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It is important to determine the activation status of Rac and Cdc42 in cancer tissues for the prediction of metastasis and patient prognosis. However, it has been impossible to detect their spatial activation on formalin-fixed paraffin embedded (FFPE) surgical specimens thus far. Here, we established a novel detection technique for activated Rac/Cdc42 in human colon cancer FFPE tissues by using a p21-activated kinase (PAK)-Rac binding domain (RBD) detection probe fused with glutathione S-transferase (GST), designated GST-PAK-RBD, and novel rapid-immunohistochemistry (R-IHC) systems using noncontact alterating-current electric field mixing, although there is a technical limitation in that it may not distinguish between Rac members and Cdc42. In 50 cases of colon cancer, various activation patterns of Rac/Cdc42 were observed, which were designated plasma membrane, cytoplasm, mixed pattern, and polarized distribution. The activity was striking in the invasive fronts of tumors and significantly correlated with tumor invasion properties evaluated by TNM classification. Of note, in tissue microarray (TMA) samples, 29 of 33 cases demonstrated higher Rac1/Cdc42 activity in the tumor area than the corresponding normal mucosa. In addition, positive correlations were detected between Rac/Cdc42 activity and clinicopathological factors such as venous and lymphatic vessel invasion. These results suggest that understanding Rac and Cdc42 activations in cancer tissues would be valuable as an option for molecular therapy as personalized medicine.

Krabbe disease is a lysosomal storage disease caused by a deficiency of the galactocerebrosidase (GALC) enzyme, which leads to demyelination of the central and peripheral nervous systems. Almost all patients with Krabbe disease are infants, and this is the first report of adult-onset cases that describe pathological findings. Here, we present two autopsy cases: a 73-year-old female and a 2-year-old male. The adult-onset case developed symptoms in her late thirties and was diagnosed by the identification of GALC D528N and L634S mutations and by T2-weighted magnetic resonance imaging; she had increased signal in the white matter along the pyramidal tract to the bilateral precentral gyrus, as well as from the triangular part to the posterior horn of the lateral ventricle. Microscopically, Kluver-Barrera staining was pale in the white matter of the precentral gyrus and occipito-thalamic radiation, and a few globoid cells were observed. The GALC mutations that were identified in the present adult-onset case do not completely inactivate GALC enzyme activity, resulting in focal demyelination of the brain.

We provide the details of the successful management of a patient with active Cushing’s disease complicated with coronavirus disease 2019 (COVID-19) pneumonia. The patient was a 27-year-old Japanese female healthcare worker who was scheduled to undergo pituitary surgery for Cushing’s disease. She had been in close contact with an undiagnosed patient infected with COVID-19 and then developed COVID-19 pneumonia. Despite a lack of known risk factors associated with severe COVID-19 infection, the patient’s dyspnea worsened and her respiratory condition deteriorated, as indicated by the need for 7 L/min oxygen supply by mask to maintain her oxygen saturation at >90%. Medical treatment was initiated to control hypercortisolism by the ‘block and replace’ regimen using steroidogenesis inhibitors and hydrocortisone. The COVID-19 pneumonia improved with multi-modal treatment including antiviral therapy. One month later, after a negative severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) test result and with appropriate protection against virus transmission to medical staff in the operating room and daily medical care nurses, trans-sphenoidal surgery was performed by our highly experienced pituitary surgeon. One month after the surgery, the patient’s basal ACTH and cortisol levels and urinary free cortisol were all under the detection limit. Surgical remission was expected. Since hypercortisolism due to active Cushing’s disease may worsen a COVID-19 infection, multi-disciplinary management that includes appropriate and prompt treatment strategies is mandatory in such cases.

Locally advanced and metastatic invasive bladder cancer (BC) has a poor prognosis, and no advanced therapies beyond cisplatin-based combination chemotherapy have been developed. Therefore, it is an urgent issue to elucidate the underlying mechanisms of tumor progression and metastasis of invasive BC for the development of new therapeutic strategies. Here, we clarified a novel role of exosomes containing ErbB2 and CRK in a formation of premetastatic niches and subsequent metastases. CRK adaptors were overexpressed in invasive UM-UC-3 BC cells. In an orthotopic xenograft model, metastases to lung, liver, and bone of UM-UC-3 cells were completely abolished by CRK elimination. Mass spectrometry analysis identified that ErbB2 was contained in UM-UC-3-derived exosomes in a CRK-dependent manner; the exosomes significantly increased proliferation and invasion properties of low-grade 5637 BC cells and HUVECs through FAK and PI3K/AKT signaling pathways. In athymic mice educated with UM-UC-3-derived exosomes, i.v. implanted UM-UC-3 cells were trapped with surrounding PKH67-labeled exosomes in lung and led to development of lung metastasis with disordered vascular proliferation. In contrast, exosomes derived from CRK-depleted BC cells failed to induce these malignant features. Taken together, we showed that CRK adaptors elevated the expression of ErbB2/3 in BC cells, and these tyrosine kinase/adaptor units were transferred from host BC cells to metastatic recipient cells by exosomes, leading to vascular leakiness and proliferation and contributing to the formation of distant metastasis. Thus, CRK intervention with ErbB2/3 blockade might be a potent therapeutic strategy for patients with ErbB2 overexpressing advanced and metastatic BC.

Locally advanced and metastatic invasive bladder cancer (BC) has a poor prognosis, and no advanced therapies beyond cisplatin-based combination chemotherapy have been developed. Therefore, it is an urgent issue to elucidate the underlying mechanisms of tumor progression and metastasis of invasive BC for the development of new therapeutic strategies. Here, we clarified a novel role of exosomes containing ErbB2 and CRK in a formation of premetastatic niches and subsequent metastases. CRK adaptors were overexpressed in invasive UM-UC-3 BC cells. In an orthotopic xenograft model, metastases to lung, liver, and bone of UM-UC-3 cells were completely abolished by CRK elimination. Mass spectrometry analysis identified that ErbB2 was contained in UM-UC-3-derived exosomes in a CRK-dependent manner; the exosomes significantly increased proliferation and invasion properties of low-grade 5637 BC cells and HUVECs through FAK and PI3K/AKT signaling pathways. In athymic mice educated with UM-UC-3-derived exosomes, i.v. implanted UM-UC-3 cells were trapped with surrounding PKH67-labeled exosomes in lung and led to development of lung metastasis with disordered vascular proliferation. In contrast, exosomes derived from CRK-depleted BC cells failed to induce these malignant features. Taken together, we showed that CRK adaptors elevated the expression of ErbB2/3 in BC cells, and these tyrosine kinase/adaptor units were transferred from host BC cells to metastatic recipient cells by exosomes, leading to vascular leakiness and proliferation and contributing to the formation of distant metastasis. Thus, CRK intervention with ErbB2/3 blockade might be a potent therapeutic strategy for patients with ErbB2 overexpressing advanced and metastatic BC.

Drug resistance, metastasis, and a mesenchymal transcriptional program are central features of aggressive breast tumors. The GTPase Arf6, often overexpressed in tumors, is critical to promote epithelial mesenchymal transition and invasiveness. The metabolic mevalonate pathway (MVP) is associated with tumor invasiveness and known to prenylate proteins, but which prenylated proteins are critical for MVP-driven cancers is unknown. We show here that MVP requires the Arf6-dependent mesenchymal program. The MVP enzyme geranylgeranyl transferase II (GGT-II) and its substrate Rab11b are critical for Arf6 trafficking to the plasma membrane, where it is activated by receptor tyrosine kinases. Consistently, mutant p53, which is known to support tumorigenesis via MVP, promotes Arf6 activation via GGT-II and Rab11b. Inhibition of MVP and GGT-II blocked invasion and metastasis and reduced cancer cell resistance against chemotherapy agents, but only in cells overexpressing Arf6 and components of the mesenchymal program. Overexpression of Arf6 and mesenchymal proteins as well as enhanced MVP activity correlated with poor patient survival. These results provide insights into the molecular basis of MVP-driven malignancy.

Acquisition of mesenchymal properties by cancer cells is critical for their malignant behaviour, but regulators of the mesenchymal molecular machinery and how it is activated remain elusive. Here we show that clear cell renal cell carcinomas (ccRCCs) frequently utilize the Arf6-based mesenchymal pathway to promote invasion and metastasis, similar to breast cancers. In breast cancer cells, ligand-activated receptor tyrosine kinases employ GEP100 to activate Arf6, which then recruits AMAP1; and AMAP1 then binds to the mesenchymal-specific protein EPB41L5, which promotes epithelial-mesenchymal transition and focal adhesion dynamics. In renal cancer cells, lysophosphatidic acid (LPA) activates Arf6 via its G-protein-coupled receptors, in which GTP-G alpha 12 binds to EFA6. The Arf6-based pathway may also contribute to drug resistance. Our results identify a specific mesenchymal molecular machinery of primary ccRCCs, which is triggered by a product of autotaxin and it is associated with poor outcome of patients.

Acquisition of mesenchymal properties by cancer cells is critical for their malignant behaviour, but regulators of the mesenchymal molecular machinery and how it is activated remain elusive. Here we show that clear cell renal cell carcinomas (ccRCCs) frequently utilize the Arf6-based mesenchymal pathway to promote invasion and metastasis, similar to breast cancers. In breast cancer cells, ligand-activated receptor tyrosine kinases employ GEP100 to activate Arf6, which then recruits AMAP1; and AMAP1 then binds to the mesenchymal-specific protein EPB41L5, which promotes epithelial-mesenchymal transition and focal adhesion dynamics. In renal cancer cells, lysophosphatidic acid (LPA) activates Arf6 via its G-protein-coupled receptors, in which GTP-G alpha 12 binds to EFA6. The Arf6-based pathway may also contribute to drug resistance. Our results identify a specific mesenchymal molecular machinery of primary ccRCCs, which is triggered by a product of autotaxin and it is associated with poor outcome of patients.