Srinivas Malladi

A CXCL1 Paracrine Network Links Cancer Chemoresistance and Metastasis

Metastasis and chemoresistance in cancer are linked phenomena, but the molecular basis for this link is unknown. We uncovered a network of paracrine signals between carcinoma, myeloid, and endothelial cells that drives both processes in breast cancer. Cancer cells that overexpress CXCL1 and 2 by transcriptional hyperactivation or 4q21 amplification are primed for survival in metastatic sites. CXCL1/2 attract CD11b(+)Gr1(+) myeloid cells into the tumor, which produce chemokines including S100A8/9 that enhance cancer cell survival. Although chemotherapeutic agents kill cancer cells, these treatments trigger a parallel stromal reaction leading to TNF-α production by endothelial and other stromal cells. TNF-α via NF-kB heightens the CXCL1/2 expression in cancer cells, thus amplifying the CXCL1/2-S100A8/9 loop and causing chemoresistance. CXCR2 blockers break this cycle, augmenting the efficacy of chemotherapy against breast tumors and particularly against metastasis. This network of endothelial-carcinoma-myeloid signaling interactions provides a mechanism linking chemoresistance and metastasis, with opportunities for intervention.

Selection of Bone Metastasis Seeds by Mesenchymal Signals in the Primary Tumor Stroma

How organ-specific metastatic traits arise in primary tumors remains unknown. Here, we show a role of the breast tumor stroma in selecting cancer cells that are primed for metastasis in bone. Cancer-associated fibroblasts (CAFs) in triple-negative (TN) breast tumors skew heterogeneous cancer cell populations toward a predominance of clones that thrive on the CAF-derived factors CXCL12 and IGF1. Limiting concentrations of these factors select for cancer cells with high Src activity, a known clinical predictor of bone relapse and an enhancer of PI3K-Akt pathway activation by CXCL12 and IGF1. Carcinoma clones selected in this manner are primed for metastasis in the CXCL12-rich microenvironment of the bone marrow. The evidence suggests that stromal signals resembling those of a distant organ select for cancer cells that are primed for metastasis in that organ, thus illuminating the evolution of metastatic traits in a primary tumor and its distant metastases.

The E3 Ubiquitin Ligase cIAP1 Binds and Ubiquitinates Caspase-3 and -7 via Unique Mechanisms at Distinct Steps in Their Processing

Inhibitor of apoptosis (IAP) proteins are widely expressed throughout nature and suppress cell death under a variety of circumstances. X-linked IAP, the prototypical IAP in mammals, inhibits apoptosis largely through direct inhibition of the initiator caspase-9 and the effector caspase-3 and -7. Two additional IAP family members, cellular IAP1 (cIAP1) and cIAP2, were once thought to also inhibit caspases, but more recent studies have suggested otherwise. Here we demonstrate that cIAP1 does not significantly inhibit the proteolytic activities of effector caspases on fluorogenic or endogenous substrates. However, cIAP1 does bind to caspase-3 and -7 and does so, remarkably, at distinct steps prior to or following the removal of their prodomains, respectively. Indeed, cIAP1 bound to an exposed IAP-binding motif, AKPD, on the N terminus of the large subunit of fully mature caspase-7, whereas cIAP1 bound to partially processed caspase-3 in a manner that required its prodomain and cleavage between its large and small subunits but did not involve a classical IAP-binding motif. As a ubiquitin-protein isopeptide ligase, cIAP1 ubiquitinated caspase-3 and -7, concomitant with binding, in a reaction catalyzed by members of the UbcH5 subfamily (ubiquitin carrier protein/ubiquitin-conjugating enzymes), and in the case of caspase-3, differentially by UbcH8. Moreover, wild-type caspase-7 and a chimeric caspase-3 (bearing the AKPD motif) were degraded in vivo in a proteasome-dependent manner. Thus, cIAPs likely suppress apoptosis, at least in part, by facilitating the ubiquitination and turnover of active effector caspases in cells.

Metastatic Latency and Immune Evasion through Autocrine Inhibition of WNT.

Metastasis frequently develops years after the removal of a primary tumor, from a minority of disseminated cancer cells that survived as latent entities through unknown mechanisms. We isolated latency competent cancer (LCC) cells from early stage human lung and breast carcinoma cell lines and defined the mechanisms that suppress outgrowth, support long-term survival, and maintain tumor-initiating potential in these cells during the latent metastasis stage. LCC cells show stem-cell-like characteristics and express SOX2 and SOX9 transcription factors, which are essential for their survival in host organs under immune surveillance and for metastatic outgrowth under permissive conditions. Through expression of the WNT inhibitor DKK1, LCC cells self-impose a slow-cycling state with broad downregulation of ULBP ligands for NK cells and evasion of NK-cell-mediated clearance. By expressing a Sox-dependent stem-like state and actively silencing WNT signaling, LCC cells can enter quiescence and evade innate immunity to remain latent for extended periods.

The Apaf‐1•procaspase‐9 apoptosome complex functions as a proteolytic‐based molecular timer

During stress‐induced apoptosis, the initiator caspase‐9 is activated by the Apaf‐1 apoptosome and must remain bound to retain significant catalytic activity. Nevertheless, in apoptotic cells the vast majority of processed caspase‐9 is paradoxically observed outside the complex. We show herein that apoptosome‐mediated cleavage of procaspase‐9 occurs exclusively through a CARD‐displacement mechanism, so that unlike the effector procaspase‐3, procaspase‐9 cannot be processed by the apoptosome as a typical substrate. Indeed, procaspase‐9 possessed higher affinity for the apoptosome and could displace the processed caspase‐9 from the complex, thereby facilitating a continuous cycle of procaspase‐9 recruitment/activation, processing, and release from the complex. Owing to its rapid autocatalytic cleavage, however, procaspase‐9 per se contributed little to the activation of procaspase‐3. Thus, the Apaf‐1 apoptosome functions as a proteolytic‐based ‘molecular timer’, wherein the intracellular concentration of procaspase‐9 sets the overall duration of the timer, procaspase‐9 autoprocessing activates the timer, and the rate at which the processed caspase‐9 dissociates from the complex (and thus loses its capacity to activate procaspase‐3) dictates how fast the timer ‘ticks’ over.

Drosophila Omi, a mitochondrial-localized IAP antagonist and proapoptotic serine protease

Although essential in mammals, in flies the importance of mitochondrial outer membrane permeabilization for apoptosis remains highly controversial. Herein, we demonstrate that Drosophila Omi (dOmi), a fly homologue of the serine protease Omi/HtrA2, is a developmentally regulated mitochondrial intermembrane space protein that undergoes processive cleavage, in situ, to generate two distinct inhibitor of apoptosis (IAP) binding motifs. Depending upon the proapoptotic stimulus, mature dOmi is then differentially released into the cytosol, where it binds selectively to the baculovirus IAP repeat 2 (BIR2) domain in Drosophila IAP1 (DIAP1) and displaces the initiator caspase DRONC. This interaction alone, however, is insufficient to promote apoptosis, as dOmi fails to displace the effector caspase DrICE from the BIR1 domain in DIAP1. Rather, dOmi alleviates DIAP1 inhibition of all caspases by proteolytically degrading DIAP1 and induces apoptosis both in cultured cells and in the developing fly eye. In summary, we demonstrate for the first time in flies that mitochondrial permeabilization not only occurs during apoptosis but also results in the release of a bona fide proapoptotic protein.

BMRP is a Bcl-2 binding protein that induces apoptosis†

Members of the Bcl‐2 family of proteins play important roles in the regulation of cell death by apoptosis. The yeast Two‐Hybrid system was utilized to identify a protein that interacts with the anti‐apoptotic protein Bcl‐2, designated BMRP. This protein corresponds to a previously known mitochondrial ribosomal protein (MRPL41). Binding experiments confirmed the interaction of BMRP to Bcl‐2 in mammalian cells. Subcellular fractionation by differential centrifugation studies showed that both Bcl‐2 and BMRP are localized to the same fractions (fractions that are rich in mitochondria). Northern blot analysis revealed a major bmrp mRNA band of approximately 0.8 kb in several human tissues. Additionally, a larger 2.2 kb mRNA species was also observed in some tissues. Western blot analysis showed that endogenous BMRP runs as a band of 16–17 kDa in SDS–PAGE. Overexpression of BMRP induced cell death in primary embryonic fibroblasts and NIH/3T3 cells. Transfection of BMRP showed similar effects to those observed by overexpression of the pro‐apoptotic proteins Bax or Bad. BMRP‐stimulated cell death was counteracted by co‐expression of Bcl‐2. The baculoviral caspase inhibitor p35 also protected cells from BMRP‐induced cell death. These findings suggest that BMRP is a mitochondrial ribosomal protein involved in the regulation of cell death by apoptosis, probably affecting pathways mediated by Bcl‐2 and caspases.

The Apaf-1 apoptosome induces formation of caspase-9 homo- and heterodimers with distinct activities

According to dogma, initiator caspases are activated through proximity-induced homodimerization, but some studies infer that during apoptosis caspase-9 may instead form a holoenzyme with the Apaf-1 apoptosome. Using several biochemical approaches, including a novel site-specific crosslinking technique, we provide the first direct evidence that procaspase-9 homodimerizes within the apoptosome, markedly increasing its avidity for the complex and inducing selective intramolecular cleavage at Asp-315. Remarkably, however, procaspase-9 could also bind via its small subunit to the NOD domain in Apaf-1, resulting in the formation of a heterodimer that more efficiently activated procaspase-3. Following cleavage, the intersubunit linker (and associated conformational changes) in caspase-9-p35/p12 inhibited its ability to form homo- and heterodimers, but feedback cleavage by caspase-3 at Asp-330 removed the linker entirely and partially restored activity to caspase-9-p35/p10. Thus, the apoptosome mediates the formation of caspase-9 homo- and heterodimers, both of which are impacted by cleavage and contribute to its overall function.

The Frequency of 1,4-Benzoquinone-Lysine Adducts in Cytochrome C Correlate With Defects in Apoptosome Activation

Electrophile-mediated post-translational modifications (PTMs) are known to cause tissue toxicities and disease progression. These effects are mediated via site-specific modifications and structural disruptions associated with such modifications. 1,4-Benzoquinone (BQ) and its quinone-thioether metabolites are electrophiles that elicit their toxicity via protein arylation and the generation of reactive oxygen species. Site-specific BQ-lysine adducts are found on residues in cytochrome c that are necessary for protein-protein interactions, and these adducts contribute to interferences in its ability to facilitate apoptosome formation. To further characterize the structural and functional impact of these BQ-mediated PTMs, the original mixture of BQ-adducted cytochrome c was fractionated by liquid isoelectric focusing to provide various fractions of BQ-adducted cytochrome c species devoid of the native protein. The fractionation process separates samples based on their isoelectric point (pI), and because BQ adducts form predominantly on lysine residues, increased numbers of BQ adducts on cytochrome c correlate with a lower protein pI. Each fraction was analyzed for structural changes, and each was also assayed for the ability to support apoptosome-mediated activation of caspase-3. Circular dichroism revealed that several of the BQ-adducted cytochrome c species maintained a slightly more rigid structure in comparison to native cytochrome c. BQ-adducted cytochrome c also failed to activate caspase-3, with increasing numbers of BQ-lysine adducts corresponding to a greater inability to activate the apoptosome. In summary, the specific site of the BQ-lysine adducts, and the nature of the adduct, are important determinants of the subsequent structural changes to cytochrome c. In particular, adducts at sites necessary for protein-protein interactions interfere with the proapoptotic function of cytochrome c.

Deletion mutational analysis of BMRP, a pro-apoptotic protein that binds to Bcl-2

Bcl-2 is an anti-apoptotic member of the Bcl-2 family of proteins that protects cells from apoptosis induced by a large variety of stimuli. The protein BMRP (MRPL41) was identified as a Bcl-2 binding partner and shown to have pro-apoptotic activity. We have performed deletion mutational analyses to identify the domain(s) of Bcl-2 and BMRP that are involved in the Bcl-2/BMRP interaction, and the region(s) of BMRP that mediate its pro-apoptotic activity. The results of these studies indicate that both the BH4 domain of Bcl-2 and its central region encompassing its BH1, BH2, and BH3 domains are required for its interaction with BMRP. The loop region and the transmembrane domain of Bcl-2 were found to be dispensable for this interaction. The Bcl-2 deletion mutants that do not interact with BMRP were previously shown to be functionally inactive. Deletion analyses of the BMRP protein delimited the region of BMRP needed for its interaction with Bcl-2 to the amino-terminal two-thirds of the protein (amino acid residues 1–92). Further deletions at either end of the BMRP(1–92) truncated protein resulted in lack of binding to Bcl-2. Functional studies performed with BMRP deletion mutants suggest that the cell death-inducing domains of the protein reside mainly within its amino-terminal two-thirds. The region of BMRP required for the interaction with Bcl-2 is very relevant for the cell death-inducing activity of the protein, suggesting that one possible mechanism by which BMRP induces cell death is by binding to and blocking the anti-apoptotic activity of Bcl-2.