Metabolic differences in breast cancer stem cells and differentiated progeny.

In general, tumor cells display a more glycolytic phenotype compared to the corresponding normal tissue. However, it is becoming increasingly clear that tumors are composed of a heterogeneous population of cells. Breast cancers are organized in a hierarchical manner, with the breast cancer stem cells (BCSCs) at the top of the hierarchy. Here, we investigate the metabolic phenotype of BCSCs and their differentiated progeny. In addition, we determine the effect of radiation on the metabolic state of these two cell populations. Luminal, basal, and claudin-low breast cancer cell lines were propagated as mammospheres enriched in BCSCs. Lactate production, glucose consumption, and ATP content were compared with differentiated cultures. A metabolic flux analyzer was used to determine the oxygen consumption, extracellular acidification rates, maximal mitochondria capacity, and mitochondrial proton leak. The effect of radiation treatment of the metabolic phenotype of each cell population was also determined. BCSCs consume more glucose, produce less lactate, and have higher ATP content compared to their differentiated progeny. BCSCs have higher maximum mitochondrial capacity and mitochondrial proton leak compared to their differentiated progeny. Radiation treatment enhances the higher energetic state of the BCSCs, while decreasing mitochondrial proton leak. Our study indicated that breast cancer cells are heterogeneous in their metabolic phenotypes and BCSCs reside in a distinct metabolic state compared to their differentiated progeny. BCSCs display a reliance on oxidative phosphorylation, while the more differentiated progeny displays a more glycolytic phenotype. Radiation treatment affects the metabolic state of BCSCs. We conclude that interfering with the metabolic requirements of BCSCs may prevent radiation-induced reprogramming of breast cancer cells during radiation therapy, thus improving treatment outcome.

Ineffectual targeting of HIV-1 Nef by cytotoxic T lymphocytes in acute infection results in no functional impairment or viremia reduction.

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef is heavily targeted by CD8(+) T lymphocytes (CTLs) during acute infection and therefore is included in many candidate vaccines. We investigated whether CTL targeting of Nef during acute infection contributes to immune control by disrupting the function of Nef. The sequence and function of Nef in parallel with CTL responses were assessed longitudinally from peak viremia until the viremia set point in a cohort of six subjects with acute infection. All but one individual had a single founder strain. Nef-specific CTL responses were detected in all subjects and declined in magnitude over time. These responses were associated with mutations, but none of the mutations were detected in important functional motifs. Nef-mediated downregulation of CD4 and major histocompatibility complex (MHC) class I molecules was better preserved in acute infection than in chronic infection. Finally, Nef-specific CTL responses were not associated with a reduction in viremia from its acute-phase peak. Our results indicate that CTLs targeting Nef epitopes outside critical functional domains have little effect on the pathogenic functions of Nef, rendering these responses ineffective in acute infection. Importance: These data indicate that using the whole Nef protein as a vaccine immunogen likely allows immunodominance that leads to targeting of CTL responses that are rapidly escaped with little effect on Nef-mediated pathogenic functions. Pursuing vaccination approaches that can more precisely direct responses to vulnerable areas would maximize efficacy. Until vaccine-induced targeting can be optimized, other approaches, such as the use of Nef function inhibitors or the pursuit of immunotherapies such as T cell receptor gene therapy or adoptive transfer, may be more likely to result in successful control of viremia.

The RNA-binding protein Musashi-1 regulates proteasome subunit expression in breast cancer- and glioma-initiating cells.

Cancer stem cells (CSCs) or tumor-initiating cells, similar to normal tissue stem cells, rely on developmental pathways, such as the Notch pathway, to maintain their stem cell state. One of the regulators of the Notch pathway is Musashi-1, a mRNA-binding protein. Musashi-1 promotes Notch signaling by binding to the mRNA of Numb, the negative regulator of Notch signaling, thus preventing its translation. CSCs have also been shown to downregulate their 26S proteasome activity in several types of solid tumors, thus making them resistant to proteasome-inhibitors used as anticancer agents in the clinic. Interestingly, the Notch pathway can be inhibited by proteasomal degradation of the Notch intracellular domain (Notch-ICD); therefore, downregulation of the 26S proteasome activity can lead to stabilization of Notch-ICD. Here, we present evidence that the downregulation of the 26S proteasome in CSCs constitutes another level of control by which Musashi-1 promotes signaling through the Notch pathway and maintenance of the stem cell phenotype of this subpopulation of cancer cells. We demonstrate that Musashi-1 mediates the downregulation of the 26S proteasome by binding to the mRNA of NF-YA, the transcriptional factor regulating 26S proteasome subunit expression, thus providing an additional route by which the degradation of Notch-ICD is prevented, and Notch signaling is sustained.

HIV-1 Nef sequence and functional compartmentalization in the gut is not due to differential cytotoxic T lymphocyte selective pressure.

The gut is the largest lymphoid organ in the body and a site of active HIV-1 replication and immune surveillance. The gut is a reservoir of persistent infection in some individuals with fully suppressed plasma viremia on combination antiretroviral therapy (cART) although the cause of this persistence is unknown. The HIV-1 accessory protein Nef contributes to persistence through multiple functions including immune evasion and increasing infectivity. Previous studies showed that Nef's function is shaped by cytotoxic T lymphocyte (CTL) responses and that there are distinct populations of Nef within tissue compartments. We asked whether Nef's sequence and/or function are compartmentalized in the gut and how compartmentalization relates to local CTL immune responses. Primary nef quasispecies from paired plasma and sigmoid colon biopsies from chronically infected subjects not on therapy were sequenced and cloned into Env(-) Vpu(-) pseudotyped reporter viruses. CTL responses were mapped by IFN-γ ELISpot using expanded CD8+ cells from blood and gut with pools of overlapping peptides covering the entire HIV proteome. CD4 and MHC Class I Nef-mediated downregulation was measured by flow cytometry. Multiple tests indicated compartmentalization of nef sequences in 5 of 8 subjects. There was also compartmentalization of function with MHC Class I downregulation relatively well preserved, but significant loss of CD4 downregulation specifically by gut quasispecies in 5 of 7 subjects. There was no compartmentalization of CTL responses in 6 of 8 subjects, and the selective pressure on quasispecies correlated with the magnitude CTL response regardless of location. These results demonstrate that Nef adapts via diverse pathways to local selective pressures within gut mucosa, which may be predominated by factors other than CTL responses such as target cell availability. The finding of a functionally distinct population within gut mucosa offers some insight into how HIV-1 may persist in the gut despite fully suppressed plasma viremia on cART.

Targeted elimination of breast cancer cells with low proteasome activity is sufficient for tumor regression.

Breast cancers are thought to be organized hierarchically with a small number of breast cancer stem cells (BCSCs), able to regrow a tumor after sublethal treatment while their progeny lack this feature. Furthermore, BCSCs are highly resistant to conventional anticancer treatments. According to the cancer stem cell hypothesis, all cancer stem cells in a tumor have to be eliminated to achieve cancer cure. In this study we tested if targeted elimination of BCSCs leads to tumor regression. Specific targeting of BCSCs was achieved via a unique imaging and targeting system that relies on their low proteasome activity. In our system breast cancer cells stably express a fluorescent fusion protein, thymidine kinase-ZsGreen-cODC, which is readily degraded after translation in cells with normal 26S proteasome activity. However, cells with low proteasome activity accumulate this fluorescent fusion protein, thus allowing for their identification, tracking, and specific elimination. Here, we show that the activity of the 26S proteasome was significantly down-regulated in MCF-7, T47D, and MDA-MB-231 cultures enriched for BCSCs. Treatment with ganciclovir resulted in abrogation of sphere formation in vitro, and tumor regression in vivo, thus demonstrating that targeted elimination of BCSCs leads to loss of self-renewal in vitro and tumor regression in vivo. We conclude that specific targeting of BCSCs could be a useful strategy to improve treatment outcome.