The Role of Serotonin in Breast Cancer Stem Cells.

Breast tumors were the first tumors of epithelial origin shown to follow the cancer stem cell model. The model proposes that cancer stem cells are uniquely endowed with tumorigenic capacity and that their aberrant differentiation yields non-tumorigenic progeny, which constitute the bulk of the tumor cell population. Breast cancer stem cells resist therapies and seed metastases; thus, they account for breast cancer recurrence. Hence, targeting these cells is essential to achieve durable breast cancer remissions. We identified compounds including selective antagonists of multiple serotonergic system pathway components required for serotonin biosynthesis, transport, activity via multiple 5-HT receptors (5-HTRs), and catabolism that reduce the viability of breast cancer stem cells of both mouse and human origin using multiple orthologous assays. The molecular targets of the selective antagonists are expressed in breast tumors and breast cancer cell lines, which also produce serotonin, implying that it plays a required functional role in these cells. The selective antagonists act synergistically with chemotherapy to shrink mouse mammary tumors and human breast tumor xenografts primarily by inducing programmed tumor cell death. We hypothesize those serotonergic proteins of diverse activity function by common signaling pathways to maintain cancer stem cell viability. Here, we summarize our recent findings and the relevant literature regarding the role of serotonin in breast cancer.

Antagonists of the serotonin receptor 5A target human breast tumor initiating cells.

BACKGROUND: Breast tumor initiating cells (BTIC) are stem-like cells that initiate and sustain tumor growth, and drive disease recurrence. Identifying therapies targeting BTIC has been hindered due primarily to their scarcity in tumors. We previously reported that BTIC frequency ranges between 15% and 50% in multiple mammary tumors of 3 different transgenic mouse models of breast cancer and that this frequency is maintained in tumor cell populations cultured in serum-free, chemically defined media as non-adherent tumorspheres. The latter enabled high-throughput screening of small molecules for their capacity to affect BTIC survival. Antagonists of several serotonin receptors (5-HTRs) were among the hit compounds. The most potent compound we identified, SB-699551, selectively binds to 5-HT5A, a Gαi/o protein coupled receptor (GPCR). METHODS: We evaluated the activity of structurally unrelated selective 5-HT5A antagonists using multiple orthogonal assays of BTIC frequency. Thereafter we used a phosphoproteomic approach to uncover the mechanism of action of SB-699551. To validate the molecular target of the antagonists, we used the CRISPR-Cas9 gene editing technology to conditionally knockout HTR5A in a breast tumor cell line. RESULTS: We found that selective antagonists of 5-HT5A reduced the frequency of tumorsphere initiating cells residing in breast tumor cell lines and those of patient-derived xenografts (PDXs) that we established. The most potent compound among those tested, SB-699551, reduced the frequency of BTIC in ex vivo assays and acted in concert with chemotherapy to shrink human breast tumor xenografts in vivo. Our phosphoproteomic experiments established that exposure of breast tumor cells to SB-699551 elicited signaling changes in the canonical Gαi/o-coupled pathway and the phosphoinositide 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) axis. Moreover, conditional mutation of the HTR5A gene resulted in the loss of tumorsphere initiating cells and BTIC thus mimicking the effect of SB-699551. CONCLUSIONS: Our data provide genetic, pharmacological and phosphoproteomic evidence consistent with the on-target activity of SB-699551. The use of such agents in combination with cytotoxic chemotherapy provides a novel therapeutic approach to treat breast cancer.

Monoamine oxidase-A activity is required for clonal tumorsphere formation by human breast tumor cells.

BACKGROUND: Breast tumor growth and recurrence are driven by an infrequent population of breast tumor-initiating cells (BTIC). We and others have reported that the frequency of BTIC is orders of magnitude higher when breast tumor cells are propagated in vitro as clonal spheres, termed tumorspheres, by comparison to adherent cells. We exploited the latter to screen > 35,000 small molecules to identify agents capable of targeting BTIC. We unexpectedly discovered that selective antagonists of serotonin signaling were among the hit compounds. To better understand the relationship between serotonin and BTIC we expanded our analysis to include monoamine oxidase-A (MAO-A), an enzyme that metabolizes serotonin. METHODS: We used the Nanostring technology and Western blotting to determine whether MAO-A is expressed in human breast tumor cell lines cultured as tumorspheres by comparison to those grown as adherent cells. We then determined whether MAO-A activity is required for tumorsphere formation, a surrogate in vitro assay for BTIC, by assessing whether selective MAO-A inhibitors affect the frequency of tumorsphere-forming cells. To learn whether MAO-A expression in breast tumor cells is associated with other reported properties of BTIC such as anticancer drug resistance or breast tumor recurrence, we performed differential gene expression analyses using publicly available transcriptomic datasets. RESULTS: Tumorspheres derived from human breast tumor cell lines representative of every breast cancer clinical subtype displayed increased expression of MAO-A transcripts and protein by comparison to adherent cells. Surprisingly, inhibition of MAO-A activity with selective inhibitors reduced the frequency of tumorsphere-forming cells. We also found that increased MAO-A expression is a common feature of human breast tumor cell lines that have acquired anticancer drug resistance and is associated with poor recurrence-free survival (RFS) in patients that experienced high-grade, ER-negative (ER-) breast tumors. CONCLUSIONS: Our data suggests that MAO-A activity is required for tumorsphere formation and that its expression in breast tumor cells is associated with BTIC-related properties. The discovery that a selective MAO-A inhibitor targets tumorsphere-forming cells with potencies in the nanomolar range provides the first evidence of this agent's anticancer property. These data warrant further investigation of the link between MAO-A and BTIC.

Serotonergic system antagonists target breast tumor initiating cells and synergize with chemotherapy to shrink human breast tumor xenografts.

Breast tumors comprise an infrequent tumor cell population, termed breast tumor initiating cells (BTIC), which sustain tumor growth, seed metastases and resist cytotoxic therapies. Hence therapies are needed to target BTIC to provide more durable breast cancer remissions than are currently achieved. We previously reported that serotonergic system antagonists abrogated the activity of mouse BTIC resident in the mammary tumors of a HER2-overexpressing model of breast cancer. Here we report that antagonists of serotonin (5-hydroxytryptamine; 5-HT) biosynthesis and activity, including US Federal Food and Drug Administration (FDA)-approved antidepressants, targeted BTIC resident in numerous breast tumor cell lines regardless of their clinical or molecular subtype. Notably, inhibitors of tryptophan hydroxylase 1 (TPH1), required for 5-HT biosynthesis in select non-neuronal cells, the serotonin reuptake transporter (SERT) and several 5-HT receptors compromised BTIC activity as assessed by functional sphere-forming assays. Consistent with these findings, human breast tumor cells express TPH1, 5-HT and SERT independent of their molecular or clinical subtype. Exposure of breast tumor cells ex vivo to sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), reduced BTIC frequency as determined by transplanting drug-treated tumor cells into immune-compromised mice. Moreover, another SSRI (vilazodone; Viibryd) synergized with chemotherapy to shrink breast tumor xenografts in immune-compromised mice by inhibiting tumor cell proliferation and inducing their apoptosis. Collectively our data suggest that antidepressants in combination with cytotoxic anticancer therapies may be an appropriate treatment regimen for testing in clinical trials.

Serotonin transporter antagonists target tumor-initiating cells in a transgenic mouse model of breast cancer.

Accumulating data suggests that the initiation and progression of human breast tumors is fueled by a rare subpopulation of tumor cells, termed breast tumor-initiating cells (BTIC), which resist radiotherapy and chemotherapy. Consequently, therapies that abrogate BTIC activity are needed to achieve durable cures for breast cancer patients. To identify such therapies we used a sensitive assay to complete a high-throughput screen of small molecules, including approved drugs, with BTIC-rich mouse mammary tumor cell populations. We found that inhibitors of the serotonin reuptake transporter (SERT) and serotonin receptors, which include approved drugs used to treat mood disorders, were potent inhibitors of mouse BTIC activity as determined by functional sphere-forming assays and the initiation of tumor formation by transplant of drug-exposed tumor cells into syngeneic mice. Moreover, sertraline (Zoloft), a selective serotonin reuptake inhibitor (SSRI), synergized with docetaxel (Taxotere) to shrink mouse breast tumors in vivo. Hence drugs targeting the serotonergic system might be repurposed to treat breast cancer patients to afford more durable breast cancer remissions.

Single unpurified breast tumor-initiating cells from multiple mouse models efficiently elicit tumors in immune-competent hosts.

The tumor-initiating cell (TIC) frequency of bulk tumor cell populations is one of the criteria used to distinguish malignancies that follow the cancer stem cell model from those that do not. However, tumor-initiating cell frequencies may be influenced by experimental conditions and the extent to which tumors have progressed, parameters that are not always addressed in studies of these cells. We employed limiting dilution cell transplantation of minimally manipulated tumor cells from mammary tumors of several transgenic mouse models to determine their tumor-initiating cell frequency. We determined whether the tumors that formed following tumor cell transplantation phenocopied the primary tumors from which they were isolated and whether they could be serially transplanted. Finally we investigated whether propagating primary tumor cells in different tissue culture conditions affected their resident tumor-initiating cell frequency. We found that tumor-initiating cells comprised between 15% and 50% of the bulk tumor cell population in multiple independent mammary tumors from three different transgenic mouse models of breast cancer. Culture of primary mammary tumor cells in chemically-defined, serum-free medium as non-adherent tumorspheres preserved TIC frequency to levels similar to that of the primary tumors from which they were established. By contrast, propagating the primary tumor cells in serum-containing medium as adherent populations resulted in a several thousand-fold reduction in their tumor-initiating cell fraction. Our findings suggest that experimental conditions, including the sensitivity of the transplantation assay, can dramatically affect estimates of tumor initiating cell frequency. Moreover, conditional on cell culture conditions, the tumor-initiating cell fraction of bulk mouse mammary tumor cell preparations can either be maintained at high or low frequency in vitro thus permitting comparative studies of tumorigenic and non-tumorigenic cancer cells.

Small molecule antagonists of the Wnt/ß-catenin signaling pathway target breast tumor-initiating cells in a Her2/Neu mouse model of breast cancer.

BACKGROUND: Recent evidence suggests that human breast cancer is sustained by a minor subpopulation of breast tumor-initiating cells (BTIC), which confer resistance to anticancer therapies and consequently must be eradicated to achieve durable breast cancer cure. METHODS/FINDINGS: To identify signaling pathways that might be targeted to eliminate BTIC, while sparing their normal stem and progenitor cell counterparts, we performed global gene expression profiling of BTIC- and mammary epithelial stem/progenitor cell- enriched cultures derived from mouse mammary tumors and mammary glands, respectively. Such analyses suggested a role for the Wnt/Beta-catenin signaling pathway in maintaining the viability and or sustaining the self-renewal of BTICs in vitro. To determine whether the Wnt/Beta-catenin pathway played a role in BTIC processes we employed a chemical genomics approach. We found that pharmacological inhibitors of Wnt/ß-catenin signaling inhibited sphere- and colony-formation by primary breast tumor cells and primary mammary epithelial cells, as well as by tumorsphere- and mammosphere-derived cells. Serial assays of self-renewal in vitro revealed that the Wnt/Beta-catenin signaling inhibitor PKF118-310 irreversibly affected BTIC, whereas it functioned reversibly to suspend the self-renewal of mammary epithelial stem/progenitor cells. Incubation of primary tumor cells in vitro with PKF118-310 eliminated their capacity to subsequently seed tumor growth after transplant into syngeneic mice. Administration of PKF118-310 to tumor-bearing mice halted tumor growth in vivo. Moreover, viable tumor cells harvested from PKF118-310 treated mice were unable to seed the growth of secondary tumors after transplant. CONCLUSIONS: These studies demonstrate that inhibitors of Wnt/ß-catenin signaling eradicated BTIC in vitro and in vivo and provide a compelling rationale for developing such antagonists for breast cancer therapy.

Scale-up of breast cancer stem cell aggregate cultures to suspension bioreactors.

It has been hypothesized that breast tumor formation results from the activity of a scarce population of cells known as Breast Cancer Stem Cells (BrCSCs) and that the development of effective breast cancer therapies may therefore ultimately rely upon the ability to effectively target these cells for eradication. The scarcity of BrCSCs in vivo severely compromises research on these populations, as analyses are restricted to those requiring small cell numbers, and has become a major impediment to the development of therapeutic strategies against breast cancer. Through the culture of murine tissue aggregates containing a population of BrCSCs, this study demonstrates the ability of propagating this scarce population in a controlled and reproducible manner, within suspension bioreactors. A rigorous theoretical framework has been developed in order to understand and characterize the implications of oxygen mass transfer within aggregates upon scale-up and thereby provide a foundation for the scale-up of aggregate cultures. A two-factor, two-level factorial experimental design was also performed in order to assess the effects of inoculation density and hydrodynamic shear upon cell yield. We discovered that the culture of the murine aggregates in a relatively low shear environment (tau(max) = 0.20 Pa) and inoculated at 3.50 x 10(4) cells/mL resulted in the best yields for the range of conditions investigated in suspension bioreactors. A detailed study on the oxygen uptake kinetics of the aggregates also revealed that the uptake rates were not significantly affected by mass transfer limitations, as uptake rates of aggregate cultures were found to be comparable to those observed in single cell cultures. Cells propagated in a process controlled 500 mL suspension bioreactor resulted in growth kinetics that were comparable to those observed in 125 mL bioreactors. Doubling times in the 500 mL vessel were found to be 23.9 h and attained a maximum cell density of 1.20 x 10(6) cells/mL. After enumerating the number of BrCSCs, this resulted in an approximately 20-fold increase in BrCSC numbers in batch suspension cultures. With greater attention being applied to BrCSCs, their propagation in suspension bioreactors makes available experimental avenues that are not currently accessible and may thereby enable the development of more effective therapeutic drugs for the treatment of breast cancer.

Large-scale expansion of mammary epithelial stem cell aggregates in suspension bioreactors.

Mutations in the pathways regulating mammary epithelial stem cell (MESC) self-renewal and differentiation are currently hypothesized to result in uncontrolled cell division and, in turn, breast tumor formation. Although research is aggressively being pursued to understand how such pathways result in breast cancer formation, current studies have been greatly limited by MESC scarcity. To address this issue, this study has successfully developed large-scale expansion protocols for MESC through the subculture of murine mammary epithelial tissue aggregates, called mammospheres, in suspension bioreactors. Growth kinetics of mammospheres cultured in 125 mL suspension bioreactors and T-flasks were found to be comparable, achieving cell densities of 3.10 x 10(5) and 2.75 x 10(5) cells/mL, respectively. This corresponded to a 4-fold expansion over 8 days. Yields were also found to be strongly affected by liquid shear forces, where high agitation rates reduced overall cell numbers. Bioreactor cultures were scaled up to 1000 mL operating volumes, resulting in the production of 4.21 x 10(8) total cells (5.6-fold expansion) from a single passage. Furthermore, intermittent replacement of culture medium with fresh medium dramatically improved maximum cell densities, resulting in an 11-fold expansion, thereby enabling the generation of stem cells in quantities sufficient for standard biochemical and genetic analyses. After being cultured in suspension bioreactors for several passages, analysis by flow cytometry of Ki-67 revealed that 85% of the population was composed of proliferating cells. The successful development of expansion protocols for MESC aggregates in suspension bioreactors makes available experimental avenues that were not previously accessible for breast cancer research, thereby facilitating future investigations into elucidating the role of MESCs in breast cancer tumorigenesis.

Gene expression profiling of neu-induced mammary tumors from transgenic mice reveals genetic and morphological similarities to ErbB2-expressing human breast cancers.

Numerous studies have shown that the overexpression and amplification of ErbB2/Neu are observed in 20-30% of patients afflicted with breast cancer. Furthermore, it has also been observed that the elevated expression of ErbB2/Neu also correlates with poor prognosis and clinical outcome. Given the prevalence of this disease, we sought to create mouse models that mimic the human condition. In this study, we compared two mouse models expressing activated neu under the control of the endogenous and mouse mammary tumor virus promoters. Although histologically similar, the latency and metastatic potential of these tumors are remarkably different. Gene expression profiling of tumor RNA from the two Neu mouse models revealed distinctive and nonoverlapping patterns of gene expression. Consistent with noninvasive nature of the mammary tumors induced by expression of neu under the endogenous promoter, these tumors expressed a number of markers characteristic of a highly differentiated state. In addition to these differences, these analyses revealed that in contrast to the mouse mammary tumor virus-based Neu model, the endogenous promoter tumors expressed elevated levels of two genes (Grb7 and Cab1) that are closely linked to ErbB2 and often coamplified in noninvasive ductal carcinoma in situ. Furthermore, this analysis has revealed several transcription factors that may be involved in ErbB2-mediated tumorigenesis. Taken together, these results illustrate the similarity of the endogenously regulated Neu tumor model to the human disease.

Myogenic specification of side population cells in skeletal muscle.

Skeletal muscle contains myogenic progenitors called satellite cells and muscle-derived stem cells that have been suggested to be pluripotent. We further investigated the differentiation potential of muscle-derived stem cells and satellite cells to elucidate relationships between these two populations of cells. FACS(R) analysis of muscle side population (SP) cells, a fraction of muscle-derived stem cells, revealed expression of hematopoietic stem cell marker Sca-1 but did not reveal expression of any satellite cell markers. Muscle SP cells were greatly enriched for cells competent to form hematopoietic colonies. Moreover, muscle SP cells with hematopoietic potential were CD45 positive. However, muscle SP cells did not differentiate into myocytes in vitro. By contrast, satellite cells gave rise to myocytes but did not express Sca-1 or CD45 and never formed hematopoietic colonies. Importantly, muscle SP cells exhibited the potential to give rise to both myocytes and satellite cells after intramuscular transplantation. In addition, muscle SP cells underwent myogenic specification after co-culture with myoblasts. Co-culture with myoblasts or forced expression of MyoD also induced muscle differentiation of muscle SP cells prepared from mice lacking Pax7 gene, an essential gene for satellite cell development. Therefore, these data document that satellite cells and muscle-derived stem cells represent distinct populations and demonstrate that muscle-derived stem cells have the potential to give rise to myogenic cells via a myocyte-mediated inductive interaction.

ErbB2 is required for muscle spindle and myoblast cell survival.

Signaling mediated by ErbB2 is thought to play a critical role in numerous developmental processes. However, due to the embryonic lethality associated with the germ line inactivation of erbB2, its role in adult tissues remains largely obscure. Given the expression of ErbB2 at the neuromuscular junction, we have created a muscle-specific knockout to assess its role there. This resulted in viable mice with a progressive defect in proprioception due to loss of muscle spindles. Interestingly, a partial reduction of ErbB2 levels also reduced the number of muscle spindles. Although histological analysis of the muscle revealed an otherwise normal architecture, induction of muscle injury revealed a defect in muscle regeneration. Consistent with these observations, primary myoblasts lacking ErbB2 exhibit extensive apoptosis upon differentiation into myofibers. Taken together, these results illustrate a dual role for ErbB2 in both muscle spindle maintenance and survival of myoblasts.