Optical imaging reveals chemotherapy-induced metabolic reprogramming of residual disease and recurrence.

Fewer than 20% of triple-negative breast cancer patients experience long-term responses to mainstay chemotherapy. Resistant tumor subpopulations use alternative metabolic pathways to escape therapy, survive, and eventually recur. Here, we show in vivo, longitudinal metabolic reprogramming in residual disease and recurrence of triple-negative breast cancer xenografts with varying sensitivities to the chemotherapeutic drug paclitaxel. Optical imaging coupled with metabolomics reported an increase in non-glucose-driven mitochondrial metabolism and an increase in intratumoral metabolic heterogeneity during regression and residual disease in resistant MDA-MB-231 tumors. Conversely, sensitive HCC-1806 tumors were primarily reliant on glucose uptake and minimal changes in metabolism or heterogeneity were observed over the tumors' therapeutic life cycles. Further, day-matched resistant HCC-1806 tumors revealed a higher reliance on mitochondrial metabolism and elevated metabolic heterogeneity compared to sensitive HCC-1806 tumors. Together, metabolic flexibility, increased reliance on mitochondrial metabolism, and increased metabolic heterogeneity are defining characteristics of persistent residual disease, features that will inform the appropriate type and timing of therapies.

APOBEC3 activity promotes the survival and evolution of drug-tolerant persister cells during acquired resistance to EGFR inhibitors in lung cancer.

APOBEC mutagenesis is one of the most common endogenous sources of mutations in human cancer and is a major source of genetic intratumor heterogeneity. High levels of APOBEC mutagenesis are associated with poor prognosis and aggressive disease across diverse cancers, but the mechanistic and functional impacts of APOBEC mutagenesis on tumor evolution and therapy resistance remain relatively unexplored. To address this, we investigated the contribution of APOBEC mutagenesis to acquired therapy resistance in a model of EGFR-mutant non-small cell lung cancer. We find that inhibition of EGFR in lung cancer cells leads to a rapid and pronounced induction of APOBEC3 expression and activity. Functionally, APOBEC expression promotes the survival of drug-tolerant persister cells (DTPs) following EGFR inhibition. Constitutive expression of APOBEC3B alters the evolutionary trajectory of acquired resistance to the EGFR inhibitor gefitinib, making it more likely that resistance arises through de novo acquisition of the T790M gatekeeper mutation and squamous transdifferentiation during the DTP state. APOBEC3B expression is associated with increased expression of the squamous cell transcription factor ΔNp63 and squamous cell transdifferentiation in gefitinib-resistant cells. Knockout of ΔNp63 in gefitinibresistant cells reduces the expression of the p63 target genes IL1a/b and sensitizes these cells to the thirdgeneration EGFR inhibitor osimertinib. These results suggest that APOBEC activity promotes acquired resistance by facilitating evolution and transdifferentiation in DTPs, and suggest that approaches to target ΔNp63 in gefitinib-resistant lung cancers may have therapeutic benefit.

Immune cells in residual disease and recurrence.

Tumor recurrence following potentially curative therapy constitutes a major obstacle to achieving cures in patients with cancer. Recurrent tumors frequently arise from a population of residual cancer cells - also referred to as minimal residual disease (RD) or persister cells - that survive therapy and persist for prolonged periods prior to tumor relapse. While there has been significant recent progress in deciphering tumor-cell-intrinsic pathways that regulate residual cancer cell survival and recurrence, much less is known about how the tumor microenvironment (TME) of residual tumors impacts persister cancer cells or tumor recurrence. In this review, we highlight recent studies exploring the regulation and function of immune cells in RD and discuss therapeutic opportunities to target immune cells in residual tumors.

In vivo metabolic imaging identifies lipid vulnerability in a preclinical model of Her2+/Neu breast cancer residual disease and recurrence.

Recurrent cancer cells that evade therapy is a leading cause of death in breast cancer patients. This risk is high for women showing an overexpression of human epidermal growth factor receptor 2 (Her2). Cells that persist can rely on different substrates for energy production relative to their primary tumor counterpart. Here, we characterize metabolic reprogramming related to tumor dormancy and recurrence in a doxycycline-induced Her2+/Neu model of breast cancer with varying times to recurrence using longitudinal fluorescence microscopy. Glucose uptake (2-NBDG) and mitochondrial membrane potential (TMRE) imaging metabolically phenotype mammary tumors as they transition to regression, dormancy, and recurrence. "Fast-recurrence" tumors (time to recurrence ~55 days), transition from glycolysis to mitochondrial metabolism during regression and this persists upon recurrence. "Slow-recurrence" tumors (time to recurrence ~100 days) rely on both glycolysis and mitochondrial metabolism during recurrence. The increase in mitochondrial activity in fast-recurrence tumors is attributed to a switch from glucose to fatty acids as the primary energy source for mitochondrial metabolism. Consequently, when fast-recurrence tumors receive treatment with a fatty acid inhibitor, Etomoxir, tumors report an increase in glucose uptake and lipid synthesis during regression. Treatment with Etomoxir ultimately prolongs survival. We show that metabolic reprogramming reports on tumor recurrence characteristics, particularly at time points that are essential for actionable targets. The temporal characteristics of metabolic reprogramming will be critical in determining the use of an appropriate timing for potential therapies; namely, the notion that metabolic-targeted inhibition during regression reports long-term therapeutic benefit.

APOBEC Mutagenesis Inhibits Breast Cancer Growth through Induction of T cell-Mediated Antitumor Immune Responses.

The APOBEC family of cytidine deaminases is one of the most common endogenous sources of mutations in human cancer. Genomic studies of tumors have found that APOBEC mutational signatures are enriched in the HER2 subtype of breast cancer and are associated with immunotherapy response in diverse cancer types. However, the direct consequences of APOBEC mutagenesis on the tumor immune microenvironment have not been thoroughly investigated. To address this, we developed syngeneic murine mammary tumor models with inducible expression of APOBEC3B. We found that APOBEC activity induced antitumor adaptive immune responses and CD4+ T cell-mediated, antigen-specific tumor growth inhibition. Although polyclonal APOBEC tumors had a moderate growth defect, clonal APOBEC tumors were almost completely rejected, suggesting that APOBEC-mediated genetic heterogeneity limits antitumor adaptive immune responses. Consistent with the observed immune infiltration in APOBEC tumors, APOBEC activity sensitized HER2-driven breast tumors to anti-CTLA-4 checkpoint inhibition and led to a complete response to combination anti-CTLA-4 and anti-HER2 therapy. In human breast cancers, the relationship between APOBEC mutagenesis and immunogenicity varied by breast cancer subtype and the frequency of subclonal mutations. This work provides a mechanistic basis for the sensitivity of APOBEC tumors to checkpoint inhibitors and suggests a rationale for using APOBEC mutational signatures and clonality as biomarkers predicting immunotherapy response in HER2-positive (HER2+) breast cancers.

Context-dependent effects of whole-genome duplication during mammary tumor recurrence.

Whole-genome duplication (WGD) generates polyploid cells possessing more than two copies of the genome and is among the most common genetic abnormalities in cancer. The frequency of WGD increases in advanced and metastatic tumors, and WGD is associated with poor prognosis in diverse tumor types, suggesting a functional role for polyploidy in tumor progression. Experimental evidence suggests that polyploidy has both tumor-promoting and suppressing effects, but how polyploidy regulates tumor progression remains unclear. Using a genetically engineered mouse model of Her2-driven breast cancer, we explored the prevalence and consequences of whole-genome duplication during tumor growth and recurrence. While primary tumors in this model are invariably diploid, nearly 40% of recurrent tumors undergo WGD. WGD in recurrent tumors was associated with increased chromosomal instability, decreased proliferation and increased survival in stress conditions. The effects of WGD on tumor growth were dependent on tumor stage. Surprisingly, in recurrent tumor cells WGD slowed tumor formation, growth rate and opposed the process of recurrence, while WGD promoted the growth of primary tumors. These findings highlight the importance of identifying conditions that promote the growth of polyploid tumors, including the cooperating genetic mutations that allow cells to overcome the barriers to WGD tumor cell growth and proliferation.

DDR2 upregulation confers ferroptosis susceptibility of recurrent breast tumors through the Hippo pathway.

Recurrent breast cancer presents significant challenges with aggressive phenotypes and treatment resistance. Therefore, novel therapeutics are urgently needed. Here, we report that murine recurrent breast tumor cells, when compared with primary tumor cells, are highly sensitive to ferroptosis. Discoidin Domain Receptor Tyrosine Kinase 2 (DDR2), the receptor for collagen I, is highly expressed in ferroptosis-sensitive recurrent tumor cells and human mesenchymal breast cancer cells. EMT regulators, TWIST and SNAIL, significantly induce DDR2 expression and sensitize ferroptosis in a DDR2-dependent manner. Erastin treatment induces DDR2 upregulation and phosphorylation, independent of collagen I. Furthermore, DDR2 knockdown in recurrent tumor cells reduces clonogenic proliferation. Importantly, both the ferroptosis protection and reduced clonogenic growth may be compatible with the compromised YAP/TAZ upon DDR2 inhibition. Collectively, these findings identify the important role of EMT-driven DDR2 upregulation in recurrent tumors in maintaining growth advantage but activating YAP/TAZ-mediated ferroptosis susceptibility, providing potential strategies to eradicate recurrent breast cancer cells with mesenchymal features.

G9a Promotes Breast Cancer Recurrence through Repression of a Pro-inflammatory Program.

Dysregulated gene expression is a common feature of cancer and may underlie some aspects of tumor progression, including tumor relapse. Here, we show that recurrent mammary tumors exhibit global changes in gene expression and histone modifications and acquire dependence on the G9a histone methyltransferase. Genetic ablation of G9a delays tumor recurrence, and pharmacologic inhibition of G9a slows the growth of recurrent tumors. Mechanistically, G9a activity is required to silence pro-inflammatory cytokines, including tumor necrosis factor (TNF), through H3K9 methylation at gene promoters. G9a inhibition induces re-expression of these cytokines, leading to p53 activation and necroptosis. Recurrent tumors upregulate receptor interacting protein kinase-3 (RIPK3) expression and are dependent upon RIPK3 activity. High RIPK3 expression renders recurrent tumors sensitive to necroptosis following G9a inhibition. These findings demonstrate that G9a-mediated silencing of pro-necroptotic proteins is a critical step in tumor recurrence and suggest that G9a is a targetable dependency in recurrent breast cancer.

Adaptation and selection shape clonal evolution of tumors during residual disease and recurrence.

The survival and recurrence of residual tumor cells following therapy constitutes one of the biggest obstacles to obtaining cures in breast cancer, but it remains unclear how the clonal composition of tumors changes during relapse. We use cellular barcoding to monitor clonal dynamics during tumor recurrence in vivo. We find that clonal diversity decreases during tumor regression, residual disease, and recurrence. The recurrence of dormant residual cells follows several distinct routes. Approximately half of the recurrent tumors exhibit clonal dominance with a small number of subclones comprising the vast majority of the tumor; these clonal recurrences are frequently dependent upon Met gene amplification. A second group of recurrent tumors comprises thousands of subclones, has a clonal architecture similar to primary tumors, and is dependent upon the Jak/Stat pathway. Thus the regrowth of dormant tumors proceeds via multiple routes, producing recurrent tumors with distinct clonal composition, genetic alterations, and drug sensitivities.

NRF2 activation promotes the recurrence of dormant tumour cells through regulation of redox and nucleotide metabolism.

The survival and recurrence of dormant tumour cells following therapy is a leading cause of death in cancer patients. The metabolic properties of these cells are likely distinct from those of rapidly growing tumours. Here we show that Her2 down-regulation in breast cancer cells promotes changes in cellular metabolism, culminating in oxidative stress and compensatory upregulation of the antioxidant transcription factor, NRF2. NRF2 is activated during dormancy and in recurrent tumours in animal models and breast cancer patients with poor prognosis. Constitutive activation of NRF2 accelerates recurrence, while suppression of NRF2 impairs it. In recurrent tumours, NRF2 signalling induces a transcriptional metabolic reprogramming to re-establish redox homeostasis and upregulate de novo nucleotide synthesis. The NRF2-driven metabolic state renders recurrent tumour cells sensitive to glutaminase inhibition, which prevents reactivation of dormant tumour cells in vitro, suggesting that NRF2-high dormant and recurrent tumours may be targeted. These data provide evidence that NRF2-driven metabolic reprogramming promotes the recurrence of dormant breast cancer.

RIPK3 upregulation confers robust proliferation and collateral cystine-dependence on breast cancer recurrence.

The molecular and genetic basis of tumor recurrence is complex and poorly understood. RIPK3 is a key effector in programmed necrotic cell death and, therefore, its expression is frequently suppressed in primary tumors. In a transcriptome profiling between primary and recurrent breast tumor cells from a murine model of breast cancer recurrence, we found that RIPK3, while absent in primary tumor cells, is dramatically reexpressed in recurrent breast tumor cells by an epigenetic mechanism. Unexpectedly, we found that RIPK3 knockdown in recurrent tumor cells reduced clonogenic growth, causing cytokinesis failure, p53 stabilization, and repressed the activities of YAP/TAZ. These data uncover a surprising role of the pro-necroptotic RIPK3 kinase in enabling productive cell cycle during tumor recurrence. Remarkably, high RIPK3 expression also rendered recurrent tumor cells exquisitely dependent on extracellular cystine and undergo necroptosis upon cystine deprivation. The induction of RIPK3 in recurrent tumors unravels an unexpected mechanism that paradoxically confers on tumors both growth advantage and necrotic vulnerability, providing potential strategies to eradicate recurrent tumors.

CCL5 promotes breast cancer recurrence through macrophage recruitment in residual tumors.

Over half of breast-cancer-related deaths are due to recurrence 5 or more years after initial diagnosis and treatment. This latency suggests that a population of residual tumor cells can survive treatment and persist in a dormant state for many years. The role of the microenvironment in regulating the survival and proliferation of residual cells following therapy remains unexplored. Using a conditional mouse model for Her2-driven breast cancer, we identify interactions between residual tumor cells and their microenvironment as critical for promoting tumor recurrence. Her2 downregulation leads to an inflammatory program driven by TNFα/NFκB signaling, which promotes immune cell infiltration in regressing and residual tumors. The cytokine CCL5 is elevated following Her2 downregulation and remains high in residual tumors. CCL5 promotes tumor recurrence by recruiting CCR5-expressing macrophages, which may contribute to collagen deposition in residual tumors. Blocking this TNFα-CCL5-macrophage axis may be efficacious in preventing breast cancer recurrence.

Optical Imaging of Glucose Uptake and Mitochondrial Membrane Potential to Characterize Her2 Breast Tumor Metabolic Phenotypes.

With the large number of women diagnosed and treated for breast cancer each year, the importance of studying recurrence has become evident due to most deaths from breast cancer resulting from tumor recurrence following therapy. To mitigate this, cellular and molecular pathways used by residual disease prior to recurrence must be studied. An altered metabolism has long been considered a hallmark of cancer, and several recent studies have gone further to report metabolic dysfunction and alterations as key to understanding the underlying behavior of dormant and recurrent cancer cells. Our group has used two probes, 2-[N-(7-nitrobenz-2-oxa-1, 3-diaxol-4-yl) amino]-2-deoxyglucose (2-NBDG) and tetramethyl rhodamine ethyl ester (TMRE), to image glucose uptake and mitochondrial membrane potential, respectively, to report changes in metabolism between primary tumors, regression, residual disease, and after regrowth in genetically engineered mouse (GEM)-derived mammospheres. Imaging revealed unique metabolic phenotypes across the stages of tumor development. Although primary mammospheres overexpressing Her2 maintained increased glucose uptake ("Warburg effect"), after Her2 downregulation, during regression and residual disease, mammospheres appeared to switch to oxidative phosphorylation. Interestingly, in mammospheres where Her2 overexpression was turned back on to model recurrence, glucose uptake was lowest, indicating a potential change in substrate preference following the reactivation of Her2, reeliciting growth. Our findings highlight the importance of imaging metabolic adaptions to gain insight into the fundamental behaviors of residual and recurrent disease. IMPLICATIONS: This study demonstrates these functional fluorescent probes' ability to report metabolic adaptations during primary tumor growth, regression, residual disease, and regrowth in Her2 breast tumors.

Epigenetic silencing of tumor suppressor Par-4 promotes chemoresistance in recurrent breast cancer.

Tumor relapse is the leading cause of death in breast cancer, largely due to the fact that recurrent tumors are frequently resistant to chemotherapy. We previously reported that downregulation of the proapoptotic protein Par-4 promotes tumor recurrence in genetically engineered mouse models of breast cancer recurrence. In the present study, we examined the mechanism and functional significance of Par-4 downregulation in recurrent tumors. We found that epithelial-to-mesenchymal transition (EMT) promotes epigenetic silencing of Par-4 in recurrent tumors. Par-4 silencing proceeded through binding of the EMT transcription factor Twist to the Par-4 promoter, where Twist induced a unique bivalent chromatin domain. This bivalent configuration conferred plasticity at the Par-4 promoter, and Par-4 silencing could be reversed with pharmacologic inhibitors of Ezh2 and HDAC1/2. Using an epigenome editing approach to reexpress Par-4 by specifically reversing the histone modifications found in recurrent tumors, we found that Par-4 reexpression sensitized recurrent tumors to chemotherapy in vitro and in vivo. Upon reexpression, Par-4 bound to the protein phosphatase PP1, caused widespread changes in phosphorylation of cytoskeletal proteins, and cooperated with microtubule-targeting drugs to induce mitotic defects. These results identify Twist-induced epigenetic silencing of Par-4 as a targetable axis that promotes chemoresistance in recurrent breast cancer.

Foxo-dependent Par-4 Upregulation Prevents Long-term Survival of Residual Cells Following PI3K-Akt Inhibition.

Tumor recurrence is a leading cause of death and is thought to arise from a population of residual cells that survive treatment. These residual cancer cells can persist, locally or at distant sites, for years or decades. Therefore, understanding the pathways that regulate residual cancer cell survival may suggest opportunities for targeting these cells to prevent recurrence. Previously, it was observed that the proapoptotic protein (PAWR/Par-4) negatively regulates residual cell survival and recurrence in mice and humans. However, the mechanistic underpinnings on how Par-4 expression is regulated are unclear. Here, it is demonstrated that Par-4 is transcriptionally upregulated following treatment with multiple drugs targeting the PI3K-Akt-mTOR signaling pathway, and identify the Forkhead family of transcription factors as mediators of this upregulation. Mechanistically, Foxo3a directly binds to the Par-4 promoter and activates its transcription following inhibition of the PI3K-Akt pathway. This Foxo-dependent Par-4 upregulation limits the long-term survival of residual cells following treatment with therapeutics that target the PI3K-Akt pathway. Taken together, these results indicate that residual breast cancer tumor cell survival and recurrence requires circumventing Foxo-driven Par-4 upregulation and suggest that approaches to enforce Par-4 expression may prevent residual cell survival and recurrence. Mol Cancer Res; 16(4); 599-609. ©2018 AACR.

Oncogene pathway activation in mammary tumors dictates FDG-PET uptake.

Increased glucose utilization is a hallmark of human cancer that is used to image tumors clinically. In this widely used application, glucose uptake by tumors is monitored by positron emission tomography of the labeled glucose analogue 2[(18)F]fluoro-2-deoxy-D-glucose (FDG). Despite its widespread clinical use, the cellular and molecular mechanisms that determine FDG uptake--and that underlie the heterogeneity observed across cancers-remain poorly understood. In this study, we compared FDG uptake in mammary tumors driven by the Akt1, c-MYC, HER2/neu, Wnt1, or H-Ras oncogenes in genetically engineered mice, correlating it to tumor growth, cell proliferation, and expression levels of gene involved in key steps of glycolytic metabolism. We found that FDG uptake by tumors was dictated principally by the driver oncogene and was not independently associated with tumor growth or cellular proliferation. Oncogene downregulation resulted in a rapid decrease in FDG uptake, preceding effects on tumor regression, irrespective of the baseline level of uptake. FDG uptake correlated positively with expression of hexokinase-2 (HK2) and hypoxia-inducible factor-1α (HIF1α) and associated negatively with PFK-2b expression and p-AMPK. The correlation between HK2 and FDG uptake was independent of all variables tested, including the initiating oncogene, suggesting that HK2 is an independent predictor of FDG uptake. In contrast, expression of Glut1 was correlated with FDG uptake only in tumors driven by Akt or HER2/neu. Together, these results demonstrate that the oncogenic pathway activated within a tumor is a primary determinant of its FDG uptake, mediated by key glycolytic enzymes, and provide a framework to interpret effects on this key parameter in clinical imaging.

SPSB1 promotes breast cancer recurrence by potentiating c-MET signaling.

UNLABELLED: Breast cancer mortality is principally due to tumor recurrence; however, the molecular mechanisms underlying this process are poorly understood. We now demonstrate that the suppressor of cytokine signaling protein SPSB1 is spontaneously upregulated during mammary tumor recurrence and is both necessary and sufficient to promote tumor recurrence in genetically engineered mouse models. The recurrence-promoting effects of SPSB1 result from its ability to protect cells from apoptosis induced by HER2/neu pathway inhibition or chemotherapy. This, in turn, is attributable to SPSB1 potentiation of c-MET signaling, such that preexisting SPSB1-overexpressing tumor cells are selected for following HER2/neu downregulation. Consistent with this, SPSB1 expression is positively correlated with c-MET activity in human breast cancers and with an increased risk of relapse in patients with breast cancer in a manner that is dependent upon c-MET activity. Our findings define a novel pathway that contributes to breast cancer recurrence and provide the first evidence implicating SPSB proteins in cancer. SIGNIFICANCE: The principal cause of death from breast cancer is recurrence. This study identifies SPSB1 as a critical mediator of breast cancer recurrence, suggests activation of the SPSB1-c-MET pathway as an important mechanism of therapeutic resistance in breast cancers, and emphasizes that pharmacologic targets for recurrence may be unique to this stage of tumor progression.

Par-4 downregulation promotes breast cancer recurrence by preventing multinucleation following targeted therapy.

Most deaths from breast cancer result from tumor recurrence, but mechanisms underlying tumor relapse are largely unknown. We now report that Par-4 is downregulated during tumor recurrence and that Par-4 downregulation is necessary and sufficient to promote recurrence. Tumor cells with low Par-4 expression survive therapy by evading a program of Par-4-dependent multinucleation and apoptosis that is otherwise engaged following treatment. Low Par-4 expression is associated with poor response to neoadjuvant chemotherapy and an increased risk of relapse in patients with breast cancer, and Par-4 is downregulated in residual tumor cells that survive neoadjuvant chemotherapy. Our findings identify Par-4-induced multinucleation as a mechanism of cell death in oncogene-addicted cells and establish Par-4 as a negative regulator of breast cancer recurrence.

Autocrine prolactin induced by the Pten-Akt pathway is required for lactation initiation and provides a direct link between the Akt and Stat5 pathways.

Extrapituitary prolactin (Prl) is produced in humans and rodents; however, little is known about its in vivo regulation or physiological function. We now report that autocrine prolactin is required for terminal mammary epithelial differentiation during pregnancy and that its production is regulated by the Pten-PI3K-Akt pathway. Conditional activation of the PI3K-Akt pathway in the mammary glands of virgin mice by either Akt1 expression or Pten deletion rapidly induced terminal mammary epithelial differentiation accompanied by the synthesis of milk despite the absence of lobuloalveolar development. Surprisingly, we found that mammary differentiation was due to the PI3K-Akt-dependent synthesis and secretion of autocrine prolactin and downstream activation of the prolactin receptor (Prlr)-Jak-Stat5 pathway. Consistent with this, Akt-induced mammary differentiation was abrogated in Prl(-/-), Prlr(-/-), and Stat5(-/-) mice. Furthermore, cells treated with conditioned medium from mammary glands in which Akt had been activated underwent rapid Stat5 phosphorylation in a manner that was blocked by inhibition of Jak2, treatment with an anti-Prl antibody, or deletion of the prolactin gene. Demonstrating a physiological requirement for autocrine prolactin, mammary glands from lactation-defective Akt1(-/-);Akt2(+/-) mice failed to express autocrine prolactin or activate Stat5 during late pregnancy despite normal levels of circulating serum prolactin and pituitary prolactin production. Our findings reveal that PI3K-Akt pathway activation is necessary and sufficient to induce autocrine prolactin production in the mammary gland, Stat5 activation, and terminal mammary epithelial differentiation, even in the absence of the normal developmental program that prepares the mammary gland for lactation. Together, these findings identify a function for autocrine prolactin during normal development and demonstrate its endogenous regulation by the PI3K-Akt pathway.

Akt is required for Stat5 activation and mammary differentiation.

INTRODUCTION: The Akt pathway plays a central role in regulating cell survival, proliferation and metabolism, and is one of the most commonly activated pathways in human cancer. A role for Akt in epithelial differentiation, however, has not been established. We previously reported that mice lacking Akt1, but not Akt2, exhibit a pronounced metabolic defect during late pregnancy and lactation that results from a failure to upregulate Glut1 as well as several lipid synthetic enzymes. Despite this metabolic defect, however, both Akt1-deficient and Akt2-deficient mice exhibit normal mammary epithelial differentiation and Stat5 activation. METHODS: In light of the overlapping functions of Akt family members, we considered the possibility that Akt may play an essential role in regulating mammary epithelial development that is not evident in Akt1-deficient mice due to compensation by other Akt isoforms. To address this possibility, we interbred mice bearing targeted deletions in Akt1 and Akt2 and determined the effect on mammary differentiation during pregnancy and lactation. RESULTS: Deletion of one allele of Akt2 in Akt1-deficient mice resulted in a severe defect in Stat5 activation during late pregnancy that was accompanied by a global failure of terminal mammary epithelial cell differentiation, as manifested by the near-complete loss in production of the three principal components of milk: lactose, lipid, and milk proteins. This defect was due, in part, to a failure of pregnant Akt1(-/-);Akt2(+/-) mice to upregulate the positive regulator of Prlr-Jak-Stat5 signaling, Id2, or to downregulate the negative regulators of Prlr-Jak-Stat5 signaling, caveolin-1 and Socs2. CONCLUSIONS: Our findings demonstrate an unexpected requirement for Akt in Prlr-Jak-Stat5 signaling and establish Akt as an essential central regulator of mammary epithelial differentiation and lactation.