Phenotypic heterogeneity drives differential disease outcome in a mouse model of triple negative breast cancer.

The triple negative breast cancer (TNBC) subtype is one of the most aggressive forms of breast cancer that has poor clinical outcome and is an unmet clinical challenge. Accumulating evidence suggests that intratumoral heterogeneity or the presence of phenotypically distinct cell populations within a tumor play a crucial role in chemoresistance, tumor progression and metastasis. An increased understanding of the molecular regulators of intratumoral heterogeneity is crucial to the development of effective therapeutic strategies in TNBC. To this end, we used an unbiased approach to identify a molecular mediator of intratumoral heterogeneity in breast cancer by isolating two tumor cell populations (T1 and T2) from the 4T1 TNBC model. Phenotypic characterization revealed that the cells are different in terms of their morphology, proliferation and self-renewal ability in vitro as well as primary tumor formation and metastatic potential in vivo. Bioinformatic analysis followed by Kaplan Meier survival analysis in TNBC patients identified Metastasis associated colon cancer 1 (Macc1) as one of the top candidate genes mediating the aggressive phenotype in the T1 tumor cells. The role of Macc1 in regulating the proliferative phenotype was validated and taken forward in a therapeutic context with Lovastatin, a small molecule transcriptional inhibitor of Macc1 to target the T1 cell population. This study increases our understanding of the molecular underpinnings of intratumoral heterogeneity in breast cancer that is critical to improve the treatment of women currently living with the highly aggressive TNBC subtype.

Targeting the Id1-Kif11 Axis in Triple-Negative Breast Cancer Using Combination Therapy.

The basic helix-loop-helix (bHLH) transcription factors inhibitor of differentiation 1 (Id1) and inhibitor of differentiation 3 (Id3) (referred to as Id) have an important role in maintaining the cancer stem cell (CSC) phenotype in the triple-negative breast cancer (TNBC) subtype. In this study, we aimed to understand the molecular mechanism underlying Id control of CSC phenotype and exploit it for therapeutic purposes. We used two different TNBC tumor models marked by either Id depletion or Id1 expression in order to identify Id targets using a combinatorial analysis of RNA sequencing and microarray data. Phenotypically, Id protein depletion leads to cell cycle arrest in the G0/G1 phase, which we demonstrate is reversible. In order to understand the molecular underpinning of Id proteins on the cell cycle phenotype, we carried out a large-scale small interfering RNA (siRNA) screen of 61 putative targets identified by using genomic analysis of two Id TNBC tumor models. Kinesin Family Member 11 (Kif11) and Aurora Kinase A (Aurka), which are critical cell cycle regulators, were further validated as Id targets. Interestingly, unlike in Id depletion conditions, Kif11 and Aurka knockdown leads to a G2/M arrest, suggesting a novel Id cell cycle mechanism, which we will explore in further studies. Therapeutic targeting of Kif11 to block the Id1-Kif11 axis was carried out using small molecular inhibitor ispinesib. We finally leveraged our findings to target the Id/Kif11 pathway using the small molecule inhibitor ispinesib in the Id+ CSC results combined with chemotherapy for better response in TNBC subtypes. This work opens up exciting new possibilities of targeting Id targets such as Kif11 in the TNBC subtype, which is currently refractory to chemotherapy. Targeting the Id1-Kif11 molecular pathway in the Id1+ CSCs in combination with chemotherapy and small molecular inhibitor results in more effective debulking of TNBC.

Id Proteins Promote a Cancer Stem Cell Phenotype in Mouse Models of Triple Negative Breast Cancer via Negative Regulation of Robo1.

Breast cancers display phenotypic and functional heterogeneity and several lines of evidence support the existence of cancer stem cells (CSCs) in certain breast cancers, a minor population of cells capable of tumor initiation and metastatic dissemination. Identifying factors that regulate the CSC phenotype is therefore important for developing strategies to treat metastatic disease. The Inhibitor of Differentiation Protein 1 (Id1) and its closely related family member Inhibitor of Differentiation 3 (Id3) (collectively termed Id) are expressed by a diversity of stem cells and are required for metastatic dissemination in experimental models of breast cancer. In this study, we show that ID1 is expressed in rare neoplastic cells within ER-negative breast cancers. To address the function of Id1 expressing cells within tumors, we developed independent murine models of Triple Negative Breast Cancer (TNBC) in which a genetic reporter permitted the prospective isolation of Id1+ cells. Id1+ cells are enriched for self-renewal in tumorsphere assays in vitro and for tumor initiation in vivo. Conversely, depletion of Id1 and Id3 in the 4T1 murine model of TNBC demonstrates that Id1/3 are required for cell proliferation and self-renewal in vitro, as well as primary tumor growth and metastatic colonization of the lung in vivo. Using combined bioinformatic analysis, we have defined a novel mechanism of Id protein function via negative regulation of the Roundabout Axon Guidance Receptor Homolog 1 (Robo1) leading to activation of a Myc transcriptional programme.

Interplay between a cytosolic and a cell surface carbonic anhydrase in pH homeostasis and acid tolerance of Leishmania.

Leishmania parasites have evolved to endure the acidic phagolysosomal environment within host macrophages. How Leishmania cells maintain near-neutral intracellular pH and proliferate in such a proton-rich mileu remains poorly understood. We report here that, in order to thrive in acidic conditions, Leishmania major relies on a cytosolic and a cell surface carbonic anhydrase, LmCA1 and LmCA2, respectively. Upon exposure to acidic medium, the intracellular pH of the LmCA1+/-, LmCA2+/- and LmCA1+/-:LmCA2+/- mutant strains dropped by varying extents that led to cell cycle delay, growth retardation and morphological abnormalities. Intracellular acidosis and growth defects of the mutant strains could be reverted by genetic complementation or supplementation with bicarbonate. When J774A.1 macrophages were infected with the mutant strains, they exhibited much lower intracellular parasite burdens than their wild-type counterparts. However, these differences in intracellular parasite burden between the wild-type and mutant strains were abrogated if, before infection, the macrophages were treated with chloroquine to alkalize their phagolysosomes. Taken together, our results demonstrate that haploinsufficiency of LmCA1 and/or LmCA2 renders the parasite acid-susceptible, thereby unravelling a carbonic anhydrase-mediated pH homeostatic circuit in Leishmania cells.

The role of cancer stem cells in tumor heterogeneity and resistance to therapy.

Cancer is a heterogenous disease displaying marked inter- and intra-tumoral diversity. The existence of cancer stem cells (CSCs) has been experimentally demonstrated in a number of cancer types as a subpopulation of tumor cells that drives the tumorigenic and metastatic properties of the entire cancer. Thus, eradication of the CSC population is critical for the complete ablation of a tumor. This is, however, confounded by the inherent resistance of CSCs to standard anticancer therapies, eventually leading to the outgrowth of resistant tumor cells and relapse in patients. The cellular mechanisms of therapy resistance in CSCs are ascribed to several factors including a state of quiescence, an enhanced DNA damage response and active repair mechanisms, up-regulated expression of drug efflux transporters, as well as the activation of pro-survival signaling pathways and inactivation of apoptotic signaling. Understanding the mechanisms underlying the acquisition of resistance to therapy may hold the key to targeting the CSC population.