Roles of CCN5 in regulating progression and therapeutic sensitivity of breast cancer

Breast cancer is one of the deadliest malignancies worldwide and also in the United States. Patients with triple negative breast cancer (TNBC), where the cancer cells do not express nuclear hormone receptors and human epidermal growth factor receptor 2 (HER2), have worse survival rate compared to the patients with luminal subtypes of cancer. Here, we have shown that Cysteine-rich 61-Connective Tissue Growth Factor-nephroblastoma-overexpressed 5 (CCN5) induces growth arrest of TNBC cells in-vitro and in xenograft tumors. Our studies show that after being secreted into the extracellular matrix, CCN5 binds to the α6β1 integrins of the cells leading to inhibition of the PI3K-AKT signaling pathway. This leads to stabilization and nuclear localization of FOXO3A resulting in transcriptional activation of the cyclin-dependent kinase inhibitor P27KIP1. Also, we found that the CCN5-induced PI3K-AKT inactivation leads to stabilization and nuclear accumulation of P27KIP1 resulting in cell cycle arrest of TNBC cells. Next, we have shown that CCN5 protein can induce expression of estrogen receptor-α (ER-α) in mammary epithelial cells. We found that mammary epithelium-specific overexpression of CCN5 in transgenic mice leads to an increase in ER-α expression and that this impact of CCN5 is not restricted to the normal cells. CCN5 treatment leads to an expression of functional ER-α in the TNBC cells, both in-vitro and in xenograft models, and sensitizes these cells to tamoxifen, commonly used for endocrine therapies. Mechanistically, transcriptional activation of ER-α by CCN5 is also mediated by FOXO3A stabilization via PI3K-AKT inhibition. Lack of ER-α expression in TNBC cells or loss of ER-α activation after endocrine treatment of luminal cancers makes these breast cancer cells resistant to tamoxifen and other endocrine therapies. Evidently, CCN5-mediated restoration of ER-α and its downstream signaling cascades renders the TNBC cells sensitive to tamoxifen. As these tumors mostly lack CCN5 expression, we anticipate that restoration of CCN5 expression might be able to provide breakthroughs in the treatment of these tumors. Finally, we discuss the effects of CCN5 expression on yet another aggressive breast cancer subtype, characterized by HER2 overexpression. Mammary-specific expression of CCN5 in HER2 overexpressing mice delays tumor progression significantly and reduces the tumor burden. Initial observations indicate that CCN5 induces expression of P16INK4A and P19ARF, resulting in cell cycle arrest of the tumor cells. Collectively, these studies suggest that CCN5 restoration can be beneficial for the better management of breast cancer progression

Interaction of Chandipura Virus N and P Proteins: Identification of Two Mutually Exclusive Domains of N Involved in Interaction with P

The nucleocapsid protein (N) and the phosphoprotein (P) of nonsegmented negative-strand (NNS) RNA viruses interact with each other to accomplish two crucial events necessary for the viral replication cycle. First, the P protein binds to the aggregation prone nascent N molecules maintaining them in a soluble monomeric (N0) form (N0-P complex). It is this form that is competent for specific encapsidation of the viral genome. Second, the P protein binds to oligomeric N in the nucleoprotein complex (N-RNA-P complex), and thereby facilitates the recruitment of the viral polymerase (L) onto its template. All previous attempts to study these complexes relied on co-expression of the two proteins in diverse systems. In this study, we have characterised these different modes of N-P interaction in detail and for the first time have been able to reconstitute these complexes individually in vitro in the chandipura virus (CHPV), a human pathogenic NNS RNA virus. Using a battery of truncated mutants of the N protein, we have been able to identify two mutually exclusive domains of N involved in differential interaction with the P protein. An unique N-terminal binding site, comprising of amino acids (aa) 1–180 form the N0-P interacting region, whereas, C-terminal residues spanning aa 320–390 is instrumental in N-RNA-P interactions. Significantly, the ex-vivo data also supports these observations. Based on these results, we suggest that the P protein acts as N-specific chaperone and thereby partially masking the N-N self-association region, which leads to the specific recognition of viral genome RNA by N0

N protein utilizes two separate domains for interacting with P in its monomeric and oligomeric forms.

<p>N-terminally His-tagged P protein (His-P) was allowed to interact with either wild-type N or different N mutants in 100 mM NaCl TET buffer containing 10 mM Imidazole for 30 minutes at 4°C. Reaction mixtures were applied to Ni-NTA column and elution profile assayed by silver staining. L- loading; F- flow through; W- 10 mM Imidazole wash; E- 250 mM Imidazole elution. (A) In the absence of 1% DOC treatment. Bovine Serum Albumin (BSA) was used as negative control. (B) Wild-type N or N mutants were pre-incubated with 1% DOC for 30 minutes, followed by dialysis in presence of His-P, before applying to Ni-NTA column. Samples were resolved in 12% SDS-PAGE and visualised by Coomasie brilliant blue staining.</p

Oligonucleotides used for the construction of the GFP fused truncated N proteins.

<p>Oligonucleotides used for the construction of the GFP fused truncated N proteins.</p

Schematic representation of the domains of CHPV N involved in interaction with P and their functional importance.

<p>Binding of P to nascent N masks the N-N self association region of CHPV N (N⁰-P complex formation) and also blocks non-specific RNA binding (upper panel). N⁰ is capable of specifically recognizing the viral leader sequence and the C-terminal 102 amino acids are essential for this recognition. Therefore, in the monomeric form, N specifically recognizes the leader RNA, to form the nucleation complex. Subsequently, the process of N-N self-association begins and P is released. Upon oligomerization, a new RNA binding cavity is formed utilizing the N-terminal arm (1–47 aa) and the central region of N (lower panel). Thus, the phase of non-specific encapsidation begins. Once nucleocapsids have formed, P can again interact with N, this time with the C-terminal region of oligomeric N, to usher the viral polymerase (L) onto its template.</p

Soluble-insoluble fractionation and sucrose density gradient centrifugation.

<p>Stoichiometry of N and P ratio is important for the N specific chaperone activity of P. (A) Total (Tot), Soluble (Sol) and Insoluble (Pel) fractionation of Vero-76 cells transfected with different ratios of pEGFP-C1 N and pCDNA 3.1 (+) P at 24 hours post-transfection. N and P proteins were detected by immunoblotting with N and P Ab respectively. It is evident that a 1∶0.5 N-P ratio is incapable of solubilising the otherwise insoluble N; however, a 1∶1 ratio can do so. GAPDH was used as a loading control. (B) Oligomerization status of soluble N. Sucrose density gradient centrifugation of the soluble fraction of cells transfected with 1∶1 ratio of GFP-N and P constructs. Fractions were collected from the bottom of the tube, and alternative fractions were immunobloted with N and P Abs. The curve shows the band intensities representing distribution of GFP-N and P against the fraction number. While majority of the soluble fraction of N is found in the monomeric form (fraction 17), a substantial amount is also found in fraction 7, indicating decameric forms. P is found to interact with both the populations of N. However, other stoichiometries of homo-oligomerization cannot be ruled out (fractions 13 through 17).</p

<i>Ex vivo</i> expression and immunoprecipitation of different N mutants with P.

<p>(A) Intra-cellular distribution of different mutants of N used in this study. Vero-76 cells were transfected with 2 µg of pEGFP-C1 constructs of each mutant. N-terminal deletants N(48–422) and N(180–422) exhibits smooth distribution. N(1–47) also exhibits smooth distribution, probably because of the large GFP-tag, which interferes with its oligomerization. The bar represents 5 µm. (B) Co-expression of wild-type N and different N mutants with P protein in Vero-76 cells. Co-expression was confirmed by immunobloting with N and P Abs (upper and lower panels, respectively). All of the mutants used for this study expresses satisfactorily, and is of the right relative size. (C) Co-immunoprecipitation of wild-type N and different N mutants with P protein. Vero-76 cells were co-transfected with 2 µg of both plasmids, labelled with L-Methionine-³⁵S 24 hours post-transfection followed by immunoprecipitation with P Ab. Except for N(180–422), all mutants co-immunoprecipitate with P.</p

CHPV N and P proteins interact differentially in transfected cells depending on their stoichiometric availabilities.

<p>(A) Vero-76 cells were transfected with 2 µg pCDNA 3.1 (+) N and immunofluorescence performed with N-Ab, 24 hours post transfection. N exhibits a punctate distribution in the cytoplasm. (B) Immunofluorescence of Vero-76 cells transfected with pCDNA 3.1 (+) P, with P-Ab. P exhibits a smooth distribution in the cytoplasm. (C) GFP fluorescence of Vero-76 cells transfected with pEGFP-C1 N. GFP-tagged N maintains its punctuated distribution. (D) GFP fluorescence of Vero-76 cells transfected with pEGFP-C1 vector alone. GFP alone shows characteristic smooth fluorescence throughout the cell. (E to G) Vero-76 cells co-transfected with pEGFP-C1 N and pCDNA 3.1 (+) P in a 1∶1 ratio. P was detected by immunofluorescence (F). Colocalization of GFP-N with P is shown in the merged image (G). Co-expression with P redistributes the otherwise punctuated N into a more homogenous fluorescence. (H to J) Co-transfection in a 1∶0.5 ratio. The lower abundance of P is insufficient to homogenise the punctuated distribution of N (H). Immunofluorescence against P reveals colocalization of P with oligomeric forms of N (I and J). All data were captured on a laser scanning confocal microscope (Carl Zeiss). All Immunofluorescence were performed with anti-rabbit TRITC conjugated secondary antibody. 2 µg of DNA was used for all transfection, except for H, I and J where 1 µg of pCDNA 3.1 (+) P was used. The bar represents 5 µm.</p

MIND model for triple-negative breast cancer in syngeneic mice for quick and sequential progression analysis of lung metastasis

<div><p>Mouse models of breast cancer with specific molecular subtypes (e.g., ER or HER2 positive) in an immunocompetent or an immunocompromised environment significantly contribute to our understanding of cancer biology, despite some limitations, and they give insight into targeted therapies. However, an ideal triple-negative breast cancer (TNBC) mouse model is lacking. What has been missing in the TNBC mouse model is a sequential progression of the disease in an essential native microenvironment. This notion inspired us to develop a TNBC-model in syngeneic mice using a mammary intraductal (MIND) method. To achieve this goal, Mvt-1and 4T1 TNBC mouse cell lines were injected into the mammary ducts via nipples of FVB/N mice and BALB/c wild-type immunocompetent mice, respectively. We established that the TNBC-MIND model in syngeneic mice could epitomize all breast cancer progression stages and metastasis into the lungs via lymphatic or hematogenous dissemination within four weeks. Collectively, the syngeneic mouse-TNBC-MIND model may serve as a unique platform for further investigation of the underlying mechanisms of TNBC growth and therapies.</p></div

Schematic representation of the timescale of the progression of the TNBC from DCIS to metastatic growth in lungs in syngeneic mouse MIND model.

<p>Schematic representation of the timescale of the progression of the TNBC from DCIS to metastatic growth in lungs in syngeneic mouse MIND model.</p