Semaphorin signaling via MICAL3 induces symmetric cell division to expand breast cancer stem-like cells.

Cancer stem-like cells (CSCs) are expanded in the CSC niche by increased frequency of symmetric cell divisions at the expense of asymmetric cell divisions. The symmetric division of CSCs is important for the malignant properties of cancer; however, underlying molecular mechanisms remain largely elusive. Here, we show a cytokine, semaphorin 3 (Sema3), produced from the CSC niche, induces symmetric divisions of CSCs to expand the CSC population. Our findings indicate that stimulation with Sema3 induced sphere formation in breast cancer cells through neuropilin 1 (NP1) receptor that was specifically expressed in breast CSCs (BCSCs). Knockdown of MICAL3, a cytoplasmic Sema3 signal transducer, greatly decreased tumor sphere formation and tumor-initiating activity. Mechanistically, Sema3 induced interaction among MICAL3, collapsin response mediator protein 2 (CRMP2), and Numb. It appears that activity of MICAL3 monooxygenase (MO) stimulated by Sema3 is required for tumor sphere formation, interaction between CRMP2 and Numb, and accumulation of Numb protein. We found that knockdown of CRMP2 or Numb significantly decreased tumor sphere formation. Moreover, MICAL3 knockdown significantly decreased Sema3-induced symmetric divisions in NP1/Numb-positive BCSCs and increased asymmetric division that produces NP1/Numb negative cells without stem-like properties. In addition, breast cancer patients with NP1-positive cancer tissues show poor prognosis. Therefore, the niche factor Sema3-stimulated NP1/MICAL3/CRMP2/Numb axis appears to expand CSCs at least partly through increased frequency of MICAL3-mediated symmetric division of CSCs.

A multicenter, observational study of metastatic breast cancer patients who were treated with eribulin mesylate or taxane-based regimens.

AIM: This multicenter, observational study aimed to investigate the survival benefit of eribulin as well as that of taxane-based regimens in Japanese patients with metastatic breast cancer (MBC) in a real-world setting. METHODS: This study enrolled women with MBC who received eribulin or taxane-based regimens with or without bevacizumab in routine clinical practice from July 2011 to March 2014. Patients were followed until September 2015. The primary endpoint was overall survival (OS). Secondary endpoints included progression-free survival (PFS), post-progression survival (PPS) and adverse events. Efficacy findings were adjusted according to demographics. RESULTS: In total, 216 patients receiving eribulin monotherapy (n = 101), taxane monotherapy (n = 73) or taxane plus bevacizumab (n = 42) were followed for a median time of 15.4 months. Median OS, PFS and PPS were 22.3, 8.1 and 14 months in the eribulin monotherapy group; 13.2, 3.6 and 7.6 months in the taxane monotherapy group; and 12.9, 5.7 and 6.3 months, in the taxane plus bevacizumab group, respectively. The incidence of neutropenia was 67.3, 41.1 and 16.7%, and the incidence of grade 4 neutropenia was 1.0, 8.2 and 7.1% in the eribulin monotherapy, taxane monotherapy and taxane plus bevacizumab groups, respectively. One patient (1.0%) discontinued eribulin and 18 patients (15.7%) discontinued taxane-based regimens because of adverse events. CONCLUSION: In Japanese MBC patients in a real-world setting, eribulin showed a survival benefit and tolerability similar to that in previous reports.

An autocrine/paracrine circuit of growth differentiation factor (GDF) 15 has a role for maintenance of breast cancer stem-like cells.

Cancer stem cells are thought to be responsible for tumor growth, recurrence, and resistance to conventional cancer therapy. However, it is still unclear how they are maintained in tumor tissues. Here, we show that the growth differentiation factor 15 (GDF15), a member of the TGFß family, may maintain cancer stem-like cells in breast cancer tissues by inducing its own expression in an autocrine/paracrine manner. We found that GDF15, but not TGFß, increased tumor sphere formation in several breast cancer cell lines and patient-derived primary breast cancer cells. As expected, TGFß strongly stimulated the phosphorylation of Smad2. GDF15 also stimulated the phosphorylation of Smad2, but the GDF15-induced tumor sphere forming efficiency was not significantly affected by treatment with SB431542, an inhibitor of the TGFß signaling. Although TGFß transiently activated ERK1/2, GDF15 induced prolonged activation of ERK1/2. Treatment with U0126, an inhibitor of the MEK-ERK1/2 signaling, greatly inhibited the GDF15-induced tumor sphere formation. Moreover, cytokine array experiments revealed that GDF15, but not TGFß, is able to induce its own expression; furthermore, it appears to form an autocrine/paracrine circuit to continuously produce GDF15. In addition, we found heterogeneous expression levels of GDF15 among cancer cells and in human breast cancer tissues using immunohistochemistry. This may reflect a heterogeneous cancer cell population, including cancer stem-like cells and other cancer cells. Our findings suggest that GDF15 induces tumor sphere formation through GDF15-ERK1/2-GDF15 circuits, leading to maintenance of GDF15high cancer stem-like cells. Targeting GDF15 to break these circuits should contribute to the eradication of tumors.

ErbB receptor tyrosine kinase/NF-κB signaling controls mammosphere formation in human breast cancer.

Breast cancer is one of the most common cancers in humans. However, our understanding of the cellular and molecular mechanisms underlying tumorigenesis in breast tissues is limited. Here, we identified a molecular mechanism that controls the ability of breast cancer cells to form multicellular spheroids (mammospheres). We found that heregulin (HRG), a ligand for ErbB3, induced mammosphere formation of a breast cancer stem cell (BCSC)-enriched population as well as in breast cancer cell lines. HRG-induced mammosphere formation was reduced by treatment with inhibitors for phosphatidyl inositol 3-kinase (PI3K) or NF-κB and by expression of IκBα-Super Repressor (IκBαSR), a dominant-negative inhibitor for NF-κB. Moreover, the overexpression of IκBαSR in breast cancer cells inhibited tumorigenesis in NOD/SCID mice. Furthermore, we found that the expression of IL8, a regulator of self-renewal in BCSC-enriched populations, was induced by HRG through the activation of the PI3K/NF-κB pathway. These findings illustrate that HRG/ErbB3 signaling appears to maintain mammosphere formation through a PI3K/NF-κB pathway in human breast cancer.

Primary non-Hodgkin's lymphoma of the breast treated nonsurgically: report of three cases.

Extranodal non-Hodgkin's lymphoma (NHL) is a rare breast disease. Here we report three cases of primary NHL of the breast. The first patient was a 29-year-old woman with a firm mass in her right breast with ipsilateral axillary lymphadenopathy. An excisional biopsy revealed NHLs. Clinical stage was IIAE. The tumor and enlarged lymph nodes had successfully been treated following the combination therapy. The second patient was a 70-year-old women with an elastic hard mass in her left breast. An excisional biopsy revealed NHLs and clinical stage was 1AE. The tumor disappeared following the combination therapy. The third patient was a 67-year-old women with a hard mass in her left breast. Core needle biopsy revealed NHLs and clinical stage was 1AE. The tumor disappeared following chemotherapy. All patients are alive with no evidence of recurrence 4-8 years after the initial treatment. Although a standard treatment has yet to be established, an initial treatment with combination therapy without surgical intervention including axillary dissection appears to be appropriate for this rare disease.

Excision of postesophageal parathyroid adenoma in posterior mediastinum with intraoperative 99mTechnetium sestamibi scanning.

Although approximately 25% of parathyroid tumors in patients with primary hyperparathyroidism are located in the mediastinum, nearly all these tumors can be removed through cervical exploration. However, 1% to 2% of the mediastinal tumors require a transthoracic approach for removal. The mediastinal tumors are usually located in the inferior parathyroid gland, and the ectopic mediastinal tumors derived from the superior glands are extremely rare. We present a case of retroesophageal mediastinal parathyroid adenoma that developed in the left superior parathyroid gland. A thoracotomy was required to remove this tumor. Radioisotope-guided surgery was effective at identifying the tumor.

Diffuse large B-cell lymphoma in the thyroid gland associated with primary hyperparathyroidism.

We report a case of primary hyperparathyroidism associated with a malignant lymphoma in the thyroid gland. A 68-year-old woman was admitted to hospital with a cervical mass. Ultrasound and computed tomography (CT) revealed a hypoechoic, multinodular tumor in the left thyroid gland. A gallium-67 citrate scintigram revealed intense radioisotope uptake in the thyroid tumor. Histological examination of biopsy specimens indicated that this tumor was a large B-cell lymphoma. The coexistence of parathyroid adenoma in this patient was revealed by a sestamibi scintigram, performed prior to chemotherapy. Following the complete remission of the lymphoma by chemotherapy, we carried out an excision of the single parathyroid adenoma. To our knowledge, this is the first report to describe a malignant thyroid lymphoma associated with primary hyperparathyroidism.

Preoperative evaluation of thyroid pathology in patients with primary hyperparathyroidism.

In parathyroidectomy, it has been recognized that a shift to a minimally invasive procedure may be accompanied by a possibility of missing thyroid pathology. However, only a few findings concerning preoperative thyroid evaluation have been reported. We investigated the prevalence of concomitant thyroid pathology by preoperative neck ultrasonography (US) in patients with primary hyperparathyroidism. There were 85 patients (66 women, 19 men; mean age 57 years) in the study group. The mean preoperative calcium level was 11.2mg/dL, and the mean intact parathyroid hormone level was 206 pg/mL. All patients underwent neck US following fine-needle aspiration biopsy (FNAB). Of the 85 patients, 21 (24.7%) had thyroid nodules. Among 21 patients with thyroid nodules, 9 (10.6%) had malignant thyroid tumors, while 12 (14.1%) patients had benign thyroid nodules including multinodular goiter. Of the 9 patients with malignant thyroid nodules, 4 had papillary carcinomas with lymph node metastases. The prevalence of thyroid disease associated with hyperparathyroidism is high, and evaluation of the thyroid pathology by US enables the shift from bilateral neck exploration to the minimally invasive parathyroid surgery.

Troglitazone, the peroxisome proliferator-activated receptor-gamma agonist, induces antiproliferation and redifferentiation in human thyroid cancer cell lines.

Troglitazone is a potent agonist for the peroxisome proliferator-activated receptor-gamma (PPARgamma) that is a ligand-activated transcription factor regulating cell differentiation and growth. PPARgamma may play a role in thyroid carcinogenesis since PAX8-PPARgamma1 chromosomal translocations are commonly found in follicular thyroid cancers. We investigated the antiproliferative and redifferentiation effects of troglitazone in 6 human thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO82-1 (anaplastic) cell lines. PPARgamma was expressed variably in these cell lines. FTC-236 and FTC-238 had a rearranged chromosome at 3p25, possibly implicating the involvement of the PPARgamma encoding gene whereas the other cell lines did not. Troglitazone significantly inhibited cell growth by cell cycle arrest and apoptotic cell death. PPARgamma overexpression did not appear to be a prerequisite for a response to treatment with troglitazone. Troglitazone also downregulated surface expression of CD97, a novel dedifferentiation marker, in FTC-133 cells and upregulated sodium iodide symporter (NIS) mRNA in TPC-1 and FTC-133 cells. Our investigations document that human thyroid cancer cell lines commonly express PPARgamma, but chromosomal translocations involving PPARgamma are uncommon. Troglitazone, a PPARgamma agonist, induced antiproliferation and redifferentiation in thyroid cancer cell lines. PPARgamma agonists may therefore be effective therapeutic agents for the treatment of patients with thyroid cancer that fails to respond to traditional treatments.

Diagnostic and prognostic value of fas and telomeric-repeat binding factor-1 genes in adrenal tumors.

It is often difficult to distinguish histologically between an adrenal cortical cancer and a benign adenoma, or to predict the prognosis of patients with adrenal cortical cancers. In this investigation, we examined whether apoptosis-regulating genes, bcl-xL and fas, and a telomere-related gene, telomeric-repeat binding factor-1 (TRF-1), differ between adrenal cortical cancers and benign adrenal tumors. Tissues from 4 adrenal cortical cancers were compared with 7 normal adrenal tissues, 17 cortical adenomas, 4 cortical hyperplasias, and 20 pheochromocytomas for expressions of bcl-xL and fas by RT-PCR, and for expressions of TRF-1 by real-time quantitative RT-PCR. All benign adrenal tissues expressed both the antiapoptosis gene, bcl-xL, and proapoptosis gene, fas, but the adrenal cortical cancers expressed only bcl-xL and not fas. TRF-1 increased by more than 30-fold in the adrenal cortical cancers, compared with benign adrenal tissues, and inversely correlated with the prognosis of patients with the adrenal cortical cancers. This lack of expression of fas in adrenal cortical cancer may help to distinguish it from benign adrenal tumors. The level of TRF-1 expression may be helpful prognostically for patients with adrenal cortical cancers.

Expression of vascular endothelial growth factor-C in benign and malignant thyroid tumors.

In contrast to vascular endothelial growth factor (VEGF), which stimulates angiogenesis, VEGF-C is thought to stimulate lymphangiogenesis. The role of VEGF-C in thyroid cancer pathogenesis has not been clarified. One might expect a different pattern of VEGF-C expression in the various types of thyroid cancer because of their different means of metastases. In this investigation, we determined whether the differential expression of VEGF-C might explain the different propensity to lymph node metastasis in thyroid cancers. One hundred eleven normal and neoplastic thyroid tissues were analyzed by real-time quantitative PCR. Papillary thyroid cancers had a higher VEGF-C expression than other thyroid malignancies (P < 0.0005 ANOVA). Among the normal thyroid tissues from patients with malignant or benign thyroid diseases, there was no significant difference in VEGF-C expression. Paired comparison of VEGF-C expression between thyroid cancers and normal thyroid tissues from the same patients showed a significant increase of VEGF-C expression in papillary thyroid cancer (1.10 +/- 0.41 vs. 0.70 +/- 0.13; P = 0.001) and a significant decrease of VEGF-C expression in medullary thyroid cancer (0.11 +/- 0.13 vs. 0.78 +/- 0.29; P = 0.001). In contrast, there was no significant difference of VEGF-C expression between cancer and normal tissues in other types of thyroid cancer. In summary, VEGF-C expression is increased in papillary thyroid cancer, compared with paired normal thyroid tissues, but not in other thyroid cancers that are also prone to lymph node metastasis. The lymphangiogenic role of VEGF-C in thyroid cancers therefore appears to be complex and other factors are likely to be also involved.

Apoptosis regulating genes, bcl-2 and bax, and human telomerase reverse transcriptase messenger RNA expression in adrenal tumors: possible diagnostic and prognostic importance.

BACKGROUND: Apoptosis-suppressing gene, bcl-2, and apoptosis-inducing gene, bax, are expressed in various tumors. Human telomerase reverse transcriptase (hTERT) expression is increased in many cancers. The purpose of this investigation was to determine whether bcl-2 or bax gene expression differs between benign and malignant adrenal tumors, and whether hTERT gene expression correlates with the prognosis of patients with adrenal cancer. METHODS: Expression of bcl-2 and bax from 52 adrenal surgical specimens was analyzed by reverse transcription-polymerase chain reaction. Expression of hTERT was analyzed by real-time quantitative reverse transcription-polymerase chain reaction, and expressed using the delta-delta threshold cycle method. RESULTS: All normal adrenal tissues (n = 7) and benign adrenal tumors (n = 41) expressed bcl-2 and bax, whereas all adrenocortical cancers (n = 4) expressed bcl-2 but not bax. Expression of hTERT was increased in adrenocortical cancers compared with that in normal tissues (P <.05), cortical adenomas (n = 17) (P <.05), cortical hyperplasias (n = 4) (P <.05), and pheochromocytomas (n = 20) (P <.05). The one patient with an adrenocortical cancer without increased hTERT expression is alive with distant metastasis 5 years after adrenalectomy. The other 3 patients with adrenocortical cancer died 5 to 24 months after adrenalectomy. CONCLUSIONS: Expression of bax gene appears to distinguish adrenal cancer from benign adrenal tumors. Gene expression of hTERT should be investigated as a prognostic marker in adrenal cortical cancers.

Consistent decrease in telomere length in parathyroid tumors but alteration in telomerase activity limited to malignancies: preliminary report.

Telomerase is known to be activated and telomere length altered in various types of malignant and benign tumors, but whether this is also the case for parathyroid lesions has hitherto been unclear. We therefore investigated telomerase activity and telomere length in 3 parathyroid metastatic cancers, 6 adenomas, 2 cases of parathyroid hyperplasia, and 16 samples of normal parathyroid tissue. Telomerase activity, assayed by the telomeric repeat amplification protocol, was detected in all of the parathyroid cancers (100%), in none of the 8 parathyroid benign lesions, and in only 1 of the 16 normal parathyroid samples (8.3%). Telomere length, determined by the terminal restriction fragment assay, was reduced in the tumor tissues with a mean telomere length of 8.23 +/- 0.86 kbp compared with the 12.61 +/- 0.81 kbp for the 16 age-matched subjects (p = 0.002). The results indicate that telomerase activity and telomere length may reflect the biologic behavior of individual parathyroid lesions.

Demonstration of human telomerase reverse transcriptase in human colorectal carcinomas by in situ hybridization.

Telomerase activity is present in most malignant tumors and provides a mechanism for unlimited replication of neoplastic cells. Expression of the gene encoding human telomerase reverse transcriptase (hTERT), the telomerase catalytic subunit gene, is associated with telomerase activity, and it is overexpressed in most colorectal carcinomas. In the present study we assayed telomerase activity by the telomeric repeat amplification protocol (TRAP) and used in situ hybridization (ISH) and the reverse transcription polymerase chain reaction (RT-PCR) to study hTERT expression in colorectal carcinomas and adjacent normal tissues. Telomerase activity was found in 30/35 (85.7%) of normal mucosae and 35/35 (100%) of adenocarcinomas, and RT-PCR detected hTERT in 33/35 (94.3%) of the carcinomas. ISH, on the other hand, detected weak but significant expression of hTERT in a significant percentage of lymphocytes infiltrating normal colorectal mucosa. hTERT gene expression was detected in the nuclei of adenocarcinoma cells in 27/35 (77.1%) of the lesions. The results of our comparison of telomerase activity and hTERT gene expression by RT-PCR-based ISH appeared contradictory, but a careful review suggested that the discrepancy was attributable to contamination by infiltrating lymphocytes. Our findings suggest that ISH-based analysis of hTERT gene expression is superior to both TRAP telomerase activity and hTERT mRNA RT-PCR analysis as a means of determining telomerase status during carcinogenesis.