Elizabeth A. Musgrove

Biological determinants of endocrine resistance in breast cancer

Endocrine therapies targeting oestrogen action (anti-oestrogens, such as tamoxifen, and aromatase inhibitors) decrease mortality from breast cancer, but their efficacy is limited by intrinsic and acquired therapeutic resistance. Candidate molecular biomarkers and gene expression signatures of tamoxifen response emphasize the importance of deregulation of proliferation and survival signalling in endocrine resistance. However, definition of the specific genetic lesions and molecular processes that determine clinical endocrine resistance is incomplete. The development of large-scale computational and genetic approaches offers the promise of identifying the mediators of endocrine resistance that may be exploited as potential therapeutic targets and biomarkers of response in the clinic.

Whole genomes redefine the mutational landscape of pancreatic cancer

Pancreatic cancer remains one of the most lethal of malignancies and a major health burden. We performed whole-genome sequencing and copy number variation (CNV) analysis of 100 pancreatic ductal adenocarcinomas (PDACs). Chromosomal rearrangements leading to gene disruption were prevalent, affecting genes known to be important in pancreatic cancer (TP53, SMAD4, CDKN2A, ARID1A and ROBO2) and new candidate drivers of pancreatic carcinogenesis (KDM6A and PREX2). Patterns of structural variation (variation in chromosomal structure) classified PDACs into 4 subtypes with potential clinical utility: the subtypes were termed stable, locally rearranged, scattered and unstable. A significant proportion harboured focal amplifications, many of which contained druggable oncogenes (ERBB2, MET, FGFR1, CDK6, PIK3R3 and PIK3CA), but at low individual patient prevalence. Genomic instability co-segregated with inactivation of DNA maintenance genes (BRCA1, BRCA2 or PALB2) and a mutational signature of DNA damage repair deficiency. Of 8 patients who received platinum therapy, 4 of 5 individuals with these measures of defective DNA maintenance responded.

Cyclin D as a therapeutic target in cancer

Cyclin D1, and to a lesser extent the other D-type cyclins, is frequently deregulated in cancer and is a biomarker of cancer phenotype and disease progression. The ability of these cyclins to activate the cyclin-dependent kinases (CDKs) CDK4 and CDK6 is the most extensively documented mechanism for their oncogenic actions and provides an attractive therapeutic target. Is this an effective means of targeting the cyclin D oncogenes, and how might the patient subgroups that are most likely to benefit be identified?

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Cyclins and Breast Cancer

The D-type and E-type cyclins control the G1 to S phase transition during normal cell cycle progression and are critical components of steroid- and growth factor-induced mitogenesis in breast epithelial cells. Mammary epithelial cell-specific overexpression of these genes leads to mammary carcinoma, while in cyclin D1-deficient mice mammary gland development is arrested prior to lobuloalveolar development. Cyclin D1 null mice are resistant to mammary carcinoma induced by the neu and ras oncogenes, indicating an essential role for cyclin D1 in the development of some mammary cancers. Cyclin D1 and E1 are commonly overexpressed in primary breast cancer, with some evidence of an association with an adverse patient outcome. This observation may result in part from their ability to confer resistance to endocrine therapies. The functional consequences of cyclin E overexpression in breast cancer are likely related to its role in cell cycle progression, whereas that of cyclin D1 may also be a consequence of a more recently defined role in transcriptional regulation.

Cyclin D1, EMS1 and 11q13 amplification in breast cancer.

Chromosome locus 11q13 is frequently amplified in a number of human cancers including carcinoma of the breast where up to 15% carry this chromosomal abnormality. Originally 11q13 amplification was thought to involve a single amplicon spanning many megabases, but more recent data have identified four core regions within 11q13 that can be amplified independently or together in different combinations. Although the region harbors several genes with known or suspected oncogenic potential, the complex structure of the amplicons and the fact that 11q13 is gene-rich have made definitive identification of specific genes that contribute to the genesis and progression of breast cancer a difficult and continuing process. To date CCND1, encoding the cell cycle regulatory gene cyclin D1, and EMS1, encoding the filamentous actin binding protein and c-Src substrate cortactin, are the favored candidates responsible for the emergence of two of the four amplification cores.

c-Myc or Cyclin D1 Mimics Estrogen Effects on Cyclin E-Cdk2 Activation and Cell Cycle Reentry

ABSTRACT Estrogen-induced progression through G1 phase of the cell cycle is preceded by increased expression of the G1-phase regulatory proteins c-Myc and cyclin D1. To investigate the potential contribution of these proteins to estrogen action, we derived clonal MCF-7 breast cancer cell lines in which c-Myc or cyclin D1 was expressed under the control of the metal-inducible metallothionein promoter. Inducible expression of either c-Myc or cyclin D1 was sufficient for S-phase entry in cells previously arrested in G1 phase by pretreatment with ICI 182780, a potent estrogen antagonist. c-Myc expression was not accompanied by increased cyclin D1 expression or Cdk4 activation, nor was cyclin D1 induction accompanied by increases in c-Myc. Expression of c-Myc or cyclin D1 was sufficient to activate cyclin E-Cdk2 by promoting the formation of high-molecular-weight complexes lacking the cyclin-dependent kinase inhibitor p21, as has been described, following estrogen treatment. Interestingly, this was accompanied by an association between active cyclin E-Cdk2 complexes and hyperphosphorylated p130, identifying a previously undefined role for p130 in estrogen action. These data provide evidence for distinct c-Myc and cyclin D1 pathways in estrogen-induced mitogenesis which converge on or prior to the formation of active cyclin E-Cdk2-p130 complexes and loss of inactive cyclin E-Cdk2-p21 complexes, indicating a physiologically relevant role for the cyclin E binding motifs shared by p130 and p21.

PI3K pathway activation in breast cancer is associated with the basal-like phenotype and cancer-specific mortality.

Breast cancer is a common malignancy with current biological therapies tailored to steroid hormone (ER, PR) and HER2 receptor status. Understanding the biological basis of resistance to current targeted therapies and the identification of new potential therapeutic targets is an ongoing challenge. The PI3K pathway is altered in a high proportion of breast cancers and may contribute to therapeutic resistance. We undertook an integrative study of mutational, copy number and expression analyses of key regulators of the PI3K pathway in a cohort of 292 invasive breast cancer patients with known treatment outcomes. The alterations identified in this cohort included PIK3CA mutations (12/168, i.e. 7%), PIK3CA copy number gain (28/209, i.e. 14%), PTEN loss (73/258, i.e. 28%) and AKT activation (62/258, i.e. 24%). Overall at least 1 parameter was altered in 72% (139/193) of primary breast cancers. PI3K pathway activation was significantly associated with ER negative (p = 0.0008) and PR negative (p = 0.006) status, high tumor grade (p = 0.032) and a “basal‐like” phenotype (p = 0.01), where 92% (25/27) of tumors had an altered pathway. In univariate analysis, PI3K pathway aberrations were associated with death from breast cancer; however, this relationship was not maintained in multivariate analysis. No association was identified between an activated pathway and outcome in tamoxifen‐ or chemotherapy‐treated patients. We concluded that >70% of breast cancers have an alteration in at least 1 component of the PI3K pathway and this might be exploited to therapeutic advantage especially in “basal‐like” cancers.

Cell cycle control in breast cancer cells

In breast cancer, cyclins D1 and E and the cyclin‐dependent kinase inhibitors p21 (Waf1/Cip1)and p27 (Kip1) are important in cell‐cycle control and as potential oncogenes or tumor suppressor genes. They are regulated in breast cancer cells following mitogenic stimuli including activation of receptor tyrosine kinases and steroid hormone receptors, and their deregulation frequently impacts on breast cancer outcome, including response to therapy. The cyclin‐dependent kinase inhibitor p16 (INK4A) also has a critical role in transformation of mammary epithelial cells. In addition to their roles in cell cycle control, some of these molecules, particularly cyclin D1, have actions that are not mediated through regulation of cyclin‐dependent kinase activity but may be important for loss of proliferative control during mammary oncogenesis.

Estrogen and progestin regulation of cell cycle progression.

Estrogens and progesterone, acting via theirspecific nuclear receptors, are essential for normalmammary gland development and differentiated function.The molecular mechanisms through which these effects are mediated are not well defined, althoughsignificant recent progress has been made in linkingsteroid hormone action to cell cycle progression. Thisreview summarizes data identifying c-myc and cyclin D1 as major downstream targets of bothestrogenand progestin-stimulated cell cycle progressionin human breast cancer cells. Additionally, estrogeninduces the formation of high specific activity forms of the cyclin E-Cdk2 enzyme complex lacking thecyclin-dependent kinase (CDK)3 inhibitor, p21. Thedelayed growth inhibitory effects of progestins, whichare likely to be prerequisites for manifestation of their function in differentiation, alsoinvolve decreases in cyclin D1 and E gene expression andrecruitment of CDK inhibitors into cyclin D1-Cdk4 andcyclin E-Cdk2 complexes. Thus estrogens and progestins affect CDK function not only by effects oncyclin abundance but also by regulating the recruitmentof CDK inhibitors and, as yet undefined, additionalcomponents which determine the activity of the CDK complexes. These effects of estrogens andprogestins are likely to be major contributors to theirregulation of mammary epithelial cell proliferation anddifferentiation.