 7. Prostate basal cells are resistant to direct oncogenic transformation

(A–B) H&E staining of K14-Pten;P53 mouse prostates 2 months after vehicle (A) or tamoxifen (B) induction. A′ and B′ show images of higher magnification. (C–D) Representative images of immunostaining of K5 and K8 of prostates from K14-Pten;P53 mice 2-month post tamoxifen induction. (E) Pten is disrupted only in basal cells in K14-Pten;P53 mice, as demonstrated by costaining of pAKT (E, E′) and P63 (E, E″). Arrowheads indicate pAKT-expressing cells that also express P63. (F–H) H&E (F) and immunostaining of K5/K8 (G) and pAKT/P63 (H) of K14-Pten;P53 mouse prostates 3 months after tamoxifen induction. Arrow in F points to a dot-circled PIN4 lesion. Arrowhead in H points to a basal cell expressing pAKT. (I–P) Two months after tamoxifen treatment, K14-Pten;P53 prostate glands were transplanted under renal capsules of immunodeficient male hosts. H&E staining (I, L, M) and immunostaining of K5/K8 (J, N) and pAKT/P63 (K,O,P) of 7-week (I–K) and 15-week (L–P) transplants. Arrowheads in O and P denote P63-expressing cells that express and do not express pAKT, respectively. See also Fig. S7.