Christine L. Clarke

Progesterone induces adult mammary stem cell expansion

Reproductive history is the strongest risk factor for breast cancer after age, genetics and breast density. Increased breast cancer risk is entwined with a greater number of ovarian hormone-dependent reproductive cycles, yet the basis for this predisposition is unknown. Mammary stem cells (MaSCs) are located within a specialized niche in the basal epithelial compartment that is under local and systemic regulation. The emerging role of MaSCs in cancer initiation warrants the study of ovarian hormones in MaSC homeostasis. Here we show that the MaSC pool increases 14-fold during maximal progesterone levels at the luteal dioestrus phase of the mouse. Stem-cell-enriched CD49fhi cells amplify at dioestrus, or with exogenous progesterone, demonstrating a key role for progesterone in propelling this expansion. In aged mice, CD49fhi cells display stasis upon cessation of the reproductive cycle. Progesterone drives a series of events where luminal cells probably provide Wnt4 and RANKL signals to basal cells which in turn respond by upregulating their cognate receptors, transcriptional targets and cell cycle markers. Our findings uncover a dynamic role for progesterone in activating adult MaSCs within the mammary stem cell niche during the reproductive cycle, where MaSCs are putative targets for cell transformation events leading to breast cancer.

Loss of Co-ordinate Expression of Progesterone Receptors A and B is an Early Event in Breast Carcinogenesis

Progesterone receptor (PR) mediates the effects of progesterone in mammary tissues and plays a crucial role in normal breast development and in breast cancer. PR proteins are expressed as two isoforms, PRA and PRB, that have different capacities to activate target genes, yet it is unknown whether progesterone action in normal and malignant breast is mediated by PRA and/or PRB. This study determines the relative expression of PRA and PRB in normal breast and in benign, premalignant and malignant archival breast lesions by dual immunofluorescent histochemistry.In normal breast and in proliferative disease without atypia (PDWA) PRA and PRB were co-expressed within the same cells in comparable amounts, implicating both isoforms in progesterone action. In atypical lesions, however, there was a significant increase in predominant expression of PRA or PRB, with lesion progression from the normal state to malignancy. PR isoform predominance, especially PRA predominance, was evident in a high proportion of ductal carcinomas in situ (DCIS) and invasive breast lesions.In the normal breast and in PDWA, the relative expression of PRA and PRB in adjacent cells was homogenous. There was a significant increase in cell-to-cell heterogeneity of PR isoform expression in ADH and DCIS lesions and in the majority of breast cancers. Heterogeneous cell-to-cell expression of PR isoforms occurred prior to overall predominant expression of one isoform in premalignant breast lesions, demonstrating that loss of control of relative PRA:PRB expression is an early event in the development of breast cancer. PRA:PRB ratios within a breast lesion are likely to be important as both markers and effectors of tumor growth and development, and progressively aberrant PR isoform expression may play a role in the etiology of breast cancer.

Analysis of cancer risk and BRCA1 and BRCA2 mutation prevalence in the kConFab familial breast cancer resource.

IntroductionThe Kathleen Cuningham Foundation Consortium for Research into Familial Breast Cancer (kConFab) is a multidisciplinary, collaborative framework for the investigation of familial breast cancer. Based in Australia, the primary aim of kConFab is to facilitate high-quality research by amassing a large and comprehensive resource of epidemiological and clinical data with biospecimens from individuals at high risk of breast and/or ovarian cancer, and from their close relatives.MethodsEpidemiological, family history and lifestyle data, as well as biospecimens, are collected from multiple-case breast cancer families ascertained through family cancer clinics in Australia and New Zealand. We used the Tyrer-Cuzick algorithms to assess the prospective risk of breast cancer in women in the kConFab cohort who were unaffected with breast cancer at the time of enrolment in the study.ResultsOf kConFabs first 822 families, 518 families had multiple cases of female breast cancer alone, 239 had cases of female breast and ovarian cancer, 37 had cases of female and male breast cancer, and 14 had both ovarian cancer as well as male and female breast cancer. Data are currently held for 11,422 people and germline DNAs for 7,389. Among the 812 families with at least one germline sample collected, the mean number of germline DNA samples collected per family is nine. Of the 747 families that have undergone some form of mutation screening, 229 (31%) carry a pathogenic or splice-site mutation in BRCA1 or BRCA2. Germline DNAs and data are stored from 773 proven carriers of BRCA1 or BRCA1 mutations. kConFabs fresh tissue bank includes 253 specimens of breast or ovarian tissue – both normal and malignant – including 126 from carriers of BRCA1 or BRCA2 mutations.ConclusionThese kConFab resources are available to researchers anywhere in the world, who may apply to kConFab for biospecimens and data for use in ethically approved, peer-reviewed projects. A high calculated risk from the Tyrer-Cuzick algorithms correlated closely with the subsequent occurrence of breast cancer in BRCA1 and BRCA2 mutation positive families, but this was less evident in families in which no pathogenic BRCA1 or BRCA2 mutation has been detected.

Imatinib Disposition and ABCB1 (MDR1, P-Glycoprotein) Genotype

The aim of this study was to explore the impact of individual variation in drug elimination on imatinib disposition. Twenty‐two patients with gastrointestinal stromal tumor or chronic myeloid leukemia initially received imatinib 600 mg daily with dosage subsequently toxicity adjusted. Pharmacokinetic parameters on day 1 and at steady‐state were compared with elimination phenotype and single‐nucleotide polymorphisms of CYP3A5 and ABCB1. A fivefold variation in estimated imatinib clearance (CL/F) was present on day 1 and mean CL/F had fallen by 26% at steady state. This reduction in imatinib CL/F was associated with ABCB1 genotype, being least apparent in thymidine homozygotes at the 1236T>C, 2677G>T/A and 3435C>T loci. Toxicity‐related dose reduction also tended to be less common in these individuals. ABCB1 genotype was associated with steady‐state CL/F due to an apparent genotype‐specific influence of imatinib on elimination. Further evaluation of ABCB1 genotype and imatinib dosage is warranted.

Detection of progesterone receptor forms A and B by immunohistochemical analysis

Aim—The measurement of progesterone receptors (PR) is recommended as part of the clinical management of breast and endometrial cancers, and immunohistochemistry on formalin fixed tissue is now the method of choice. PR is expressed as two isoforms, PRA and PRB, and although both these proteins are expressed in hormone dependent cancers, there is evidence that a large proportion of tumours express a predominance of one isoform. Therefore, it is essential to document the individual detection of PRA and PRB by the presently available anti-PR antibodies. The aim of this study is to investigate the detection of PR isoforms A and B in formalin fixed, paraffin wax embedded cell lines and tissue sections by immunohistochemistry, using a panel of commercial and in house antibodies to human PR. Methods—PR negative cell lines stably transfected to express only PRA (MCF-7Mll/PRA) or PRB (MDA-MB-231/PRB), and tissue sections of human breast carcinoma and normal endometrium were stained using an immunoperoxidase method. A panel of primary PR specific antibodies was evaluated for ability to detect both PRA and PRB proteins, and for intensity and distribution of positive staining under optimal conditions. Results—Of the 11 antibodies assessed, only four recognised PRA and PRB similarly. Six recognised PRA proteins but were unable to detect PRB expression in the cell lines expressing only PRA or PRB. In tissues expressing high amounts of PRA and PRB, all antibodies tested demonstrated positive PR staining. However, in tissues expressing a predominance of PRB, differential staining patterns were observed, with variations in staining intensity and in the proportion of cells positive for PR. Conclusions—Most PR specific antibodies tested failed to detect PRB in formalin fixed tissue by immunohistochemical techniques, despite their ability to do so by immunoblot analysis. These observations suggest that there are conformational differences between PRA and PRB that mask epitopes on the PRB protein recognised by most anti-PR antibodies. The selection of antibodies that recognise both PRB and PRA in formalin fixed tissue is essential for the accurate evaluation of PR positivity in clinical specimens.

Progesterone receptors - animal models and cell signaling in breast cancer Expression and transcriptional activity of progesterone receptor A and progesterone receptor B in mammalian cells

Progesterone is an essential regulator of normal female reproductive function. Its effects are mediated by two nuclear progesterone receptor (PR) proteins, PRA and PRB, which are identical except for an additional 164 amino acids at the N-terminal end of PRB. Transcriptional analyses of the two receptor forms have assigned strikingly distinct functional signatures to the two PRs, despite their apparent physical similarity. The basis of these differences is yet to be fully understood. Furthermore, these differences are strongly influenced by the cell type and the promoter used. We review the mammalian transcriptional studies of PRA and PRB, and compare them with what is known about their expression and function in target tissues.

Progesterone action in human tissues: regulation by progesterone receptor (PR) isoform expression, nuclear positioning and coregulator expression.

Progesterone is a critical regulator of normal female reproductive function, with diverse tissue-specific effects in the human. The effects of progesterone are mediated by its nuclear receptor (PR) that is expressed as two isoforms, PRA and PRB, which are virtually identical except that PRA lacks 164 amino acids that are present at the N-terminus of PRB. Considerable in vitro evidence suggests that the two PRs are functionally distinct and in animals, tissue-specific distribution patterns of PRA and PRB may account for some of the diversity of progesterone effects. In the human, PRA and PRB are equivalently expressed in most target cells, suggesting that alternative mechanisms control the diversity of progesterone actions. PR mediates the effects of progesterone by association with a range of coregulatory proteins and binding to specific target sequences in progesterone-regulated gene promoters. Ligand activation of PR results in redistribution into discrete subnuclear foci that are detectable by immunofluorescence, probably representing aggregates of multiple transcriptionally active PR-coregulator complexes. PR foci are aberrant in cancers, suggesting that the coregulator composition and number of complexes is altered. A large family of coregulators is now described and the range of proteins known to bind PR exceeds the complement required for transcriptional activation, suggesting that in the human, tissue-specific coregulator expression may modulate progesterone response. In this review, we examine the role of nuclear localization of PR, coregulator association and tissue-specific expression in modulating progesterone action in the human.

CYP3A5 Genotype and Midazolam Clearance in Australian Patients Receiving Chemotherapy

Cytochrome P450 (CYP) 3A enzymes are key metabolizing enzymes for many chemotherapeutic agents, and detection of functionally significant CYP3A genetic variants may be useful in predicting interpatient variation of drug clearance. We have examined the significance of CYP3A5*3 single‐nucleotide polymorphism to overall CYP3A activity in vivo in a predominantly Caucasian Australian cancer population.

Common Breast Cancer Susceptibility Loci Are Associated with Triple-Negative Breast Cancer

Triple-negative breast cancers are an aggressive subtype of breast cancer with poor survival, but there remains little known about the etiologic factors that promote its initiation and development. Commonly inherited breast cancer risk factors identified through genome-wide association studies display heterogeneity of effect among breast cancer subtypes as defined by the status of estrogen and progesterone receptors. In the Triple Negative Breast Cancer Consortium (TNBCC), 22 common breast cancer susceptibility variants were investigated in 2,980 Caucasian women with triple-negative breast cancer and 4,978 healthy controls. We identified six single-nucleotide polymorphisms, including rs2046210 (ESR1), rs12662670 (ESR1), rs3803662 (TOX3), rs999737 (RAD51L1), rs8170 (19p13.1), and rs8100241 (19p13.1), significantly associated with the risk of triple-negative breast cancer. Together, our results provide convincing evidence of genetic susceptibility for triple-negative breast cancer.

Progesterone receptor A and B protein expression in human breast cancer

The human progesterone receptor (PR) is a ligand-activated nuclear transcription factor which mediates progesterone action in target tissues. Two PR proteins, PR A (81-83 kDa) and PR B (116-120 kDa), have been described and different physiological activities ascribed to each on the basis of in vitro studies, suggesting that their ratio of expression may control progesterone responsiveness in target cells. Presence of PR in breast tumors is an important indicator of likely responsiveness to endocrine agents. However, the relative expression of PR A and B in breast cancer has not been described and its clinical significance has not been addressed. We have examined the expression of PR A and B in PR-positive breast tumors and found that while in most tumors PR A and B were expressed in similar amounts there was a broad overall distribution of PR A:B ratio which deviated significantly from a normal log distribution with tumors containing a PR A:B ration greater than 4 being over-represented in the group. Linear regression analysis revealed that high PR A:B ratios, in general, derived from a low concentration of PR B rather than high expression of PR A. PR A:B protein ratios were not correlated with the age of the patient or with total PR concentration. A third PR protein band (PR 78 kDa) was detected which comprised greater than 20% of total PR protein in a quarter of the tumor samples examined. The characteristics of tumors containing PR 78 kDa were not different from the overall group. In summary, in PR-positive breast tumors the ratio of expression of PR A and B proteins is close to unity as is seen in a number of other progestin target tissues. However, a significant proportion of tumors expressed very low levels of PR B and a consequently high PR A:B ration. Although the clinical consequence of this observation is not known, the in vitro findings that PR A may act as a repressor for PR B suggests that tumors containing primarily PR A may identify a subset of patients with low or aberrant response to endocrine agents.