Tumour cell heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers.

Cancer genome sequencing studies indicate that a single breast cancer typically harbours multiple genetically distinct subclones. As carcinogenesis involves a breakdown in the cell-cell cooperation that normally maintains epithelial tissue architecture, individual subclones within a malignant microenvironment are commonly depicted as self-interested competitors. Alternatively, breast cancer subclones might interact cooperatively to gain a selective growth advantage in some cases. Although interclonal cooperation has been shown to drive tumorigenesis in fruitfly models, definitive evidence for functional cooperation between epithelial tumour cell subclones in mammals is lacking. Here we use mouse models of breast cancer to show that interclonal cooperation can be essential for tumour maintenance. Aberrant expression of the secreted signalling molecule Wnt1 generates mixed-lineage mammary tumours composed of basal and luminal tumour cell subtypes, which purportedly derive from a bipotent malignant progenitor cell residing atop a tumour cell hierarchy. Using somatic Hras mutations as clonal markers, we show that some Wnt tumours indeed conform to a hierarchical configuration, but that others unexpectedly harbour genetically distinct basal Hras mutant and luminal Hras wild-type subclones. Both subclones are required for efficient tumour propagation, which strictly depends on luminally produced Wnt1. When biclonal tumours were challenged with Wnt withdrawal to simulate targeted therapy, analysis of tumour regression and relapse revealed that basal subclones recruit heterologous Wnt-producing cells to restore tumour growth. Alternatively, in the absence of a substitute Wnt source, the original subclones often evolve to rescue Wnt pathway activation and drive relapse, either by restoring cooperation or by switching to a defector strategy. Uncovering similar modes of interclonal cooperation in human cancers may inform efforts aimed at eradicating tumour cell communities.

Tumor escape in a Wnt1-dependent mouse breast cancer model is enabled by p19Arf/p53 pathway lesions but not p16 Ink4a loss.

Breast cancers frequently progress or relapse during targeted therapy, but the molecular mechanisms that enable escape remain poorly understood. We elucidated genetic determinants underlying tumor escape in a transgenic mouse model of Wnt pathway-driven breast cancer, wherein targeted therapy is simulated by abrogating doxycycline-dependent Wnt1 transgene expression within established tumors. In mice with intact tumor suppressor pathways, tumors typically circumvented doxycycline withdrawal by reactivating Wnt signaling, either via aberrant (doxycycline-independent) Wnt1 transgene expression or via acquired somatic mutations in the gene encoding beta-catenin. Germline introduction of mutant tumor suppressor alleles into the model altered the timing and mode of tumor escape. Relapses occurring in the context of null Ink4a/Arf alleles (disrupting both the p16 Ink4a and p19 Arf tumor suppressors) arose quickly and rarely reactivated the Wnt pathway. In addition, Ink4a/Arf-deficient relapses resembled p53-deficient relapses in that both displayed morphologic and molecular hallmarks of an epithelial-to-mesenchymal transition (EMT). Notably, Ink4a/Arf deficiency promoted relapse in the absence of gross genomic instability. Moreover, Ink4a/Arf-encoded proteins differed in their capacity to suppress oncogene independence. Isolated p19 Arf deficiency mirrored p53 deficiency in that both promoted rapid, EMT-associated mammary tumor escape, whereas isolated p16 Ink4a deficiency failed to accelerate relapse. Thus, p19 Arf/p53 pathway lesions may promote mammary cancer relapse even when inhibition of a targeted oncogenic signaling pathway remains in force.

Dormant Wnt-initiated mammary cancer can participate in reconstituting functional mammary glands.

The minimal residual disease foci that beget breast cancer relapse after a period of disease dormancy remain uncharacterized despite their enormous clinical importance. To model dormant breast cancer in vivo, we employed a transgenic mouse model in which Wnt1-initiated mammary cancer is doxycycline dependent. After regression of Wnt-dependent cancers, subclinical disease lesions were propagated in vivo using classical tissue recombination techniques. Surprisingly, outgrowths derived from dormant malignant tissue reconstituted morphologically normal ductal trees in wild-type mammary fat pads. Whereas hyperplasia-derived outgrowths remained benign, outgrowths derived from dormant malignancy underwent a morphological transition suggesting single-step transformation following reactivation of Wnt signaling and rapidly yielded invasive mammary tumors. Remarkably, outgrowths derived from dormant malignancy could be serially propagated in vivo and retained the potential to undergo lobuloalveolar differentiation in response to hormones of pregnancy. Matching somatic H-Ras mutations shared by antecedent tumors and descendant mammary ductal outgrowths confirmed their clonal relatedness. Thus, propagation of epithelium that possesses a latent malignant growth program reveals impressive regenerative and developmental potential, supporting the notion that dormant mammary cancers harbor transformed mammary progenitor cells. Our results define an experimental paradigm for elucidating biological properties of dormant malignancy.