Basal but not luminal mammary epithelial cells require PI3K/mTOR signaling for Ras-driven overgrowth

Abstract

The mammary ducts of humans and mice are comprised of two main mammary epithelial cell (MEC) subtypes: a surrounding layer of basal MECs and an inner layer of luminal MECs. Breast cancer subtypes show divergent clinical behavior that may reflect properties inherent in their MEC compartment of origin. How the response to a cancer-initiating genetic event is shaped by MEC subtype remains largely unexplored. Using the mouse mammary gland, we designed organotypic three-dimensional culture models that permit challenge of discrete MEC compartments with the same oncogenic insult. Mammary organoids were prepared from mice engineered for compartment-restricted coexpression of oncogenic H-RAS(G12V) together with a nuclear fluorescent reporter. Monitoring of H-RAS(G12V)-expressing MECs during extended live cell imaging permitted visualization of Ras-driven phenotypes via video microscopy. Challenging either basal or luminal MECs with H-RAS(G12V) drove MEC proliferation and survival, culminating in aberrant organoid overgrowth. In each compartment, Ras activation triggered modes of collective MEC migration and invasion that contrasted with physiologic modes used during growth factor-initiated branching morphogenesis. Although basal and luminal Ras activation produced similar overgrowth phenotypes, inhibitor studies revealed divergent use of Ras effector pathways. Blocking either the phosphoinositide 3-kinase or the mammalian target of rapamycin pathway completely suppressed Ras-driven invasion and overgrowth of basal MECs, but only modestly attenuated Ras-driven phenotypes in luminal MECs. We show that MEC subtype defines signaling pathway dependencies downstream of Ras. Thus, cells-of-origin may critically determine the drug sensitivity profiles of mammary neoplasia.

Figure 1. MMTV-rtTA and K5-rtTA restrict transgene expression to opposing MEC compartments

Organoids prepared from Dox-naïve bi-transgenic mice engineered for basal MEC-restricted labeling (K5-rtTA/TGFP; Basal^GFP) or luminal MEC-restricted labeling (MMTV-rtTA/TGFP; Luminal^GFP) were cultured in 3D. (A) Compartment-restricted MEC labeling. Live cell images (20X) of representative organoids captured before and after Dox-induced reporter gene expression are shown. Bar, 100 μm. (B). Confirmation of compartment-restricted labeling by confocal microscopy. Organoids were recovered after 7 d Dox treatment in 3D culture. Equatorial confocal images (63X) of representative Basal^GFP and Luminal^GFP organoids are shown. Bar, 20 μm. (C) Live cell imaging of MEC proliferation and death. Time-lapse images (20X) depict typical MEC mitosis and cell death events captured during extended imaging of a Dox-treated Basal^GFP organoid. Bar, 20 μm.

Figure 2. H-RAS^G12V expressed in either MEC compartment drives organoid overgrowth

(A) Normal histomorphology of Dox-naïve transgenic mammary glands. Representative images of camine-stained mammary gland whole mounts and immunohistochemically stained tissue sections are shown. White bar, 500 μm; black bar, 100 μm. (B) Time-lapse imaging of organoid overgrowth. Organoids prepared from Dox-naïve mice engineered for Ras pathway activation in either basal MECs (K5-rtTA/TGFP/TRAS; Basal^RAS/GFP) or luminal MECs (MMTV-rtTA/TGFP/TRAS; Luminal^RAS/GFP) were cultured concurrently in 3D along with genetic control organoids lacking the TRAS transgene (Basal^GFP and Luminal^GFP). Sequential images of representative organoids of each genotype captured during a one week live-cell imaging experiment are shown. Times denote hrs after Dox addition. Bar, 50 μm. (C) Ras-mediated changes in organoid size and shape. 2D organoid images captured after 6 d Dox treatment were subjected to morphometric analysis to determine mean organoid area (left) and circularity (right). Mean values reflect analysis of 23 to 111 organoids for each genotype and treatment condition. (D) Ras-mediated increases in MEC proliferation and survival. Organoid videos were viewed frame-by-frame to score mitotic (left) and cell death (right) events occurring during the first 2 days of Dox treatment. Mean values reflect indices (events per organoid, normalized to a 24 hour acquisition period) derived from analyzing videos from 8 to 38 organoids of each genotype. *, p < 0.01; **, p < 0.05; Kruskal-Wallis analysis of variance, Tukey-Kramer method. All error bars indicate SEM.

Figure 3. H-RAS^G12V, but not Fgf2, triggers focal invasion into Matrigel

(A) Fgf2-induced multilayered epithelial elongation. Still image from a live cell imaging experiment depict a representative luminal^GFP organoid 7 d after initiating Fgf2-induced branching morphogenesis. (B) Ras-mediated focal invasion. Time-lapse images depict leader cells initiating invasion. Arrowheads mark protrusions emanating from leader cells indicated by white arrows. Red Arrows indicate follower “stalk” cells. Bar, 25μm. (C) Quantification of Ras-mediated focal invasion. Organoids of the indicated genotypes were cultured in the presence and absence of Dox for 6 d, then scored for the presence of focal invasion as described in Materials and Methods. Mean values reflect analysis of 20 to 77 organoids for each genotype and treatment condition. (D) Contrasting modes of collective MEC invasion triggered by Fgf2 versus H-RAS^G12V. Organoids of the indicated genotypes underwent Fgf2-induced branching prior to Dox treatment. Panels depict morphology of a representative organoid before and after Dox-induced transgene expression.

Figure 4. MEK inhibition partially blocks Ras phenotypes in both MEC compartments

Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions for 6 d. PD denotes MEK inhibitor PD0325901 at 100 nM. Left panels depict day 6 images (10X) of representative organoids for each genotype and treatment condition (see also Videos 5 and 6). Morphometry of 2D images captured at days 0, 2, 4 and 6 were used to determine the prevalence of focal invasion, as well as the mean organoid circularity and area at each time point. Each data point reflects the analysis of 37 to 55 organoids. Error bars denote SEM.

Figure 5. PI3K/mTOR inhibition fully blocks Ras phenotypes in basal but not luminal MECs

Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions for 6 d. Left panels depict day 6 images (10X) of representative organoids for each genotype and treatment condition (see also Videos 7–10). Morphometry of 2D images captured at days 0, 2, 4 and 6 were used to determine the prevalence of focal invasion, as well as the mean organoid circularity and area at each time point. (A) PI3K pathway inhibition. LY denotes PI3K inhibitor LY294002 at 10 μM. Each data point reflects the analysis of 24 to 47 organoids. (B) mTOR pathway inhibition. Rap denotes rapamycin at 5nM. Each data point reflects the analysis of 18 to 114 organoids. Error bars denote SEM.

Figure 6. Suppressing Ras-driven phenotypes involves decreased proliferation and preservation of MEC compartments

Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions in 3D. Mitotic and cell death events were scored during the first 2 days of Dox treatment. (A) MEK inhibitor effects on MEC proliferation and survival. PD denotes MEK inhibitor PD0325901 at 100 nM. (B) PI3K/mTOR inhibitor effects on MEC proliferation and survival. Mean values reflect indices (events per organoid, normalized to a 24 hour acquisition period) derived from analyzing videos from 11 to 34 organoids of each genotype. Error bars indicate SEM. (C) PI3K/mTOR inhibitor effects on MEC compartmentalization. Organoids were recovered after 3D culture for 7 d, then fixed and stained with Hoechst and phalloidin. Panels depict equatorial confocal microscopy images (63X) of representative organoids of each indicated genotype and treatment condition. Bars, 50 μm. LY denotes PI3K inhibitor LY294002 at 10 μM. Rap denotes rapamycin at 5nM.

Figure 7. Pi3k/mTOR blockade abrogates basal but not luminal MEC overgrowth across a range of Ras activation levels and inhibitor doses

Basal^RAS/GFP and luminal^RAS/GFP organoids were cultured under the indicated treatment conditions for 6 d. 2D image morphometry was used to determine mean organoid area. (A) LY effects on organoid size across varied Dox doses. When administered, the LY dose was held constant at 10 μM. (B) Inhibitor effects on organoid size across varied drug doses. When administered, the Dox dose was held constant at 1000 ng/ml. Mean values reflect analysis of 18 to 34 organoids for each genotype and treatment condition. Error bars denote SEM.