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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., (©2014 American Association for Cancer Research.)

Recurrent breast cancer is typically an incurable disease and, as such, is disproportionately responsible for deaths from this disease. Recurrent breast cancers arise from the pool of disseminated tumor cells (DTC) that survive adjuvant or neoadjuvant therapy, and patients with detectable DTCs following therapy are at substantially increased risk for recurrence. Consequently, the identification of pathways that contribute to the survival of breast cancer cells following therapy could aid in the development of more effective therapies that decrease the burden of residual disease and thereby reduce the risk of breast cancer recurrence. We now report that ceramide kinase (Cerk) is required for mammary tumor recurrence following HER2/neu pathway inhibition and is spontaneously upregulated during tumor recurrence in multiple genetically engineered mouse models for breast cancer. We find that Cerk is rapidly upregulated in tumor cells following HER2/neu downregulation or treatment with Adriamycin and that Cerk is required for tumor cell survival following HER2/neu downregulation. Consistent with our observations in mouse models, analysis of gene expression profiles from more than 2,200 patients revealed that elevated CERK expression is associated with an increased risk of recurrence in women with breast cancer. In addition, although CERK expression is associated with aggressive subtypes of breast cancer, including those that are estrogen receptor-negative, HER2(+), basal-like, or high grade, its association with poor clinical outcome is independent of these clinicopathologic variables. Together, our findings identify a functional role for Cerk in breast cancer recurrence and suggest the clinical utility of agents targeted against this prosurvival pathway., (©2014 American Association for Cancer Research.)

Tumor development relies upon essential contributions from the tumor microenvironment and host immune alterations. These contributions may inform the plasma proteome in a manner that could be exploited for cancer diagnosis and prognosis. In this study, we employed a systems biology approach to characterize the plasma proteome response in the inducible HER2/neu mouse model of breast cancer during tumor induction, progression, and regression. Mass spectrometry data derived from approximately 1.6 million spectra identified protein networks involved in wound healing, microenvironment, and metabolism that coordinately changed during tumor development. The observed alterations developed prior to cancer detection, increased progressively with tumor growth and reverted toward baseline with tumor regression. Gene expression and immunohistochemical analyses suggested that the cancer-associated plasma proteome was derived from transcriptional responses in the noncancerous host tissues as well as the developing tumor. The proteomic signature was distinct from a nonspecific response to inflammation. Overall, the developing tumor simultaneously engaged a number of innate physiologic processes, including wound repair, immune response, coagulation and complement cascades, tissue remodeling, and metabolic homeostasis that were all detectable in plasma. Our findings offer an integrated view of tumor development relevant to plasma-based strategies to detect and diagnose cancer.

Women who have their first child early in life have a substantially lower lifetime risk of breast cancer. The mechanism for this is unknown. Similar to humans, rats exhibit parity-induced protection against mammary tumorigenesis. To explore the basis for this phenomenon, we identified persistent pregnancy-induced changes in mammary gene expression that are tightly associated with protection against tumorigenesis in multiple inbred rat strains. Four inbred rat strains that exhibit marked differences in their intrinsic susceptibilities to carcinogen-induced mammary tumorigenesis were each shown to display significant protection against methylnitrosourea-induced mammary tumorigenesis following treatment with pregnancy levels of estradiol and progesterone. Microarray expression profiling of parous and nulliparous mammary tissue from these four strains yielded a common 70-gene signature. Examination of the genes constituting this signature implicated alterations in transforming growth factor-beta signaling, the extracellular matrix, amphiregulin expression, and the growth hormone/insulin-like growth factor I axis in pregnancy-induced alterations in breast cancer risk. Notably, related molecular changes have been associated with decreased mammographic density, which itself is strongly associated with decreased breast cancer risk. Our findings show that hormone-induced protection against mammary tumorigenesis is widely conserved among divergent rat strains and define a gene expression signature that is tightly correlated with reduced mammary tumor susceptibility as a consequence of a normal developmental event. Given the conservation of this signature, these pathways may contribute to pregnancy-induced protection against breast cancer.

A conditional tetracycline-responsive transgenic mouse model with deregulated estrogen receptor alpha expression in mammary epithelial cells developed ductal hyperplasia (DH), lobular hyperplasia, and ductal carcinoma in situ (DCIS) by 4 months of age. Higher proliferative rates were found in both normal and abnormal ductal and lobular structures. DH and DCIS but not normal ductal structures showed an increased percentage of cells with nuclear-localized cyclin D1. No differences in either the prevalence or extent of these phenotypes following exogenous 17beta-estradiol treatment were found suggesting that alteration of ERalpha expression was the rate-limiting factor in initiation of DH, lobular hyperplasia, and DCIS.

To address the role of transforming growth factor (TGF) beta in the progression of established tumors while avoiding the confounding inhibitory effects of TGF-beta on early transformation, we generated doxycycline (DOX)-inducible triple transgenic mice in which active TGF-beta1 expression could be conditionally regulated in mouse mammary tumor cells transformed by the polyomavirus middle T antigen. DOX-mediated induction of TGF-beta1 for as little as 2 weeks increased lung metastases >10-fold without a detectable effect on primary tumor cell proliferation or tumor size. DOX-induced active TGF-beta1 protein and nuclear Smad2 were restricted to cancer cells, suggesting a causal association between autocrine TGF-beta and increased metastases. Antisense-mediated inhibition of TGF-beta1 in polyomavirus middle T antigen-expressing tumor cells also reduced basal cell motility, survival, anchorage-independent growth, tumorigenicity, and metastases. Therefore, induction and/or activation of TGF-beta in hosts with established TGF-beta-responsive cancers can rapidly accelerate metastatic progression.

While screening for protein kinases expressed in the murine mammary gland, we identified previously a Ca2+/calmodulin-dependent kinase, Pnck, that is most closely related to CaMKI. In this report, we show that Pnck is temporally regulated during murine mammary development with highest levels of expression observed late in pregnancy, concomitant with the decreased cellular proliferation and terminal differentiation of the mammary epithelium. Consistent with this finding, Pnck is up-regulated in confluent mammary epithelial cells and is down-regulated as serum-starved cells are stimulated to reenter the cell cycle. In the mammary gland, Pnck is expressed in an epithelial-specific and markedly heterogeneous manner, suggesting that the expression of this kinase may be restricted to a particular mammary epithelial cell type. Potentially related to its heterogeneous in vivo expression pattern, Pnck expression is oncogene-associated in murine epithelial cell lines derived from mammary tumors arising in different transgenic mouse models of breast cancer; cell lines derived from mammary tumors initiated by c-myc or int-2/Fgf3 express Pnck, whereas cell lines initiated by neu or H-ras do not. In an analogous manner, expression of the human homologue of Pnck is restricted to a subset of human breast cancer cell lines. Moreover, PNCK was found to be highly overexpressed in a subset of human primary human breast cancers compared with benign mammary tissue. Together, our data suggest that Pnck may play a role in mammary development, and that expression of this kinase may be restricted to a mammary epithelial cell type that is transformed in a subset of human breast cancers.

The observation that normal pathways of differentiation and development are invariably altered during the process of carcinogenesis implies an intrinsic relationship between these processes. This relationship is particularly evident in the breast, as exemplified by the existence of endocrine risk factors for breast cancer that are related to the timing of normal developmental events. Understanding the mechanisms by which normal developmental events alter breast cancer risk is a central focus of our laboratory. Herein, we describe three approaches being taken in our laboratory toward defining the molecular basis of this relationship. These include: determining the roles played by the tumor suppressor genes, BRCA1 and BRCA2, in the normal differentiation and development of the breast; studying the function of three novel protein kinases identified in our laboratory in mammary epithelial development; and defining the molecular and cellular changes that occur in the breast as a result of reproductive events known to influence breast cancer risk.