Metabolic memory underlying minimal residual disease in breast cancer.

Funder: European Molecular Biology LaboratoryFunder: European Molecular Biology Laboratory (EMBL)Tumor relapse from treatment-resistant cells (minimal residual disease, MRD) underlies most breast cancer-related deaths. Yet, the molecular characteristics defining their malignancy have largely remained elusive. Here, we integrated multi-omics data from a tractable organoid system with a metabolic modeling approach to uncover the metabolic and regulatory idiosyncrasies of the MRD. We find that the resistant cells, despite their non-proliferative phenotype and the absence of oncogenic signaling, feature increased glycolysis and activity of certain urea cycle enzyme reminiscent of the tumor. This metabolic distinctiveness was also evident in a mouse model and in transcriptomic data from patients following neo-adjuvant therapy. We further identified a marked similarity in DNA methylation profiles between tumor and residual cells. Taken together, our data reveal a metabolic and epigenetic memory of the treatment-resistant cells. We further demonstrate that the memorized elevated glycolysis in MRD is crucial for their survival and can be targeted using a small-molecule inhibitor without impacting normal cells. The metabolic aberrances of MRD thus offer new therapeutic opportunities for post-treatment care to prevent breast tumor recurrence

Cell cycle regulation of embryonic stem cells and mouse embryonic fibroblasts lacking functional Pax7

The transcription factor Pax7 plays a key role during embryonic myogenesis and in adult organisms in that it sustains the proper function of satellite cells, which serve as adult skeletal muscle stem cells. Recently we have shown that lack of Pax7 does not prevent the myogenic differentiation of pluripotent stem cells. In the current work we show that the absence of functional Pax7 in differentiating embryonic stem cells modulates cell cycle facilitating their proliferation. Surprisingly, deregulation of Pax7 function also positively impacts at the proliferation of mouse embryonic fibroblasts. Such phenotypes seem to be executed by modulating the expression of positive cell cycle regulators, such as cyclin E

Tumor cell dormancy

Metastasis is the primary cause of death in cancer patients and current treatments fail to provide durable responses. Efforts to treat metastatic disease are hindered by the fact that metastatic cells often remain dormant for prolonged intervals of years, or even decades. Tumor dormancy reflects the capability of disseminated tumor cells (DTCs), or micrometastases, to evade treatment and remain at low numbers after primary tumor resection. Unfortunately, dormant cells will eventually produce overt metastasis. Innovations are needed to understand metastatic dormancy and improve cancer detection and treatment. Currently, few models exist that faithfully recapitulate metastatic dormancy and metastasis to clinically relevant tissues, such as the bone. Herein, we discuss recent advances describing genetic cell-autonomous and systemic or local changes in the microenvironment that have been shown to endow DTCs with properties to survive and eventually colonize distant organs

Identification and functional analysis of molecular mechanisms involved in the latency of ER positive breast cancer

[eng] Breast cancer is the most frequently diagnosed cancer and remains the second leading cause of death among women in Europe and United States. In this malignancy, metastasis remains to be an incurable condition, and therefore the major cause of death. Metastatic lesions can appear within a wide time ranging from months to years or decades after primary tumor resection. In particular, in the estrogen receptor (ER) positive breast cancer subgroup metastatic latency continues to be a major challenge for the researchers, clinicians and patients. This thesis reports the identification and functional analysis of molecular mechanisms involved in the latency of ER positive breast cancer. For that purpose we based our research on a comprehensive approach that relies on genetically engineered human breast cancer cells, experimental mouse models, unbiased whole- genome screen and clinical data. The first part of the thesis describes a novel mouse model of breast cancer dormancy. We showed that metastatic cells home the bone and enter the latency phase as micrometastatic lesions where tumor growth is restricted mainly due to the equilibrated ratios of cell proliferation and cell death. This experimental mouse model was used to identify genes relevant for long- latent relapse. To this end, we performed in vivo loss-of- function shRNA screening. In the screening we challenged a whole-genome library of shRNA to uncover genes whose depletion negatively regulates dormancy. Among the candidate genes revealed by the screen we focused on MSK1 as a long-latent metastasis regulator. The in vivo and in vitro validation results indicate that MSK1 plays a role in homing and differentiation of metastatic cells. We showed that MSK1 promotes the expression luminal transcription factors - FOXA1 and GATA-3. Therefore, MSK1 depletion is beneficial for metastatic cells leading to a partial phenotype shift towards a more aggressive and poorly differentiated basal population. Furthermore, our data suggest that MSK1 may be involved in metastatic cell plasticity by remodeling the chromatin. Importantly, low MSK1 gene expression levels associate with early metastasis in ER positive breast cancer.[spa] El cáncer de mama es el tipo de cáncer más frecuentemente diagnosticado, siendo la segunda causa de muerte entre las mujeres de Europa y Estados Unidos. En esta enfermedad, la metástasis sigue siendo incurable, y por ello es la principal causa de muerte. Las lesiones metastásicas pueden aparecer dentro de un amplio periodo de tiempo que va desde meses hasta años o incluso décadas después de la extirpación del tumor primario. Concretamente, en el subgrupo de cáncer de mama RE positivo, este largo periodo de latencia es el principal desafío para investigadores, médicos y pacientes. En esta tesis se muestra la identificación y el análisis funcional de mecanismos moleculares implicados en la latencia del cáncer de mama RE positivo. Para este propósito, nuestros estudios se han llevado a cabo mediante una estrategia experimental basada en líneas celulares de cáncer de mama genéticamente modificadas, modelos experimentales de ratón, análisis global del genoma y datos clínicos. La primera parte de la tesis describe un novedoso modelo de ratón de dormancia de cáncer de mama. Observamos que, en nuestro modelo, las células metastásicas llegan al hueso y entran en una fase de latencia en forma de lesiones micrometastásicas en la que el crecimiento del tumor se ve impedido, principalmente debido a que la tasa de proliferación celular se iguala a la tasa de muerte celular. Este modelo experimental de ratón se usó para identificar genes relevantes en el proceso de latencia y por tanto en la recurrencia a largo plazo. Para ello, llevamos a cabo un análisis in vivo de pérdida de función con shRNA. En este análisis utilizamos una amplia librería de shRNA para descubrir genes cuya eliminación regula la dormancia de manera negativa. Entre los genes candidatos identificados en este análisis nos focalizamos en MSK1 como un regulador de la metástasis latente. La validación in vitro e in vivo indica que MSK1 juega un papel en el anidamiento y la diferenciación de las células metastásica

Regulation of Mammary Luminal Cell Fate and Tumorigenesis by p38α

Summary: Mammary stem and progenitor cells are essential for mammary gland homeostasis and are also candidates for cells of origin of mammary tumors. Here, we have investigated the function of the protein kinase p38α in the mammary gland using mice that delete this protein in the luminal epithelial cells. We show that p38α regulates the fate of luminal progenitor cells through modulation of the transcription factor RUNX1, an important controller of the estrogen receptor-positive cell lineage. We also provide evidence that the regulation of RUNX1 by p38α probably involves the kinase MSK1, which phosphorylates histone H3 at the RUNX1 promoter. Moreover, using a mouse model for breast cancer initiated by luminal cells, we show that p38α downregulation in mammary epithelial cells reduces tumor burden, which correlates with decreased numbers of tumor-initiating cells. Collectively, our results define a key role for p38α in luminal progenitor cell fate that affects mammary tumor formation

Enhanced MAF Oncogene Expression and Breast Cancer Bone Metastasis.

BACKGROUND: There are currently no biomarkers for early breast cancer patient populations at risk of bone metastasis. Identification of mediators of bone metastasis could be of clinical interest. METHODS: A de novo unbiased screening approach based on selection of highly bone metastatic breast cancer cells in vivo was used to determine copy number aberrations (CNAs) associated with bone metastasis. The CNAs associated with bone metastasis were examined in independent primary breast cancer datasets with annotated clinical follow-up. The MAF gene encoded within the CNA associated with bone metastasis was subjected to gain and loss of function validation in breast cancer cells (MCF7, T47D, ZR-75, and 4T1), its downstream mechanism validated, and tested in clinical samples. A multivariable Cox cause-specific hazard model with competing events (death) was used to test the association between 16q23 or MAF and bone metastasis. All statistical tests were two-sided. RESULTS: 16q23 gain CNA encoding the transcription factor MAF mediates breast cancer bone metastasis through the control of PTHrP. 16q23 gain (hazard ratio (HR) for bone metastasis = 14.5, 95% confidence interval (CI) = 6.4 to 32.9, P < .001) as well as MAF overexpression (HR for bone metastasis = 2.5, 95% CI = 1.7 to 3.8, P < .001) in primary breast tumors were specifically associated with risk of metastasis to bone but not to other organs. CONCLUSIONS: These results suggest that MAF is a mediator of breast cancer bone metastasis. 16q23 gain or MAF protein overexpression in tumors may help to select patients at risk of bone relapse.MP and SG is supported by “La Caixa” PhD fellowship program. AAE and AB hold PhD fellowships from the Spanish Ministerio de Ciencia e Innovación (MICINN). MTS is supported by the IRB Barcelona PhD program. JU is a “Juan de la Cierva” Researcher (MICINN). FIS PI12/00680, PI12/01552, and PI12/01421 supported JA, ALl, and FR, respectively. JA, ALl, and AP were part of RD12/0036/0051, RD12/0036/0070, and RD12/0036/0042, and FR is part of biobanc RD/09/0076/00101. JA and FR are recipients of intensification grant ISCIII, 2009SGR321, XBTC, MARBiobanc, and Cellex. RRG was supported by the Institució Catalana de Recerca i Estudis Avançats. Support and structural funds were provided by the BBVA Foundation, the Generalitat de Catalunya (2014 SGR 535), and the Spanish Ministerio de Ciencia e Innovación (MICINN) (SAF2013-46196) to RRG

Enhanced MAF Oncogene Expression and Breast Cancer Bone Metastasis.

BACKGROUND: There are currently no biomarkers for early breast cancer patient populations at risk of bone metastasis. Identification of mediators of bone metastasis could be of clinical interest. METHODS: A de novo unbiased screening approach based on selection of highly bone metastatic breast cancer cells in vivo was used to determine copy number aberrations (CNAs) associated with bone metastasis. The CNAs associated with bone metastasis were examined in independent primary breast cancer datasets with annotated clinical follow-up. The MAF gene encoded within the CNA associated with bone metastasis was subjected to gain and loss of function validation in breast cancer cells (MCF7, T47D, ZR-75, and 4T1), its downstream mechanism validated, and tested in clinical samples. A multivariable Cox cause-specific hazard model with competing events (death) was used to test the association between 16q23 or MAF and bone metastasis. All statistical tests were two-sided. RESULTS: 16q23 gain CNA encoding the transcription factor MAF mediates breast cancer bone metastasis through the control of PTHrP. 16q23 gain (hazard ratio (HR) for bone metastasis = 14.5, 95% confidence interval (CI) = 6.4 to 32.9, P < .001) as well as MAF overexpression (HR for bone metastasis = 2.5, 95% CI = 1.7 to 3.8, P < .001) in primary breast tumors were specifically associated with risk of metastasis to bone but not to other organs. CONCLUSIONS: These results suggest that MAF is a mediator of breast cancer bone metastasis. 16q23 gain or MAF protein overexpression in tumors may help to select patients at risk of bone relapse.MP and SG is supported by “La Caixa” PhD fellowship program. AAE and AB hold PhD fellowships from the Spanish Ministerio de Ciencia e Innovación (MICINN). MTS is supported by the IRB Barcelona PhD program. JU is a “Juan de la Cierva” Researcher (MICINN). FIS PI12/00680, PI12/01552, and PI12/01421 supported JA, ALl, and FR, respectively. JA, ALl, and AP were part of RD12/0036/0051, RD12/0036/0070, and RD12/0036/0042, and FR is part of biobanc RD/09/0076/00101. JA and FR are recipients of intensification grant ISCIII, 2009SGR321, XBTC, MARBiobanc, and Cellex. RRG was supported by the Institució Catalana de Recerca i Estudis Avançats. Support and structural funds were provided by the BBVA Foundation, the Generalitat de Catalunya (2014 SGR 535), and the Spanish Ministerio de Ciencia e Innovación (MICINN) (SAF2013-46196) to RRG

Enhanced MAF Oncogene Expression and Breast Cancer Bone Metastasis

Background: There are currently no biomarkers for early breast cancer patient populations at risk of bone metastasis. Identification of mediators of bone metastasis could be of clinical interest. Methods: A de novo unbiased screening approach based on selection of highly bone metastatic breast cancer cells in vivo was used to determine copy number aberrations (CNAs) associated with bone metastasis. The CNAs associated with bone metastasis were examined in independent primary breast cancer datasets with annotated clinical follow-up. The MAF gene encoded within the CNA associated with bone metastasis was subjected to gain and loss of function validation in breast cancer cells (MCF7, T47D, ZR-75, and 4T1), its downstream mechanism validated, and tested in clinical samples. A multivariable Cox cause-specific hazard model with competing events (death) was used to test the association between 16q23 or MAF and bone metastasis. All statistical tests were two-sided. Results: 16q23 gain CNA encoding the transcription factor MAF mediates breast cancer bone metastasis through the control of PTHrP. 16q23 gain (hazard ratio (HR) for bone metastasis = 14.5, 95% confidence interval (CI) = 6.4 to 32.9, P < .001) as well as MAF overexpression (HR for bone metastasis = 2.5, 95% CI = 1.7 to 3.8, P < .001) in primary breast tumors were specifically associated with risk of metastasis to bone but not to other organs. Conclusions: These results suggest that MAF is a mediator of breast cancer bone metastasis. 16q23 gain or MAF protein overexpression in tumors may help to select patients at risk of bone relapse