Distinct sub-assemblies (modules) of mitochondrial complex I (CI) are assembled with the assistance of CI Assembly Factors (CIAFs) through mechanisms that are incompletely defined. Here, using genetic analyses in Drosophila, we report that when either of the CIAFs - NDUFAF3 or NDUFAF4 - is disrupted, biogenesis of the Q-, N-, and PP-b-modules of CI is impaired. This is due, at least in part, to the compromised integration of NDUFS3 and NDUFS5 into the Q-, and PP-b-modules, respectively, coupled with a destabilization of another CIAF, TIMMDC1, in assembly intermediates. Notably, forced expression of NDUFAF4 rescues the biogenesis defects in the Q-module and some aspects of the defects in the PP-b-module of CI when NDUFAF3 is disrupted. Altogether, our studies furnish new fundamental insights into the mechanism by which NDUFAF3 and NDUFAF4 regulate CI assembly and raises the possibility that certain point mutations in NDUFAF3 may be rescued by overexpression of NDUFAF4.

Recently, we observed that the TGF-β pathway is altered in 39% of HCCs. The alterations are correlated with a raised HMGA2 level. Therefore, we compared genetic alterations of HMGA2 and 43 TGF-β pathway core genes in HCC patients from TCGA database. Genetic alterations of 15 genes, including INHBE, INHBC, GDF11, ACVRL and TGFB2 out of 43 core genes, highly-moderately matched that of HMGA2. Co-occurrences of mutation amplification, gains, deletions and high/low mRNA of HMGA2 with those of the core genes were highly significant in INHBE, INHBC, ACVR1B, ACVRL and GDF11. Mass spectrometry studies revealed that HMGA2 interacted with an E3 ligase, PJA1, and that this interaction is enhanced by TGF-β treatment in the nuclear of HCC cells. Co-localization of nuclear PJA1 and HMGA2 in HCC cells increased upon TGF-β treatment. Raised HMGA2 levels that occur with alterations in the TGF-β signaling pathway may reflect an altered activity of E3 ligases, such as PJA1, and potentially contribute to the tumor-promoting roles of TGF-β signaling. Here, we report that the co-occurrence of genetic alterations in HMGA2 and TGF-β pathway core genes is implicated in HCC progression, and propose that HMGA2 and PJA1 may be potential novel targets in dysfunctional TGF-β signaling in HCC.

Mutational processes and signatures that drive early tumorigenesis are centrally important for early cancer prevention. Yet, to date, biomarkers and risk factors for polyps (adenomas) that inordinately and rapidly develop into colon cancer remain poorly defined. Here, we describe surprisingly high mutational profiles through whole-genome sequence (WGS) analysis in 2 of 4 pairs of benign colorectal adenoma tissue samples. Unsupervised hierarchical clustered transcriptomic analysis of a further 7 pairs of adenomas reveals distinct mutational signatures regardless of adenoma size. Transitional single nucleotide substitutions of C:G>T:A predominate in the adenoma mutational spectrum. Strikingly, we observe mutations in the TGF-β pathway and CEA-associated genes in 4 out of 11 adenomas, overlapping with the Wnt pathway. Immunohistochemical labeling reveals a nearly 5-fold increase in CEA levels in 23% of adenoma samples with a concomitant loss of TGF-β signaling. We also define a functional role by which the CEA B3 domain interacts with TGFBR1, potentially inactivating the tumor suppressor function of TGF-β signaling. Our study uncovers diverse mutational processes underlying the transition from early adenoma to cancer. This has broad implications for biomarker-driven targeting of CEA/TGF-β in high-risk adenomas and may lead to early detection of aggressive adenoma to CRC progression.

PRAJA, a RING-H2 E3 ligase, is abundantly expressed in brain tissues such as the cerebellum and frontal cortex, amongst others, and more specifically in neural progenitor cells as well as in multiple cancers that include glioblastomas. However, the specific role that Praja plays in neural development and gliomas remains unclear. In this investigation, we performed bioinformatic analyses to examine Praja1 and Praja2 expression across 29 cancer types, and observed raised levels of Praja1 and Praja2 in gliomas with an inverse relationship between Praja1 and apoptotic genes and Praja substrates such as Smad3. We analyzed the role of Praja in the developing brain through loss of function studies, using morpholinos targeting Praja1 in embryonic zebrafish, and observed that Praja1 is expressed prominently in regions enriched with neural precursor cell subtypes. Antisense Praja morpholinos resulted in multiple embryonic defects including delayed neural development likely through increased apoptosis. Further studies revealed high levels of Cdk1 with loss of Praja1 in TGF-β or insulin treated cells, supporting the link between Praja1 and cell cycle regulation. In summary, these studies underscore Praja's role in mammalian brain development and Praja1 deregulation may lead to gliomas possibly through the regulation of cell cycle and/or apoptosis.

Disturbances in endoplasmic reticulum (ER) Ca2+ homeostasis have been associated with many diseases including loss of salivary glands. Although significant progress has been accomplished which led to the increase in our understanding of the cellular responses to ER stress, the factors/ion channels that could inhibit ER stress are not yet identified. Here we show that TRPC1 (transient receptor potential canonical 1) is involved in regulating Ca2+ homeostasis and loss of TRPC1 decreased ER Ca2+ levels, inhibited the unfolded protein response (UPR), that induced loss of salivary gland cells. We provide further evidence that ER stress inducing agents (Tunicamycin and Brefeldin A) disrupts Ca2+ homeostasis by directly inhibiting TRPC1-mediated Ca2+ entry, which led to ER stress in salivary gland cells. Moreover, induction of ER stress lead to an increase in CHOP expression, which decreased TRPC1 expression and subsequently attenuated autophagy along with increased apoptosis. Importantly, TRPC1-/- mice showed increased ER stress, increased immune cell infiltration, loss of Ca2+ homeostasis, decreased saliva secretion, and decreased salivary gland survival. Finally, restoration of TRPC1 not only maintained Ca2+ homeostasis, but inhibited ER stress that induced cell survival. Overall these results suggest a significant role of TRPC1 Ca2+ channels in ER stress and homeostatic function/survival of salivary gland cells.

Disruption of the TGF-β pathway is associated with liver fibrosis and suppression of liver tumorigenesis, conditions associated with low Vitamin D (VD) levels. However, potential contributions of VD to liver tumor progression in the context of TGF-β signaling remain unexplored. Our analyses of VD deprivation (VDD) in in vivo models of liver tumor formation revealed striking three-fold increases in tumor burden in Smad3(+/-) mice, with a three-fold increase in TLR7 expression compared to controls. ChIP and transcriptional assays confirm Smad3 binding at two TLR7 promoter SBE sites. Molecular interactions between TGF-β pathway and VDD were validated clinically, where an absence of VD supplementation was associated with low TGF-β pathway member expression levels and β-catenin activation in fibrotic/cirrhotic human liver tissues. Subsequent supplementing VD led to restoration of TGF-β member expression with lower β-catenin levels. Bioinformatics analysis provides positive supportive correlation between somatic mutations for VD-related genes and the TGF-β pathway. We conclude that VDD promotes tumor growth in the context of Smad3 disruption, potentially through regulation of TLR7 expression and β-catenin activation. VD could therefore be a strong candidate for liver cancer prevention in the context of aberrant Smad3 signaling.

The boot-shaped respiratory complex I (CI) consists of a mitochondrial matrix and membrane domain organized into N-, Q- and P-modules. The N-module is the most distal part of the matrix domain, whereas the Q-module is situated between the N-module and the membrane domain. The proton-pumping P-module is situated in the membrane domain. We explored the effect of aging on the disintegration of CI and its constituent subcomplexes and modules in Drosophila flight muscles. We find that the fully-assembled complex remains largely intact in aged flies. And while the effect of aging on the stability of many Q- and N-module subunits in subcomplexes was stochastic, NDUFS3 was consistently down-regulated in subcomplexes with age. This was associated with an accumulation of many P-module subunits in subcomplexes. The potential significance of these studies is that genetic manipulations aimed at boosting, perhaps, a few CI subunits may suffice to restore the whole CI biosynthesis pathway during muscle aging.