Increased understanding of the molecular heterogeneity that is intrinsic to the various subtypes of breast cancer will likely shape the future of breast cancer diagnosis, prognosis, and treatment. Advances in the field over the last several decades have been remarkable and have clearly translated into better patient care as evidenced by the earlier detection, better prognosis, and new targeted therapies. There have been two recent advances in the breast cancer research field that have lead to paradigm shifts: first, the identification of intrinsic breast tumor subtypes, which has changed the way we think about breast cancer and second, the recent characterization of cancer stem cells (CSCs), which are suspected to be responsible for tumor initiation, recurrence and resistance to therapy, have opened new exciting avenues to think about breast cancer therapeutic strategies. While these advances constitute major paradigm shifts within the research realm, the clinical arena has yet to adopt and apply our understanding of the molecular basis of the disease to early diagnosis, prognosis and therapy of breast cancers. Here, we will review the current clinical approach to classification of breast cancers, newer molecular-based classification schemes, and potential future of biomarkers representing a functional classification of breast cancer.

Background: Epidermal growth factor receptor (EGFR) controls a wide range of cellular processes, and altered EGFR signaling contributes to human cancer. EGFR kinase domain mutants found in non-small cell lung cancer (NSCLC) are constitutively active, a trait critical for cell transformation through activation of downstream pathways. Endocytic trafficking of EGFR is a major regulatory mechanism as ligand-induced lysosomal degradation results in termination of signaling. While numerous studies have examined mutant EGFR signaling, the endocytic traffic of mutant EGFR within the NSCLC milieu remains less clear. Results: This study shows that mutant EGFRs in NSCLC cell lines are constitutively endocytosed as shown by their colocalization with the early/recycling endosomal marker transferrin and the late endosomal/lysosomal marker LAMP1. Notably, mutant EGFRs, but not the wild-type EGFR, show a perinuclear accumulation and colocalization with recycling endosomal markers such as Rab11 and EHD1 upon treatment of cells with endocytic recycling inhibitor monensin, suggesting that mutant EGFRs preferentially traffic through the endocytic recycling compartments. Importantly, monensin treatment enhanced the mutant EGFR association and colocalization with Src, indicating that aberrant transit through the endocytic recycling compartment promotes mutant EGFR-Src association. Conclusion: The findings presented in this study show that mutant EGFRs undergo aberrant traffic into the endocytic recycling compartment which allows mutant EGFRs to engage in a preferential interaction with Src, a critical partner for EGFR-mediated oncogenesis.

There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types—a K5 ⁺ /K19 ⁻ type and a K5 ⁺ /K19 ⁺ type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

Background: The C-terminal Eps15 homology domain-containing protein 1 (EHD1) is ubiquitously expressed and regulates the endocytic trafficking and recycling of membrane components and several transmembrane receptors. To elucidate the function of EHD1 in mammalian development, we generated Ehd1 ^-/- mice using a Cre/loxP system. Results: Both male and female Ehd1 ^-/- mice survived at sub-Mendelian ratios. A proportion of Ehd1 ^-/- mice were viable and showed smaller size at birth, which continued into adulthood. Ehd1 ^-/- adult males were infertile and displayed decreased testis size, whereas Ehd1 ^-/- females were fertile. In situ hybridization and immunohistochemistry of developing wildtype mouse testes revealed EHD1 expression in most cells of the seminiferous epithelia. Histopathology revealed abnormal spermatogenesis in the seminiferous tubules and the absence of mature spermatozoa in the epididymides of Ehd1 ^-/- males. Seminiferous tubules showed disruption of the normal spermatogenic cycle with abnormal acrosomal development on round spermatids, clumping of acrosomes, misaligned spermatids and the absence of normal elongated spermatids in Ehd1 ^-/- males. Light and electron microscopy analyses indicated that elongated spermatids were abnormally phagocytosed by Sertoli cells in Ehd1 ^-/- mice. Conclusions: Contrary to a previous report, these results demonstrate an important role for EHD1 in pre- and post-natal development with a specific role in spermatogenesis.

3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition 3D hydrogel breast cancer models for studying the effects of hypoxia on epithelial to mesenchymal transition

The integration of HPV DNA into human chromosomes plays a pivotal role in the onset of papillomavirus-related cancers. HPV DNA integration often occurs by linearizing the viral DNA in the E1/E2 region, resulting in the loss of a critical viral early polyadenylation signal (PAS), which is essential for the polyadenylation of the E6E7 bicistronic transcripts and for the expression of the viral E6 and E7 oncogenes. Here, we provide compelling evidence that, despite the presence of numerous integrated viral DNA copies, virus-host fusion transcripts originate from only a single integrated HPV DNA in HPV16 and HPV18 cervical cancers and cervical cancer-derived cell lines. The host genomic elements neighboring the integrated HPV DNA are critical for the efficient expression of the viral oncogenes that leads to clonal cell expansion. The fusion RNAs that are produced use a host RNA polyadenylation signal downstream of the integration site, and almost all involve splicing to host sequences. In cell culture, siRNAs specifically targeting the host portion of the virus-host fusion transcripts effectively silenced viral E6 and E7 expression. This, in turn, inhibited cell growth and promoted cell senescence in HPV16+ CaSki and HPV18+ HeLa cells. Showing that HPV E6 and E7 expression from a single integration site is instrumental in clonal cell expansion sheds new light on the mechanisms of HPV-induced carcinogenesis and could be used for the development of precision medicine tailored to combat HPV-related malignancies. IMPORTANCE: Persistent oncogenic HPV infections lead to viral DNA integration into the human genome and the development of cervical, anogenital, and oropharyngeal cancers. The expression of the viral E6 and E7 oncogenes plays a key role in cell transformation and tumorigenesis. However, how E6 and E7 could be expressed from the integrated viral DNA which often lacks a viral polyadenylation signal in the cancer cells remains unknown. By analyzing the integrated HPV DNA sites and expressed HPV RNAs in cervical cancer tissues and cell lines, we show that HPV oncogenes are expressed from only one of multiple chromosomal HPV DNA integrated copies. A host polyadenylation signal downstream of the integrated viral DNA is used for polyadenylation and stabilization of the virus-host chimeric RNAs, making the oncogenic transcripts targetable by siRNAs. This observation provides further understanding of the tumorigenic mechanism of HPV integration and suggests possible therapeutic strategies for the development of precision medicine for HPV cancers.