Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Histone deacetylase activity mediates acquired resistance towards structurally diverse HSP90 inhibitors.

Heat shock protein 90 (HSP90) regulates multiple signalling pathways critical for tumour growth. As such, HSP90 inhibitors have been shown to act as effective anticancer agents in preclinical studies but, for a number of reasons, the same effect has not been observed in the clinical trials to date. One potential reason for this may be the presence of de novo or acquired resistance within the tumours. To investigate mechanisms of resistance, we generated resistant cell lines through gradual dose escalation of the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG). The resultant resistant cell lines maintained their respective levels of resistance (7-240×) in the absence of 17-AAG and were also cross-resistant with other benzoquinone ansamycin HSP90 inhibitors. Expression of members of the histone deacetylase family (HDAC 1, 5, 6) was altered in the resistant cells. To determine whether HDAC activity contributed to resistance, pan-HDAC inhibitors (TSA and LBH589) and the class II HDAC-specific inhibitor SNDX275 were found to resensitize resistant cells towards 17-AAG and 17-dimethylaminoethylamino-17-demethoxygeldanamycin. Most significantly, resistant cells were also identified as cross-resistant towards structurally distinct HSP90 inhibitors such as radicicol and the second-generation HSP90 inhibitors CCT018159, VER50589 and AUY922. HDAC inhibition also resensitized resistant cells towards these classes of HSP90 inhibitors. In conclusion, we report that prolonged 17-AAG treatment results in acquired resistance of cancer cells towards not just 17-AAG but also to a spectrum of structurally distinct HSP90 inhibitors. This acquired resistance can be inhibited using clinically relevant HDAC inhibitors. This work supports the potential benefit of using HSP90 and HDAC inhibitors in combination within the clinical setting.

SCA-1 Labels a Subset of Estrogen-Responsive Bipotential Repopulating Cells within the CD24+ CD49fhi Mammary Stem Cell-Enriched Compartment.

Estrogen stimulates breast development during puberty and mammary tumors in adulthood through estrogen receptor-α (ERα). These effects are proposed to occur via ERα+ luminal cells and not the mammary stem cells (MaSCs) that are ERαneg. Since ERα+ luminal cells express stem cell antigen-1 (SCA-1), we sought to determine if SCA-1 could define an ERα+ subset of EpCAM+/CD24+/CD49fhi MaSCs. We show that the MaSC population has a distinct SCA-1+ population that is abundant in pre-pubertal mammary glands. The SCA-1+ MaSCs have less stem cell markers and less in vivo repopulating activity than their SCA-1neg counterparts. However, they express ERα and specifically enter the cell cycle at puberty. Using estrogen-deficient aromatase knockouts (ArKO), we showed that the SCA-1+ MaSC could be directly modulated by estrogen supplementation. Thus, SCA-1 enriches for an ERα+, estrogen-sensitive subpopulation within the CD24+/CD49fhi MaSC population that may be responsible for the hormonal sensitivity of the developing mammary gland.

The Inositol Polyphosphate 5-Phosphatase PIPP Regulates AKT1-Dependent Breast Cancer Growth and Metastasis.

Metastasis is the major cause of breast cancer mortality. Phosphoinositide 3-kinase (PI3K) generated PtdIns(3,4,5)P3 activates AKT, which promotes breast cancer cell proliferation and regulates migration. To date, none of the inositol polyphosphate 5-phosphatases that inhibit PI3K/AKT signaling have been reported as tumor suppressors in breast cancer. Here, we show depletion of the inositol polyphosphate 5-phosphatase PIPP (INPP5J) increases breast cancer cell transformation, but reduces cell migration and invasion. Pipp ablation accelerates oncogene-driven breast cancer tumor growth in vivo, but paradoxically reduces metastasis by regulating AKT1-dependent tumor cell migration. PIPP mRNA expression is reduced in human ER-negative breast cancers associated with reduced long-term outcome. Collectively, our findings identify PIPP as a suppressor of oncogenic PI3K/AKT signaling in breast cancer.

Molecular stress-inducing compounds increase osteoclast formation in a heat shock factor 1 protein-dependent manner.

Many anticancer therapeutic agents cause bone loss, which increases the risk of fractures that severely reduce quality of life. Thus, in drug development, it is critical to identify and understand such effects. Anticancer therapeutic and HSP90 inhibitor 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) causes bone loss by increasing osteoclast formation, but the mechanism underlying this is not understood. 17-AAG activates heat shock factor 1 (Hsf1), the master transcriptional regulator of heat shock/cell stress responses, which may be involved in this negative action of 17-AAG upon bone. Using mouse bone marrow and RAW264.7 osteoclast differentiation models we found that HSP90 inhibitors that induced a heat shock response also enhanced osteoclast formation, whereas HSP90 inhibitors that did not (including coumermycin A1 and novobiocin) did not affect osteoclast formation. Pharmacological inhibition or shRNAmir knockdown of Hsf1 in RAW264.7 cells as well as the use of Hsf1 null mouse bone marrow cells demonstrated that 17-AAG-enhanced osteoclast formation was Hsf1-dependent. Moreover, ectopic overexpression of Hsf1 enhanced 17-AAG effects upon osteoclast formation. Consistent with these findings, protein levels of the essential osteoclast transcription factor microphthalmia-associated transcription factor were increased by 17-AAG in an Hsf1-dependent manner. In addition to HSP90 inhibitors, we also identified that other agents that induced cellular stress, such as ethanol, doxorubicin, and methotrexate, also directly increased osteoclast formation, potentially in an Hsf1-dependent manner. These results, therefore, indicate that cellular stress can enhance osteoclast differentiation via Hsf1-dependent mechanisms and may significantly contribute to pathological and therapeutic related bone loss.

Heat-shock factor 1 both positively and negatively affects cellular clonogenic growth depending on p53 status.

HSF1 (heat-shock factor 1) is the master regulator of the heat-shock response; however, it is also activated by cancer-associated stresses and supports cellular transformation and cancer progression. We examined the role of HSF1 in relation to cancer cell clonogenicity, an important attribute of cancer cells. Ectopic expression or HSF1 knockdown demonstrated that HSF1 positively regulated cancer cell clonogenic growth. Furthermore, knockdown of mutant p53 indicated that HSF1 actions were mediated via a mutant p53-dependent mechanism. To examine this relationship more specifically, we ectopically co-expressed mutant p53(R273H) and HSF1 in the human mammary epithelial cell line MCF10A. Surprisingly, within this cellular context, HSF1 inhibited clonogenicity. However, upon specific knockdown of endogenous wild-type p53, leaving mutant p53(R273H) expression intact, HSF1 was observed to greatly enhance clonogenic growth of the cells, indicating that HSF1 suppressed clonogenicity via wild-type p53. To confirm this we ectopically expressed HSF1 in non-transformed and H-Ras(V12)-transformed MCF10A cells. As expected, HSF1 significantly reduced clonogenicity, altering wild-type p53 target gene expression levels consistent with a role of HSF1 increasing wild-type p53 activity. In support of this finding, knockdown of wild-type p53 negated the inhibitory effects of HSF1 expression. We thus show that HSF1 can affect clonogenic growth in a p53 context-dependent manner, and can act via both mutant and wild-type p53 to bring about divergent effects upon clonogenicity. These findings have important implications for our understanding of HSF1's divergent roles in cancer cell growth and survival as well as its disparate effect on mutant and wild-type p53.