PGC-1α drives small cell neuroendocrine cancer progression towards an ASCL1-expressing subtype with increased mitochondrial capacity.

Adenocarcinomas from multiple tissues can converge to treatment-resistant small cell neuroendocrine (SCN) cancers comprised of ASCL1, POU2F3, NEUROD1, and YAP1 subtypes. We investigated how mitochondrial metabolism influences SCN cancer (SCNC) progression. Extensive bioinformatics analyses encompassing thousands of patient tumors and human cancer cell lines uncovered enhanced expression of PGC-1α, a potent regulator of mitochondrial oxidative phosphorylation (OXPHOS), across several SCNC types. PGC-1α correlated tightly with increased expression of the lineage marker ASCL1 through a positive feedback mechanism. Analyses using a human prostate tissue-based SCN transformation system showed that the ASCL1 subtype has heightened PGC-1α expression and OXPHOS activity. PGC-1α inhibition diminished OXPHOS, reduced SCNC cell proliferation, and blocked SCN prostate tumor formation. PGC-1α overexpression enhanced OXPHOS, tripled the SCN prostate tumor formation rate, and promoted commitment to the ASCL1 lineage. These findings reveal the metabolic heterogeneity among SCNC subtypes and identify PGC-1α-induced OXPHOS as a regulator of SCNC lineage plasticity.

Wild-type C-Raf gene dosage and dimerization drive prostate cancer metastasis.

Mutated Ras and Raf kinases are well-known to promote cancer metastasis via flux through the Ras/Raf/MEK/ERK (mitogen-activated protein kinase [MAPK]) pathway. A role for non-mutated Raf in metastasis is also emerging, but the key mechanisms remain unclear. Elevated expression of any of the three wild-type Raf family members (C, A, or B) can drive metastasis. We utilized an in vivo model to show that wild-type C-Raf overexpression can promote metastasis of immortalized prostate cells in a gene dosage-dependent manner. Analysis of the transcriptomic and phosphoproteomic landscape indicated that C-Raf-driven metastasis is accompanied by upregulated MAPK signaling. Use of C-Raf mutants demonstrated that the dimerization domain, but not its kinase activity, is essential for metastasis. Endogenous Raf monomer knockouts revealed that C-Raf's ability to form dimers with endogenous Raf molecules is important for promoting metastasis. These data identify wild-type C-Raf heterodimer signaling as a potential target for treating metastatic disease.

Temporal evolution reveals bifurcated lineages in aggressive neuroendocrine small cell prostate cancer trans-differentiation.

Trans-differentiation from an adenocarcinoma to a small cell neuroendocrine state is associated with therapy resistance in multiple cancer types. To gain insight into the underlying molecular events of the trans-differentiation, we perform a multi-omics time course analysis of a pan-small cell neuroendocrine cancer model (termed PARCB), a forward genetic transformation using human prostate basal cells and identify a shared developmental, arc-like, and entropy-high trajectory among all transformation model replicates. Further mapping with single cell resolution reveals two distinct lineages defined by mutually exclusive expression of ASCL1 or ASCL2. Temporal regulation by groups of transcription factors across developmental stages reveals that cellular reprogramming precedes the induction of neuronal programs. TFAP4 and ASCL1/2 feedback are identified as potential regulators of ASCL1 and ASCL2 expression. Our study provides temporal transcriptional patterns and uncovers pan-tissue parallels between prostate and lung cancers, as well as connections to normal neuroendocrine cell states.

Hydrogel Arrays Enable Increased Throughput for Screening Effects of Matrix Components and Therapeutics in 3D Tumor Models.

Cell-matrix interactions mediate complex physiological processes through biochemical, mechanical, and geometrical cues, influencing pathological changes and therapeutic responses. Accounting for matrix effects earlier in the drug development pipeline is expected to increase the likelihood of clinical success of novel therapeutics. Biomaterial-based strategies recapitulating specific tissue microenvironments in 3D cell culture exist but integrating these with the 2D culture methods primarily used for drug screening has been challenging. Thus, the protocol presented here details the development of methods for 3D culture within miniaturized biomaterial matrices in a multi-well plate format to facilitate integration with existing drug screening pipelines and conventional assays for cell viability. Since the matrix features critical for preserving clinically relevant phenotypes in cultured cells are expected to be highly tissue- and disease-specific, combinatorial screening of matrix parameters will be necessary to identify appropriate conditions for specific applications. The methods described here use a miniaturized culture format to assess cancer cell responses to orthogonal variation of matrix mechanics and ligand presentation. Specifically, this study demonstrates the use of this platform to investigate the effects of matrix parameters on the responses of patient-derived glioblastoma (GBM) cells to chemotherapy.

Fis1 ablation in the male germline disrupts mitochondrial morphology and mitophagy, and arrests spermatid maturation.

Male germline development involves choreographed changes to mitochondrial number, morphology and organization. Mitochondrial reorganization during spermatogenesis was recently shown to require mitochondrial fusion and fission. Mitophagy, the autophagic degradation of mitochondria, is another mechanism for controlling mitochondrial number and physiology, but its role during spermatogenesis is largely unknown. During post-meiotic spermatid development, restructuring of the mitochondrial network results in packing of mitochondria into a tight array in the sperm midpiece to fuel motility. Here, we show that disruption of mouse Fis1 in the male germline results in early spermatid arrest that is associated with increased mitochondrial content. Mutant spermatids coalesce into multinucleated giant cells that accumulate mitochondria of aberrant ultrastructure and numerous mitophagic and autophagic intermediates, suggesting a defect in mitophagy. We conclude that Fis1 regulates mitochondrial morphology and turnover to promote spermatid maturation.

Mitochondrial fission factor (Mff) is required for organization of the mitochondrial sheath in spermatids.

BACKGROUND: Mitochondrial fission counterbalances fusion to maintain organelle morphology, but its role during development remains poorly characterized. Mammalian spermatogenesis is a complex developmental process involving several drastic changes to mitochondrial shape and organization. Mitochondria are generally small and spherical in spermatogonia, elongate during meiosis, and fragment in haploid round spermatids. Near the end of spermatid maturation, small mitochondrial spheres line the axoneme, elongate, and tightly wrap around the midpiece to form the mitochondrial sheath, which is critical for fueling flagellar movements. It remains unclear how these changes in mitochondrial morphology are regulated and how they affect sperm development. METHODS: We used genetic ablation of Mff (mitochondrial fission factor) in mice to investigate the role of mitochondrial fission during mammalian spermatogenesis. RESULTS: Our analysis indicates that Mff is required for mitochondrial fragmentation in haploid round spermatids and for organizing mitochondria in the midpiece in elongating spermatids. In Mff mutant mice, round spermatids have aberrantly elongated mitochondria that often show central constrictions, suggestive of failed fission events. In elongating spermatids and spermatozoa, mitochondrial sheaths are disjointed, containing swollen mitochondria with large gaps between organelles. These mitochondrial abnormalities in Mff mutant sperm are associated with reduced respiratory chain Complex IV activity, aberrant sperm morphology and motility, and reduced fertility. CONCLUSIONS: Mff is required for organization of the mitochondrial sheath in mouse sperm. GENERAL SIGNIFICANCE: Mitochondrial fission plays an important role in regulating mitochondrial organization during a complex developmental process.

Mitochondrial dynamics during spermatogenesis.

Mitochondrial fusion and fission (mitochondrial dynamics) are homeostatic processes that safeguard normal cellular function. This relationship is especially strong in tissues with constitutively high energy demands, such as brain, heart and skeletal muscle. Less is known about the role of mitochondrial dynamics in developmental systems that involve changes in metabolic function. One such system is spermatogenesis. The first mitochondrial dynamics gene, Fuzzy onions (Fzo), was discovered in 1997 to mediate mitochondrial fusion during Drosophila spermatogenesis. In mammals, however, the role of mitochondrial fusion during spermatogenesis remained unknown for nearly two decades after discovery of Fzo Mammalian spermatogenesis is one of the most complex and lengthy differentiation processes in biology, transforming spermatogonial stem cells into highly specialized sperm cells over a 5-week period. This elaborate differentiation process requires several developmentally regulated mitochondrial and metabolic transitions, making it an attractive model system for studying mitochondrial dynamics in vivo We review the emerging role of mitochondrial biology, and especially its dynamics, during the development of the male germ line.

Mitochondrial fusion is required for spermatogonial differentiation and meiosis.

Differentiating cells tailor their metabolism to fulfill their specialized functions. We examined whether mitochondrial fusion is important for metabolic tailoring during spermatogenesis. Acutely after depletion of mitofusins Mfn1 and Mfn2, spermatogenesis arrests due to failure to accomplish a metabolic shift during meiosis. This metabolic shift includes increased mitochondrial content, mitochondrial elongation, and upregulation of oxidative phosphorylation (OXPHOS). With long-term mitofusin loss, all differentiating germ cell types are depleted, but proliferation of stem-like undifferentiated spermatogonia remains unaffected. Thus, compared with undifferentiated spermatogonia, differentiating spermatogonia and meiotic spermatocytes have cell physiologies that require high levels of mitochondrial fusion. Proteomics in fibroblasts reveals that mitofusin-null cells downregulate respiratory chain complexes and mitochondrial ribosomal subunits. Similarly, mitofusin depletion in immortalized spermatocytes or germ cells in vivo results in reduced OXPHOS subunits and activity. We reveal that by promoting OXPHOS, mitofusins enable spermatogonial differentiation and a metabolic shift during meiosis.

Hierarchical and stage-specific regulation of murine cardiomyocyte maturation by serum response factor.

After birth, cardiomyocytes (CM) acquire numerous adaptations in order to efficiently pump blood throughout an animal's lifespan. How this maturation process is regulated and coordinated is poorly understood. Here, we perform a CRISPR/Cas9 screen in mice and identify serum response factor (SRF) as a key regulator of CM maturation. Mosaic SRF depletion in neonatal CMs disrupts many aspects of their maturation, including sarcomere expansion, mitochondrial biogenesis, transverse-tubule formation, and cellular hypertrophy. Maintenance of maturity in adult CMs is less dependent on SRF. This stage-specific activity is associated with developmentally regulated SRF chromatin occupancy and transcriptional regulation. SRF directly activates genes that regulate sarcomere assembly and mitochondrial dynamics. Perturbation of sarcomere assembly but not mitochondrial dynamics recapitulates SRF knockout phenotypes. SRF overexpression also perturbs CM maturation. Together, these data indicate that carefully balanced SRF activity is essential to promote CM maturation through a hierarchy of cellular processes orchestrated by sarcomere assembly.

Mitochondrial Dynamics is a Distinguishing Feature of Skeletal Muscle Fiber Types and Regulates Organellar Compartmentalization.

Skeletal muscle fibers differentiate into specific fiber types with distinct metabolic properties determined by their reliance on oxidative phosphorylation (OXPHOS). Using in vivo approaches, we find that OXPHOS-dependent fibers, compared to glycolytic fibers, contain elongated mitochondrial networks with higher fusion rates that are dependent on the mitofusins Mfn1 and Mfn2. Switching of a glycolytic fiber to an oxidative IIA type is associated with elongation of mitochondria, suggesting that mitochondrial fusion is linked to metabolic state. Furthermore, we reveal that mitochondrial proteins are compartmentalized to discrete domains centered around their nuclei of origin. The domain dimensions are dependent on fiber type and are regulated by the mitochondrial dynamics proteins Mfn1, Mfn2, and Mff. Our results indicate that mitochondrial dynamics is tailored to fiber type physiology and provides a rationale for the segmental defects characteristic of aged and diseased muscle fibers.

Huntington disease skeletal muscle is hyperexcitable owing to chloride and potassium channel dysfunction.

Huntington disease is a progressive and fatal genetic disorder with debilitating motor and cognitive defects. Chorea, rigidity, dystonia, and muscle weakness are characteristic motor defects of the disease that are commonly attributed to central neurodegeneration. However, no previous study has examined the membrane properties that control contraction in Huntington disease muscle. We show primary defects in ex vivo adult skeletal muscle from the R6/2 transgenic mouse model of Huntington disease. Action potentials in diseased fibers are more easily triggered and prolonged than in fibers from WT littermates. Furthermore, some action potentials in the diseased fibers self-trigger. These defects occur because of decreases in the resting chloride and potassium conductances. Consistent with this, the expression of the muscle chloride channel, ClC-1, in Huntington disease muscle was compromised by improper splicing and a corresponding reduction in total Clcn1 (gene for ClC-1) mRNA. Additionally, the total Kcnj2 (gene for the Kir2.1 potassium channel) mRNA was reduced in disease muscle. The resulting muscle hyperexcitability causes involuntary and prolonged contractions that may contribute to the chorea, rigidity, and dystonia that characterize Huntington disease.