A single hematopoietic stem cell (HSC) is capable of reconstituting hematopoiesis and maintaining homeostasis by balancing self-renewal and cell differentiation. The mechanisms of HSC division balance, however, are not yet defined. Here we demonstrate, by characterizing at the single-cell level a purified and minimally heterogeneous murine Tie2+ HSC population, that these top hierarchical HSCs preferentially undergo symmetric divisions. The induction of mitophagy, a quality control process in mitochondria, plays an essential role in self-renewing expansion of Tie2+ HSCs. Activation of the PPAR (peroxisome proliferator–activated receptor)–fatty acid oxidation pathway promotes expansion of Tie2+ HSCs through enhanced Parkin recruitment in mitochondria. These metabolic pathways are conserved in human TIE2+ HSCs. Our data thus identify mitophagy as a key mechanism of HSC expansion and suggest potential methods of cell-fate manipulation through metabolic pathways.

SUMMARY pRb and p53 are two major tumor suppressors. Here, we found that p53 activates expression of Pirh2 and KPC1, two of the three ubiquitin ligases for p27. Loss of p53 in the absence of Skp2, the third ubiquitin ligase for p27, shrinks the cellular pool of p27 ubiquitin ligases to accumulate p27 protein. In the absence of pRb and p53, p27 was unable to inhibit DNA synthesis in spite of its abundance, but could inhibit division of cells that maintain DNA replication with re-replication. This mechanism blocked pRb and p53 doubly deficient pituitary and prostate tumorigenesis lastingly coexistent with BrdU-labeling neoplastic lesions, revealing an unconventional cancer cell vulnerability when pRb and p53 are inactivated.

Clostridium botulinum type B strain produces two forms of progenitor toxin, 16S and 12S. The 12S toxin is formed by association of a neurotoxin (NTX) and a non-toxic non-haemagglutinin (NTNH), and the 16S toxin is formed by conjugation of the 12S toxin with a haemagglutinin (HA). HA consists of four subcomponents designated HA1, HA2, HA3a and HA3b. When mice were immunized with formalin-detoxified NTX, 12S or 16S, a significantly greater amount of anti-NTX antibody (Ab) was produced in the mice injected with 16S than in NTX-or 12S-injected mice. Immunization with NTX mixed with HA1 and/or HA3b also increased the anti-NTX Ab production, whereas NTX mixed with HA2 did not, indicating that HA1 and HA3b have adjuvant activity. This was further confirmed by immunizing mice with human albumin (Alb) alone or Alb mixed with either HA1 or HA3b. When mouse-spleen cells were stimulated with NTX, 16S or different HA subcomponents, 16S, HA1, HA3b and the mixture of HA1 and HA3 significantly increased interleukin 6 (IL6) production compared with NTX alone. Transcription of IL6 mRNA was low after stimulation with NTX alone, but increased to 16S-stimulation levels when NTX was mixed with HA1 or HA3b. In flow cytometry using labelled Abs against CD3 and CD19, the percentage of CD19 cells was higher following stimulation with 16S or NTX mixed with HA1 or HA3b compared with stimulation with NTX. The percentage of CD3 cells remained unchanged. These results suggest strongly that HA1 and HA3b demonstrate adjuvant activity via increasing IL6 production.

One mechanism of tumor suppression by pRb is repressing E2F1. Hence, E2f1 deletion diminishes tumorigenesis following Rb1 loss. However, E2F1 promotes both proliferation and apoptosis. It therefore remains unclear how de-repressed E2F1 promotes tumorigenesis. Another mechanism of pRb function is repressing Skp2 to elevate p27 to arrest proliferation. However, Skp2 deletion induced apoptosis, not proliferation arrest, in Rb1 deficient pituitary tumorigenesis. Here, we show that Rb1 deletion induces higher expression of E2F1 target genes in the absence of Skp2. E2F1 binds less cyclin A but more target promoters when Rb1 is deleted with Skp2 knockout or p27T187A knockin, suggesting that stabilized p27 prevents cyclin A from binding and inhibiting E2F1. In Rb1 deficient pituitary tumorigenesis, Skp2 deletion or p27T187A mutation converts E2F1’s role from proliferative to apoptotic. These findings delineate a pRb-Skp2-p27-cyclin A-E2F1 pathway that determines whether E2F1 is proliferative or apoptotic in Rb1 deficient tumorigenesis.

Immune responses against heat shock protein 60 (HSP60) of pathogen-origin are thought to be defensive events which, due to molecular mimicry, misdirect to a human counterpart. Therefore, atherosclerosis may be serologically predicted by anti-HSP60 antibodies (Abs). In the present study, we analyzed the clinical prevalence of the serum IgG Abs against Helicobacter pylori (Hp)-derived HSP60 (Hp-HSP60) or its peptide fragments in patients with cardiovascular disease (CVD; n=250), as compared to those in age- and gender-matched non-CVD patients (n=293). Anti-Hp cell lysate Abs frequently appeared in Hp-infected patients who were not associated with CVD. In contrast, Abs against the particular amino acid sequence Hp-HSP60(II3) (II3 region, Glu(141)-Leu(160), in Hp-HSP60) predominantly appeared in CVD patients, as well as IgG anti-human HSP60 (Hu-HSP60(w)). Furthermore, neither titer of anti-Hp-HSP60(II3) nor anti-Hu-HSP60(w) Abs was correlated with the levels of high sensitivity C-reactive protein (hsCRP). This data strongly suggested that IgG anti-Hp-HSP60(II3) Abs cross-reacted with Hu-HSP60(w) were independent diagnostic markers relevant to CVD. Further, the 20 amino acid residues (Glu(141)-Leu(160)) might be predominant CVD-associated epitopes that induce anti-Hu-HSP60 auto-Abs, whose location was predicted in the tertiary structure of Hu-HSP60.

SCFSkp2/Cks1 ubiquitinates Thr187-phosphorylated p27 for degradation. Over-expression of Skp2 coupled with under-expression of p27 are frequent characteristics of cancer cells. When the role of SCFSkp2/Cks1 mediated p27 ubiquitination in cancer was specifically tested by p27 Thr187-to-Ala knockin (p27T187A KI), it was found dispensable for KrasG12D induced lung tumorigenesis but essential for Rb1 deficient pituitary tumorigenesis. Here we identify pRb and p53 doubly deficient (DKO) prostate tumorigenesis as a context in which p27 ubiquitination by SCFSkp2/Cks1 is required for p27 down-regulation. p27 protein accumulated in prostate when p27T187A KI mice underwent DKO prostate tumorigenesis. p27T187A KI or Skp2 knockdown (KD) induced similar degrees of p27 protein accumulation in DKO prostate cells, and Skp2 KD did not further increase p27 protein in DKO prostate cells that contained p27T187A KI (AADKO prostate cells). p27T187A KI activated an E2F1-p73-apoptosis axis in DKO prostate tumorigenesis, slowed disease progression, and significantly extended survival. Querying co-occurrence relationships among RB1, TP53, PTEN, NKX3-1, and MYC in TCGA of prostate cancer identified co-inactivation of RB1 and TP53 as the only statistically significant co-occurrences in metastatic castration resistant prostate cancer (mCRPC). Together, our study identifies Skp2/Cks1 pocket inhibitors as potential therapeutics for mCRPC. Procedures for establishing mCRPC organoid cultures from contemporary patients were recently established. An Skp2/Cks1 pocket inhibitor preferentially collapsed DKO prostate tumor organoids over AADKO organoids, which spontaneously disintegrated over time when DKO prostate tumor organoids grew larger, setting the stage to translate mouse model findings to precision medicine in the clinic on the organoid platform.

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Background: p27T187A knockin mice facilitate studying p27Kip1 protein in physiology. Results: p27T187A knockin prevented pituitary tumorigenesis in Rb1 ϩ/Ϫ mice and enhanced humoral response to immuniza-