In vivo CRISPRi screen reveals the differential requirement for mitochondrial respiratory chain function between in vivo and in vitro tumor growth

The Warburg effect, aerobic glycolysis, is a hallmark feature of cancer cells grown in culture. However, the relative roles of glycolysis and respiratory metabolism in supporting in vivo tumor growth and significant processes such as tumor dissemination and metastases remain poorly understood, particularly on a systems level. Using a CRISPRi mini-library enriched for mitochondrial ribosomal protein and respiratory chain genes in multiple human lung cancer cell lines we analyzed in vivo metabolic requirements in xenograft tumors grown in distinct anatomic contexts. While knockdown of mitochondrial ribosomal protein and respiratory chain genes (mito-respiratory genes) has little impact on growth in vitro, tumor cells depend heavily on these genes when grown in vivo as either flank or primary orthotopic lung tumor xenografts. In contrast, respiratory function is comparatively dispensable for metastatic tumor growth. RNA-Seq and metabolomics analysis of tumor cells expressing individual sgRNAs against mito-respiratory genes indicate overexpression of glycolytic genes and increased sensitivity of glycolytic inhibition compared to control when grown in vitro, but when grown in vivo as primary tumors these cells downregulate glycolytic mechanisms. These studies demonstrate that discrete perturbations of mitochondrial metabolism impact in vivo tumor growth in a context-specific manner and provides systems-level evidence that respiratory function modulates tumor growth in vivo, suggesting that ATP limits growth and metastatic potential.

Differential expression of mitochondrial ribosomal protein S34 in cancers of the breast.

Breast cancer affects women at relatively high frequency (1). We mined published microarray datasets (2, 3) to determine in an unbiased fashion and at the systems level genes most differentially expressed in the primary tumors of patients with breast cancer. We report here significant differential expression of the gene encoding the mitochondrial ribosomal protein S34, MRPS34, when comparing primary tumors of the breast to the tissue of origin, the normal breast. MRPS34 was also differentially expressed in the tumor cells of patients with triple negative breast cancer. MRPS34 mRNA was present at significantly higher quantities in tumors of the breast as compared to normal breast tissue. Analysis of human survival data revealed that expression of MRPS34 in primary tumors of the breast was correlated with overall survival in patients with luminal B subtype cancer. MRPS34 may be of relevance to initiation, maintenance or progression of cancers of the female breast.

Polymorphisms in mitochondrial ribosomal protein S5 (MRPS5) are associated with leprosy risk in Chinese

Leprosy is an infectious disease caused by Mycobacterium leprae (M. leprae), with about 210,000 new cases per year worldwide. Although numerous risk loci have been uncovered by genome-wide association studies, the effects of common genetic variants are relatively modest. To identify possible new genetic locus involved in susceptibility to leprosy, whole exome sequencing was performed for 28 subjects including 14 patients and 12 unaffected members from 8 leprosy-affected families as well as another case and an unrelated control, and then the follow-up SNP genotyping of the candidate variants was studied in case-control sample sets. A rare missense variant in mitochondrial ribosomal protein S5 (MRPS5), rs200730619 (c. 95108402T>C [p. Tyr137Cys]) was identified and validated in 369 cases and 270 controls of Chinese descent (Padjusted = 0.006, odds ratio [OR] = 2.74) as a contributing factor to leprosy risk. Moreover, the mRNA level of MRPS5 was downregulated in M. leprae sonicate-stimulated peripheral blood mononuclear cells. Our results indicated that MRPS5 may be involved in leprosy pathogenesis. Further studies are needed to determine if defective MRPS5 could lead to impairment of energy metabolism of host immune cells, which could further cause defect in clearing M. leprae and increase susceptibility to infection.

Cell stemness is maintained upon concurrent expression of RB and the mitochondrial ribosomal protein S18-2

Stemness encompasses the capability of a cell for self-renewal and differentiation. The stem cell maintains a balance between proliferation, quiescence, and regeneration via interactions with the microenvironment. Previously, we showed that ectopic expression of the mitochondrial ribosomal protein S18-2 (MRPS18-2) led to immortalization of primary fibroblasts, accompanied by induction of an embryonic stem cell (ESC) phenotype. Moreover, we demonstrated interaction between S18-2 and the retinoblastoma-associated protein (RB) and hypothesized that the simultaneous expression of RB and S18-2 is essential for maintaining cell stemness. Here, we experimentally investigated the role of S18-2 in cell stemness and differentiation. Concurrent expression of RB and S18-2 resulted in immortalization ofRb1−/−primary mouse embryonic fibroblasts and in aggressive tumor growth in severe combined immunodeficiency mice. These cells, which express both RB and S18-2 at high levels, exhibited the potential to differentiate into various lineages in vitro, including osteogenic, chondrogenic, and adipogenic lineages. Mechanistically, S18-2 formed a multimeric protein complex with prohibitin and the ring finger protein 2 (RNF2). This molecular complex increased the monoubiquitination of histone H2ALys119, a characteristic trait of ESCs, by enhanced E3-ligase activity of RNF2. Furthermore, we found enrichment of KLF4 at theS18-2promoter region and that theS18-2expression is positively correlated withKLF4levels. Importantly, knockdown of S18-2 in zebrafish larvae led to embryonic lethality. Collectively, our findings suggest an important role for S18-2 in cell stemness and differentiation and potentially also in cancerogenesis.